Universidade de Aveiro Departamento de Biologia
2011
Sónia Marina Pinto
Nunes da Silva
TOXICIDADE DO ALUMÍNIO EM TRIGO E CENTEIO
ALUMINIUM TOXICITY IN WHEAT AND RYE
Universidade de Aveiro Departamento de Biologia
2011
Sónia Marina Pinto
Nunes da Silva
TOXICIDADE DO ALUMÍNIO EM TRIGO E CENTEIO
ALUMINIUM TOXICITY IN WHEAT AND RYE
Tese apresentada à Universidade de Aveiro para cumprimento dos requisitos
necessários à obtenção do grau de Doutor em Biologia, realizada sob a
orientação científica da Professora Doutora Conceição Santos, Professora
Associada com Agregação do Departamento de Biologia da Universidade de
Aveiro e co-orientação científica da Professora Doutora Olinda Pinto Carnide,
Professora Catedrática do Departamento de Genética e Biotecnologia da
Universidade de Trás-os-Montes e Alto Douro.
Apoio financeiro da FCT e do
POPH/FSE.
Dedico esta Tese ao meu verdadeiro companheiro e à minha mãe.
o júri
presidente
Prof. Doutor Anibal Guimarães da Costa
Professor Catedrático do Departamento de Engenharia Civil da Universidade de Aveiro
Prof. Doutor Olinda da Conceição Pinto Carnide
Professora Catedrática do Departamento de Genética e Biotecnologia da Universidade de Trás-osMontes e Alto Douro
(co-orientadora)
Prof. Doutor Amadeu Mortágua Velho da Maia Soares
Professor Catedrático do Departamento de Biologia da Universidade de Aveiro
Prof. Doutor António Carlos Matias Correia
Professor Catedrático do Departamento de Biologia da Universidade de Aveiro
Prof. Doutor Anabela Maria Lopes Romano
Professora Associada com Agregação da Faculdade de Ciências e Tecnologia da Universidade do
Algarve
Prof. Doutor Maria da Conceição Lopes Vieira dos Santos
Professora Associada com Agregação do Departamento de Biologia da Universidade de Aveiro
(orientadora)
Prof. Doutor Jorge Manuel Pataca Leal Canhoto
Professor Auxiliar com Agregação da Faculdade de Ciências e Tecnologia da Universidade de
Coimbra
Prof. Doutor Maria Manuela Outeiro Correia de Matos
Professora Auxiliar do Departamento de Genética e Biotecnologia da Universidade de Trás-osMontes e Alto Douro
agradecimentos/
acknowledgements
This PhD Thesis is in part the result of a FCT Project
(FCT/POCI/AGR/58174/04) between the Institute of Genetics and
Biotechnology/Centre of Genetics and Biotechnology-UTAD and the Laboratory
of Biotechnology and Cytomics (Department of Biology)/CESAM.
I would like to express my gratitude to my major professors, Conceição Santos
(University of Aveiro/CESAM) and Olinda Carnide (University of Trás-osMontes and Alto Douro) for giving me the necessary guidance, support and
encouragement.
I wish to express my gratefulness to all those friends that have been by my side
giving me encouragement through all the years, special the colleagues from the
Laboratory of Biotechnology and Cytomics.
In special I would like to express my deepest gratitude to Glória Pinto, which
was always available to help, hear and advise me. Thanks for the friendship
and for always been present.
My gratitude goes also to Armando Costa, for his technical support and help in
the laboratory, but especially for his patience.
The foundation for Science and technology (FCT) is thanked for supporting this
work providing the PhD fellowship no. SFRH/BD/32257/2006.
palavras-chave
Alumínio (Al), Al-tolerância, Al-sensibilidade, calose, centeio, ciclo celular,
exposição, fotossíntese, histologia, nutrientes, raiz, Secale cereale, stress
oxidativo, trigo, Triticum aestivum.
resumo
Com o presente trabalho pretendeu-se determinar e compreender melhor
quais os alvos do Alumínio (Al) nas plantas, e contribuir para um melhor
entendimento dos mecanismos de tolerância presentes em genótipos com
elevado grau de tolerância ao Al.
O Al é um dos maiores constituintes do solo e torna-se biodisponível em solos
com baixo pH. Nesses casos, a exposição ao Al afecta negativamente o
crescimento das plantas conduzindo a uma diminuição da produção. Estes
factos são especialmente visíveis nos cereais, sendo a exposição ao Al uma
das principais causas das quebras de produção nestas espécies.
O Capítulo I consiste numa revisão geral sobre a toxicidade do Al nas plantas,
apontando os seus principais alvos. Apresenta também os mecanismos de
resistência, que inclui Al-destoxificação externa e interna, em diferentes
espécies.
O Capítulo II aborda os estudos sobre a exposição de curto prazo ao Al em
duas espécies de cereais: Triticum aestivum L. e Secale cereale L., tendo-se
sempre utilizado um genótipo Al-tolerante e um Al-sensível para cada espécie.
Este capítulo está dividido em três estudos: no Capítulo II.1 realça-se o efeito
da exposição a 185 µM de Al no equilíbrio nutricional em trigo. Verificou-se que
em ambos os genótipos (sensível e tolerante) o perfil de macro e micro
nutrientes se alterou, tendo uma interferência negativa, sobretudo no nível de
P, Mg e K. Além disso, registaram-se diferenças na diferenciação da
endoderme consoante o grau de tolerância/sensibilidade do genótipo. No
Capítulo II.2 apresenta-se uma visão mais abrangente dos efeitos da
exposição a 185 µM de Al em trigo, incluindo parâmetros fisiológicos,
estruturais, citológicos e genotóxicos. Demonstra-se, pela primeira vez, que a
progressão do ciclo celular é diferentemente regulada, dependendo da
tolerância/sensibilidade do genótipo e que, mesmo em zonas já diferenciadas
da raiz a exposição ao Al leva à deposição de calose. O Capítulo II.3 aborda
os efeitos da exposição de 1.1 mM de Al em centeio, numa perspectiva
bastante alargada. Apresenta-se o desequilíbrio nutricional, sobretudo no
genótipo sensível, assim como a translocação de Al para a parte aérea nesse
mesmo genótipo. Analisa-se também o comportamento de ambos os genótipos
no que se refere ao ciclo celular, diferenciação da endoderme, crescimento
radicular, reservas de hidratos de carbono, entre outros. Os resultados
apontam para estratégias bem definidas adoptadas pelo genótipo tolerante de
forma a minimizar a acção do Al no sistema radicular.
resumo (cont.)
O Capítulo III compreende a exposição longa ao Al. Dois genótipos de centeio
com diferentes graus de tolerância ao Al foram expostos a 1.11 mM e 1.85 mM
de Al durante 21 dias, tendo sido usados dois pontos de amostragem (15 e 21
dias). Este capítulo está dividido em dois estudos: No Capítulo III. 1 analisamse os mecanismos antioxidantes (folhas e raízes) como resposta à exposição
ao Al, dando-se especial atenção ao ciclo do ascorbato-glutationas. A
exposição ao Al levou a stress oxidativo e a alterações na actividade de
enzimas antioxidantes e no conteúdo de antioxidantes não-enzimáticos.
Demonstra-se que os dois órgãos apresentam respostas diferentes à
exposição ao Al e que a capacidade de sobreviver em ambientes ricos em Al
depende da eficácia da resposta antioxidante. Para além disso, a resposta do
ciclo ascorbato-glutationas parece estar dependente do tipo de órgão, grau de
tolerância e do tempo de exposição ao Al. No Capítulo III. 2 analisam-se os
efeitos da exposição ao Al na fotossíntese. Verificou-se que o Al afecta
negativamente a taxa fotossintética em ambos os genótipos, embora as
alterações que o Al provoca nas trocas gasosas e no Ciclo de Calvin sejam
dependentes do genótipo. Verificou-se também que os danos no genótipo
sensível surgem mais cedo do que no genótipo tolerante, mas que ambos
apresentam susceptibilidade ao Al após exposição de longo termo.
Por fim, no Capítulo IV são apresentadas as conclusões da Tese de
Doutoramento.
Keywords
Aluminium (Al), Al-tolerance, Al-sensitivity, cell cycle, exposure, histology,
nutrients, oxidative stress, photosynthesis, root, rye, Secale cereale,Triticum
aestivum , wheat.
abstract
The present work aims to better understand which are the targets of aluminium
(Al) in plants, and contribute to a better understanding of the tolerance
mechanisms present in genotypes with high Al tolerance.
Al is a major constituent of the soil and become bioavailable in soils with low
pH. In such cases, exposure to Al adversely affects plant growth and leads to a
decrease in production. These facts are especially evident in crops and
exposure to Al is a major reason for reduction in crops production.
Chapter I present a general review about Al toxicity in plants, pointing out their
main targets. In addition, also presents the mechanisms of resistance, including
external and internal Al-detoxification, shown in different species.
Chapter II focuses on short term Al exposure in two species of cereals: Triticum
aestivum L. and Secale cereal L., having always used one Al-tolerant and one
Al-sensitive genotype for each species. This chapter is divided into three
studies: In Chapter II.1 is enhanced the effect of exposure to 185μM of Al in the
nutrient balance in wheat. It was found that in both genotypes (sensitive and
tolerant) the profile of macro and micronutrients changed, with a negative
interference on the level of P, Mg and K. Also, presents differences in
endoderm differentiation depending on the degree of tolerance / sensitivity of
the genotype. In Chapter II.2 a more comprehensive view of the effects of
exposure to 185 μM Al in wheat is presented. This section includes
physiological, structural, cytological and genotoxic effects. For the first time is
demonstrated that the cell cycle progression is differently regulated depending
on the tolerance / sensitivity of the genotype, and that even in differentiated
root zones Al exposure leads to callose deposition. Chapter II.3 focuses on the
effects of 1.1mm Al exposure in rye, in a very broad perspective. It is presented
nutritional imbalances observed mainly in sensitive genotype and the Al
translocation to shoots in the same genotype. It is also referred the different
behaviour of both genotypes in relation to cell cycle, differentiation of
endodermis, root growth, carbohydrate reserves, among others. All that points
to well-defined strategies taken up by the tolerant genotype, in order to
minimize the action of Al in roots.
abstract (cont.)
Chapter III focuses in long-term Al exposure. Two rye genotypes with different
Al tolerance were exposed to 1.11 mM and 1.85 mM Al during 21 days, having
been used two sampling points (15 and 21 days).This chapter is divided into
two studies: In Chapter III. 1, are analyse the antioxidant mechanisms (leaves
and roots) in response to Al exposure, with particular focus on the ascorbateglutathione cycle. Al exposure leaded to oxidative stress and to changes in the
activity of antioxidant enzymes and in the content of non-enzymatic
antioxidants. Is demonstrated that the two organs have different responses to
Al exposure and that the ability to survive in environments rich in Al depends on
the effectiveness of the antioxidant response. Furthermore, the response of the
ascorbate-glutathione cycle seems to be dependent on organ type, Al-tolerance
and time of Al-exposure. Chapter III. 2 analyze the effects of Al-exposure in
photosynthesis. It is presented that Al negatively affects the photosynthetic rate
in both genotypes, however the Al-induced alterations on gas exchange
parameters and in Calvin cycle are genotype dependent. In this chapter, is
demonstrated that Al induce earlier damages in the sensitive genotype, but
both genotypes showed long-term susceptibility to Al.
Finally, in Chapter IV the general conclusions of the present PhD Thesis are
presented.
Abbreviations:
1
O2: Singlet oxygen
A: Net photosynthetic rate
Al: Aluminium
ALA: 5-aminolevulinic acid
AlCl3: Aluminium chloride
AlCl3·6H2O: Aluminium chloride hexahydride
ALMT1: Aluminium-activated malate transporter
APX: Ascorbate peroxidase
AS6: Rye Al sensitive of Barbela line
AsA: Ascorbate
AT6: Rye Al tolerant of Barbela line
AtALMT1: Arabidopsis homolog of the ALMT1
AtBCB: Arabidopsis blue-copper-binding protein gene
B: Borum
BSA: Bovine serum albumin
Ca: Calcium
CAT: Catalase
Chl a: Chlorophyll a
Chl b: Chlorophyll b
CHM: Nonspecific protein-synthesis inhibitor
Ci/Ca: Intercellular to atmospheric CO2 concentration ratio
CS: Citrate synthase
Cu: Copper
CV: Coefficient of variation
DHA: Dehydroascorbate
DHAR: Dehydroascorbate reductase
DNA: Deoxyribonucleic acid
DTNB: 5,5’-dithiobis-(2-nitrobenzoic acid)
DTT: Dithiothreitol
DTZ: Distal transition zone
DW: Dry weight
E: Transpiration rate
EDTA: Ethylenediamine tetraacetic acid
ETR: Electron transport rate
EZ: Elongation zone
F0: Minimal fluorescence
Fe: Iron
FeCl3: Iron chloride
FCM: Flow cytometry
Fm: Maximal fluorescence
FPCV: Full peak coefficient of variation
Fv: Variable fluorescence
FW: Fresh weight
G-POX: Guaiacol peroxidase
GR: Gluthatione reductase
gs: stomatal conductance
GSH: Gluthatione
GSHt: Total gluthatione
GSSG: Oxized glutathione
GST: Glutathione-S-transferase
H+: Hydrogen
HCL: Chloride acid
H2O2: Hydrogen peroxide
H2SO4: Sulfuric acid
HZ: Hairy root zone
IRGA: Infra-red gas analyser
K+: Potassium
KCl: Potassium chloride
MATE: Mmultidrug and toxic compound exudation
MDA: malondialdehyde
MDH: Malate dehydrogenase
MDHAR: Monodehydroascorbate reductase
Mg2+: Magnesium
MgCl2: Magnesium chloride
Mn: Manganese
MZ: Meristematic zone
N2: Liquid nitrogen
NAD(P)H: Nicotinamide adenine (phosphate) dinucleotide
NsHCO3: Sodium bicarbonate
NBT: Nitro-blue tetrazolium chloride
NH4+: Ammonium
NtGDI1: Tobacco GDP-dissociation inhibitor gene
NtPox: Tobacco peroxidase gene
O2-: Superoxide radical
.
OH: Hydroxyl radical
P: Phosphorus
parB: Tobacco glutathione S-transferase gene
PAS: Periodic acid-Schiff reaction
PEPC: Phosphoenolpyruvate carboxylase
PGI: Phosphoglucose isomerase
PI: Propidium iodide
PIPES: Piperazine-N,N’-bis-2-ethanesulfonic
qp: Photochemical quenching
qN: Non-photochemical quenching
RO.: Alkoxyl radical
ROS: Reactive oxygen species
RuBisCo: Ribulose-1,5-bisphosphate carboxylase/oxigenase
RWC: Relative water content
SD: Standard deviation
SE: Standard error
sFBPase: Stromal fructose-1,6-bisphosphatase
Si: Silicon
SOD: Superoxide dismutase
TCA: Trichloroacetic acid
Zn: Zinc
ΦPSII: Effective photochemical efficiency of PSII
INDEX
Jury
Acknowledgments
Resumo
Abstract
Abbreviations
CHAPTER I
ALUMINIUM TOXICITY IN PLANTS: A GENERAL APPROACH
I.1 Aluminium Chemistry in the Soil
3
I.2 Aluminium Toxicity
4
I.2.1 Root Growth
4
I.2.2 Oxidative Stress
6
I.2.3 Cell Wall, Plasma Membrane and Nutrient Unbalances
9
I.2.4 Cytoplasmatic Ca2+
11
I.2.5 Callose
12
I.2.6 Others
12
I.3 Aluminium Resistance
13
I.3.1 External Detoxification
13
I.3.1.1 Organic Acids
13
I.3.1.2 Phenolic Compounds
16
I.3.1.3 Root Mucilage
17
I.3.2 Internal Detoxification
18
I.4 Model Species
19
I.5 Aims
21
References
22
CHAPTER II
SHORT-TERM ALUMINIUM EXPOSURE
II.1: Differential aluminium changes on nutrient accumulation and root
differentiation in Al sensitive vs tolerant wheat
Abstract
39
Introduction
41
Material and Methods
42
Results
44
Discussion and Conclusions
50
References
54
II.2: Different structural and functional responses of sensitive vs tolerant wheat
roots during Al exposure and recovery
Abstract
61
Introduction
63
Material and Methods
65
Results
68
Discussion and Conclusions
78
References
83
II.3: Al toxicity mechanisms in tolerant and sensitive rye genotypes: a
comprehensive physiological and histological study
Abstract
91
Introduction
93
Material and Methods
95
Results
98
Discussion and Conclusions
108
References
112
CHAPTER III
LONG-TERM ALUMINIUM EXPOSURE
III.1: Antioxidant mechanisms in response to Al of tolerant and sensitive rye
genotypes
Abstract
121
Introduction
123
Material and Methods
125
Results
129
Discussion and Conclusions
138
References
142
III.2: Aluminium long-term stress differently affects photosynthesis in rye
genotypes
Abstract
149
Introduction
151
Material and Methods
153
Results
156
Discussion and Conclusions
163
References
167
CHAPTER IV
CONCLUDING REMARKS
Short-term Al Exposure
173
Long-term Al Exposure
175
Challenges for the Future
177
Aluminium toxicity in wheat and rye
CHAPTER I
ALUMINIUM TOXICITY IN PLANTS: A GENERAL APPROACH
1
Aluminium toxicity in wheat and rye
2
Aluminium toxicity in wheat and rye
I.1 Aluminium Chemistry in the Soil
Aluminium (Al) ranks third in abundance among the Earth’s crust elements, after oxygen
and silicon, and is the most abundant metallic element. A large amount of Al is
incorporated into aluminosilicate soil minerals and very small quantities appear in the
soluble form, capable of influencing biological systems (May and Nordstrom, 1991).
Al bioavailability and, in consequence toxicity, is mainly restricted to acid environments.
Acid soils (with a pH of 5.5 or lower) are among the most important limitations to
agricultural production. The production of staple food crops, in particular grain crops, is
negatively influenced by acid soils (Kochian et al., 2005). It has been estimated that 15%
of the world´s soil is acidic (http://www.fao.org/ag/agl/agll/terrastat/#terrastatlinks)
and that over 50% of the world’s potentially arable lands are acidic (von Uexküll and
Mutert, 1995; Panda et al., 2009). In Portugal, 80% of soils have pH lower than 6.5 and
more than 40% lower than 5.5 (Almeida, 1955) and are located mostly in the Northern
regions (Fig. 1). Furthermore, 60% of acid soils in the world are located in the humid
tropics and subtropics. Thus, in many developing countries, acid soils limit crop
production. In developed countries, some agricultural practices, as removal of products
from the farm or paddock, leaching of nitrogen below the plant root zone, inappropriate
use of nitrogenous fertilizers and build up in organic matter are causing further
acidification of agricultural soils (http://www.dpi.nsw.gov.au/).
When pH drops below 5.5, aluminosilicate clays and aluminium hydroxide minerals begin
to dissolve, releasing aluminium-hydroxy cations and Al(H2O)63+ (Al3+), that then
exchange with other cations. The chemistry of Al3+ in soil solution is complicated by the
fact that soluble inorganic (such sulfate and fluoride) and organic ligands form complexes
with Al3+. Whether a ligand increases or decrease aluminium solubility depends on the
particular aluminium-ligand complex and its tendency to remain in solution or precipitate
(McBride, 1994; Panda and Matsumoto, 2007). On that conditions, Al3+ also forms the
mononuclear species AlOH2+, Al(OH)2+, Al(OH)3 and Al(OH)4 (Panda and Matsumoto,
2007). For wheat roots Al species are toxic in the following order: AlF2+ ˂ AlF2+ ˂ Al3+ ˂
3
Aluminium toxicity in wheat and rye
Al13 (AlO4Al12(OH)24(H2O)127+) (Massor-Pietraszewska, 2001). The mononuclear Al3+
species and Al13 are considered as the most toxic forms (Kinraide, 1991; Kochian, 1995).
Fig. 1. Distribution of acid soils in Portugal
(http://www.iambiente.pt/)
I.2 Aluminium Toxicity
I.2.1 Root Growth
A major consequence of Al toxicity is the inhibition of root growth, and this outcome has
been reported during the last century (e.g. Eisenmenger, 1935) for innumerous species
(Jan, 1991; Ryan et al., 1993; Shen et al., 1993; Crowford and Wilkens, 1998; Ahn et al.,
2002; Ma et al., 2002; Kikui et al., 2005; Abdel-Basset et al., 2010). Consequently, root
growth inhibition has been widely used to assess Al toxicity.
Root growth is the combination of cell division and elongation. Only during the last
decade, researchers started to look at the cell cycle (de)regulation induced by Al, with
some works focusing unbalances on mitosis phase, and very few on other interphase
phases (e.g. Silva et al., 2000). Decrease of mitotic activity was reported as a consequence
of Al exposure in root tips of several species as: wheat (Frantzios et al., 2001; Li et al.,
2008), maize (Doncheva et al., 2005), barley (Budikova and Durcekova, 2004), and oat
(Marienfeld et al., 1995). Some authors defended that inhibition of cell elongation was the
primary mechanism leading to root growth inhibition (Čiamporová, 2002; Zheng and
4
Aluminium toxicity in wheat and rye
Yang, 2005). The reason for that is that root growth inhibition could occur within a short
time period - 30 min in Al-sensitive maize (Llugany et al., 1995) - and that cell division is
a slow process (cell cycle takes usually several hours to be completed). However,
Doncheva et al (2005) reported inhibition of cell division (decrease of S-phase cells) in the
proximal meristem after 5 min Al exposure and inhibition of root cell division in the apical
meristem within 10 or 30 minutes. Furthermore, Al can accumulate in the nuclei of cells in
the meristematic region of the root tip within 30 minutes (Silva et al., 2000). So, although
root elongation inhibition is a primary target of Al toxicity, it does not preclude fast Al
effects on root cell division (Doncheva et al., 2005). Therefore, whereas inhibition of cell
elongation or cell division is the primary mechanism leading to root growth inhibition is
still unclear. Recently, Yi et al (2009) reported that Al exposure leaded to abnormal
progress through mitosis and induced micronuclei formation in Vicia faba roots, which is
in agreement with Al-induced chromosome aberrations found in wheat roots (Bulanova et
al., 2001) and Al-induced chromosome stickness and breacks in Oryza sativa (Mohanty et
al., 2004). From the literature review, it is evident that Al leads to cell cycle unbalances,
but many questions still remain to clarify. For example it still remains unclear how and
where, Al exerts its influence throughout the cell cycle, if these changes are species and
region dependent (most studies are performed in root apices), how the putative changes are
exerted through time and/or if they may be reversible after Al removal.
The root growth inhibition and increase in root diameter observed in roots exposed to Al
(Zobel et al., 2007) suggested that plant cytoskeleton could be a cellular target of Al
phytotoxicity (Blancaflor et al., 1998). Blancaflor et al (1998) and Horst et al (1999)
studied Al-induced effects on microtubules and actin microfilaments and showed that
microtubules and microfilaments are altered, in their stability, organization and
polymerization, when exposed to Al. Also, in Triticum turgidum Al treatment leaded to
disorganization of actin filaments and formation of actin deposits (Frantzios, 2005).
Recently, Zhang et al (2007a) showed that Al inhibited actin and profilin genes. Profilin, as
an actin-binding protein, provides cells with the ability to remodel the cytoskeleton
(Krishnan and Moens, 2009). In Arabidopsis thaliana a decrease in profilin expression
resulted in an elongation defect (Ramachandran et al., 2000). Furthermore, Sivaguru et al
(1999) and Čiamporová (2002) showed that organization of cytoskeleton is most sensitive
in the distal transition zone of the root apex, providing evidence that this zone represents a
potential target with respect to Al toxicity.
5
Aluminium toxicity in wheat and rye
The most sensitive root zone to Al toxicity is under great attention. Earlier, it was
hypothesized that root cap played a major role in the mechanism of Al toxicity/protection
(Bennet and Breen, 1991). However, Ryan et al (1993) demonstrated that the removal of
the root cap had no effect on the Al-induced inhibition of root growth in maize.
Furthermore, the same authors also suggested that the meristem is the primary site of Altoxicity. Later, Sivaguru and Horst (1998), applying Al to 1mm root segments, reported
that Al accumulation in the distal transition zone (DTZ: 1-2mm) led to a rapid inhibition of
the root elongation and suggested that this root zone is the primary target of Al in an Alsensitive maize cultivar.
I.2.2 Oxidative Stress
Al-induced oxidative stress and changes in cell wall properties have been suggested as the
two major factors leading to Al toxicity (Yamamoto et al., 2003; Zheng and Yang, 2005).
Oxidative stress occurs when any condition disrupt the cellular redox homeostasis.
Reactive oxygen species (ROS) include the superoxide radical (O2-), the hydroxyl radical
(.OH), the alkoxyl radical (RO.), hydrogen peroxide (H2O2), and singlet oxygen (1O2).
These ROS have the capacity to oxidize cellular components such as lipids, proteins,
enzymes and nucleic acids, leading to cell death. Metals are known to act as catalysts in
ROS production and to induce oxidative damage in plants. Al itself is not a transition metal
and cannot catalyze redox reactions, however, Al exposure leads to oxidative stress
(Yamamoto et al., 2001, 2003; Boscolo et al., 2003; Kuo and Kao, 2003; Jones et al., 2006;
Liu et al., 2008a, b). Because aluminium ions form electrostatic bonds preferentially with
oxygen donor ligands (e.g. carboxylate or phosphate groups), cell wall pectin and the outer
surface of the plasma membrane seem to be major targets of aluminium (Yamamoto et al.,
2001). Al binding to biomembranes lead to rigidification (Jones et al., 2006), which seems
to facilitate the radical chain reactions by iron (Fe) ions and enhance the peroxidation of
lipids (Yamamoto et al., 2003).
Al induction lipid peroxidation has been reported for some species, including barley (Guo
et al., 2004), sorghum (Peixoto et al., 1999), triticale (Liu et al., 2008a), rice (Kuo and Kao,
2003), greengram (Panda et al., 2003) and wheat (Hossain et al., 2005). Yamamoto et al
(2001) found that, for Pisum sativum seedlings treated with Al in a simple Ca solution, Al
accumulation, lipid peroxidation and callose production had a similar distribution on the
6
Aluminium toxicity in wheat and rye
root apex surface and was accompanied by root growth inhibition. However, the loss of
membrane integrity was only detected at the periphery of the cracks on the surface of the
root apex. Furthermore, Yamamoto et al (2003) concluded that the Al enhancement of lipid
peroxidation is an early symptom of Al accumulation and appears to cause partly callose
production, but not root growth inhibition. Later, however, in maize, Al treatment didn´t
induced lipid peroxidation, indicating that lipids are not the primary cellular target of
oxidative stress in maize (Boscolo et al., 2003). So, it seems that cellular target of
oxidative stress depend on plant species.
Plant cells are equipped with a defensive system composed by enzymatic antioxidants
(catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (G-POX), superoxide
dismutase (SOD), monodehydroascorbate reductase (MDHAR), dehydroascorbate
reductase (DHAR), glutathione-S-transferase (GST), and gluthatione reductase (GR) and
nonenzymatic
antioxidants
(ascorbate
(AsA),
glutathione
(GSH),
α-tocopherol,
carotenoids) that help to detoxify the ROS. Some works reported ROS production and
alterations in the antioxidant system as a consequence of Al exposure. In pea seedlings,
ROS production is detected in root apex after two hours of Al exposure and increase with
time exposure (Yamamoto et al., 2003). Furthermore, Yamamoto et al (2002) found, in
pea, that ATP content, respiration and root elongation decreased as consequence of Al
treatment. In maize roots, Al-treatment also lead to increase in ROS production rate in all
epidermal cells, only within 10 min of Al exposure and continued to increase during Al
exposure (Jones et al., 2006). APX and SOD activity increased in roots of both Al-resistant
and Al-sensitive triticale cultivars (with higher magnitude in the sensitive one), but
changes were detected first in the sensitive cultivar (6h) and then in the resistant (12h) (Liu
et al., 2008a). Boscolo et al (2003) reported for maize root tips an increase of SOD and
APX activities, mostly in the sensitive line, and suggested that Al exposure lead to
formation of O2- in a greater quantity than the pre-existent SOD can remove and therefore
induce the cells to initiate SOD synthesis. Furthermore, these authors found that SOD and
APX activity are inversely proportional to root growth rate and, therefore, suggested that
the increase of O2- and H2O2 production are related to Al toxicity. An increase in SOD,
APX and GR activities was reported for greengram seedlings, whereas a decrease in CAT
activity and glutathione and ascorbate contents was also found at higher Al concentrations
(Panda et al., 2003). These authors justified the decrease in CAT activity due to the fact
that this enzyme is photosensitive and, therefore, needs constant synthesis and suggested
7
Aluminium toxicity in wheat and rye
that glutathione and ascorbate may be enable to detoxify the ROS directly (Panda et al.,
2003). Devi et al (2003) found an increase in manganese superoxide dismutase (MnSOD)
activity in both sensitive and tolerant cell lines of tobacco, and in AsA and GSH contents,
mostly in the tolerant line. These data indicated that AsA and GSH seem to be in part
responsible for the tolerance mechanisms of the tolerant line to Al. Activities of SOD,
CAT and APX also increased in roots of tea plants and in cultured tea cells exposed to Al
(Ghanati et al., 2005). That increase seemed to result in increased membrane integrity,
since lipid peroxidation reduced with Al exposure (Ghanati et al., 2005).
These findings reporting increase of antioxidants (enzymatic and nonenzymatic), are
accompanied with others that prove gene regulation associated with oxidative stress. A
large range of genes induced by Al stress have been isolated from a variety of plant
species, and some of them are related to oxidative stress. For example, Ezaki et al (2000)
expressed nine genes derived from Arabidopsis, tobacco, wheat, and yeast in Arabidopsis
ecotype Landsberg. An Arabidopsis blue-copper-binding protein gene (AtBCB), a tobacco
glutathione-S-transferase gene (parB), a tobacco peroxidase gene (NtPox), and a tobacco
GDP-dissociation inhibitor gene (NtGDI1) conferred a degree of resistance to Al:
significative differences in relative root growth, and decrease in Al content and oxidative
damages. They also showed that overexpression of three Al-induced genes in plants
conferred oxidative stress resistance. Furthermore, overexpression of the parB gene
simultaneously conferred resistance to both Al and oxidative stresses. Therefore, Ezaki and
coworkers concluded that some of the genes induced during Al-exposure and oxidative
stresses play protective roles against both stresses. Cancado et al (2005) identified a maize
Al-inducible cDNA encoding a glutathione-S-transferase (GST). Expression of that gene
(GST27.2) was up-regulated in response to various Al concentrations in both Al-tolerant
and Al-sensitive maize lines. Recently, using Al-sensitive Medicago truncatula cultivar
Jemalong genotype A17, 324 genes were up-regulated and 267 genes were down-regulated
after Al exposure (Chandran et al., 2008). Up-regulated genes were enriched in transcripts
involved in cell-wall modification and abiotic and biotic stress responses, while downregulated genes were enriched in transcripts involved in primary metabolism, secondary
metabolism, protein synthesis and processing, and the cell cycle. Known markers of Alinduced gene expression including genes associated with oxidative stress and cell wall
stiffening, were differentially regulated in that study (Chandran et al., 2008). For maize
plants, Al exposure leaded to alteration in gene expression, mostly in the Al sensitive
8
Aluminium toxicity in wheat and rye
genotype. Although in Al-sensitive genotype was altered the expression of more genes,
several Al regulated genes exhibited higher expression in the tolerant genotype (Marom et
al., 2008). So, it is clear that expression of some genes confers Al resistance and
contributes to reduce oxidative stress.
I.2.3 Cell Wall, Plasma Membrane and Nutrient Unbalances
Al accumulation is primarily and predominantly in the root apoplast (30-90% of the total
absorved Al (e.g. Rengel and Reid, 1997; Liu et al., 2008a) of peripherical cells, and is
only very slowly translocated to more central tissues (Marienfeld and Stelzer, 1993;
Marienfeld et al., 2000; Schmohl and Horst, 2000). The primary binding of Al3+ in the
apoplast is probably the pectin matrix, with its negatively charged carboxylic groups
(Chang et al., 1999; Schmohl and Horst, 2000).
Several works reported increases of pectin levels in Al-sensitive genotypes (Van et al.,
1994; Chang et al., 1999; Horst et al., 1999; Schmohl and Horst, 2000; Hossain et al.,
2006; Liu et al., 2008b) and, some, also detected increase in Al contents in the same
sensitive genotypes (Horst et al., 1999; Schmohl and Horst, 2000; Hossain at al, 2006).
These findings indicate that pectin plays a major role in the binding of Al, and suggest that
some of the additional Al accumulation in sensitive genotypes, bound in the newly formed
cell wall pectin (Chang et al., 1999; Schmohl and Horst, 2000; Liu et al., 2008b). Binding
of Al to the pectin matrix and other cell wall constituents could alter cell wall
characteristics and functions such as extensibility (Tabuchi and Matsumoto, 2001),
porosity and enzyme activities thus leading to inhibition of root growth (Schmohl and
Horst, 2000). Another mechanism for Al toxicity targeted to the apoplast invokes a rapid
and irreversible displacement of Ca2+ from cell wall components by Al ions (Tabuchi and
Matsumoto, 2001; Zheng and Yang, 2005).
Accumulation of Al occurs predominantly in the root apoplast. Nevertheless, Al
accumulates also in the symplast and with a fast rate (Marienfeld et al., 2000). Recently,
Xia et al (2010) reported a transporter, Nrat1 (Nramp aluminium transporter 1), specific
for Al3+ localized at the plasma membrane of all rice root tips cells, except epidermal cells.
Those authors referred that the elimination of the Nrat1 enhanced Al-sensitivity, decreased
Al uptake, increased Al binding to cell wall and concluded that this transporter is required
for prior step of final Al detoxification through sequestration of Al into vacuoles.
9
Aluminium toxicity in wheat and rye
Furthermore, given its physicochemical properties, Al can interact strongly with the
negatively charged plasma membrane. Plasma membrane usually possesses two types of
membrane potential. One reflects the unbalance in ions concentration inside the cell and
the other is caused by the net concentration of fixe ions on the membrane surface (Zheng
and Yang, 2005). Al seems to be able to affect both types of potentials (Ahn et al., 2001;
2002). For instance, Al can displace other cations (e.g. Ca2+) that may form bridges
between the phospholipid head groups of the membrane bilayer (Akeson and Munns,
1989). Furthermore, Al interaction with plasma membrane could lead to depolarization of
the transmembranar potential (e.g. Kinraide et al., 1992) and/or reduction of H+-ATPase
(e.g. Ahn et al., 2001; 2002) which, in turn, can alter the activities of ions near the plasma
membrane surface and impede the formation and maintenance of the trans-membrane H+
gradient (Kochian et al., 2005). Moreover, Al changes in plasma membrane can modify the
uptake of several cations (e.g. Ca2+, Mg2+, K+, NH4+) (Jan, 1991; Nichol et al., 1993;
Poschenrieder et al., 1995; Mariano and Keltjens, 2005). These changes are related to
direct Al3+ interactions with plasma membrane ion channels (Piñeros and Kochian, 2001)
and changes in membrane potential.
Nutrional unbalances induced by Al exposure were reported for several plant species.
Eleven families of pteridophytes presented different nutritional unbalances (mostly in Ca,
Mg, P, K) depending on Al accumulation (Olivares et al., 2009) and in maize, Al had
negative effects on the uptake of macro and micronutrients, being Ca and Mg the macroand Mn and Zn the micronutrients more affected (Mariano and Keltjens, 2005). Also, the
maize Al-tolerant genotypes accumulated higher concentration of Ca, Mg (Mariano and
Keltjens, 2005) and K (Giannakoula et al., 2008) than the sensitive genotypes. However,
others studies reported different results in Al-induced nutritional imbalances in maize:
Lidon et al (2000) referred that all elements in roots, except K, Mn and Zn, increased in
Al-treated roots and that in shoots Ca and Mg had little variation. Poschenrieder et al.
(1995) reported that only the specific absorption rate of B was correlated to the Al-induced
root growth inhibition. Al exposure leaded to decrease in K, Mg, Ca and P contents and
uptake in rice plants and, as observed in maize, the tolerant cultivar presented less negative
effects in nutrient content than the sensitive one (de Mendonça et al., 2003). In tomato
cultivars, Al exposure decreased the content of Ca, K, Mg, Mn, Fe and Zn in roots, stems
and leaves (Simon et al., 1994). Zobel et al (2007) related changes in fine root diameter
with changes in concentration of some nutrients, as N, P and Al. It seems that the
10
Aluminium toxicity in wheat and rye
differential tolerance to Al may be due to their differences in uptake, ability to keep
adequate concentrations and to use the nutrients efficiently. Differences in nutrient uptake,
accumulation and translocation are evident between plant species and within each species.
Furthermore, since each author utilized different Al concentrations, variated nutritive
solutions and time exposures is difficult to make a general and accurate model of Alinduced nutritional imbalances.
I.2.4 Cytoplasmic Ca2+
Disturbance of cytoplasmic Ca2+ homeostasis is believed to be the primary target of Al
toxicity (Rengel and Zhang, 2003) and may be involved in the inhibition of the cell
division or root elongation by causing potentially disruptions of Ca2+-dependent
biochemical and physiological processes (Sivaguru et al., 1999; Rengel and Zhang, 2003).
In wheat root apices, Jones and Kochian (1995; 1997) found that Al inhibit Ca2+dependent phospholipase C, which acts on the lipid substrate phosphatidylinositol-4,5biphosphate. The authors hypothesized that phosphoinosite signilling pathway might be the
initial target of Al. In accordance, Zhang et al (2007a) found Al-induced inhibition of
genes related to phosphoinosite signilling pathway and hypothesized that the gene
inhibition could result in disruption of this pathway. Also, it was reported that components
of the actin-based cytoskeleton interact directly with phospholipase C in oat (Huang and
Crain, 2009).
Most works reported an increase in cytoplasmic Ca2+ when plants were exposed to Al
(Zhang and Rengel, 1999; Ma et al., 2002; Bhuja et al., 2004). However, Jones et al (1998)
reported a decrease in cytoplasmic Ca2+ in tobacco cell cultures in the presence of Al.
Furthermore, Zhang and Rengel (1999) reported an increase in cytoplasmic Ca2+ in two
lines with different tolerance to Al and correlated it with the inhibition of root growth in
both lines. As well, Ma et al (2002) correlated cytoplasmic Ca2+ to root growth response.
Moreover, alteration in cytoplasmic Ca2+ homeostasis can occur within few minutes (20-30
minutes) in root hair tips of Arabidopsis thaliana (Jones et al., 1998).
It is certain that Al exposure influences cytoplasmic Ca2+ homeostasis, but it is still unclear
if it is a primary cause of Al-induced inhibition of root growth or a secondary effect. The
source of Ca2+ for the increase if cytosolic Ca2+ activity could be extracellular and/or
11
Aluminium toxicity in wheat and rye
intracellular, but is still insufficiently documented, as well the effects on increased
cytosolic Ca2+ (for review see Rengel and Zhang, 2003).
I.2.5 Callose
The induction of callose (1,3-β-D-glucan) formation in Al exposed roots has been reported
in many plant species (e.g. Jorns et al., 1991; Schereiner et al., 1994; Poschenrieder et al.,
1995; Budikova and Durcekova, 2004; Eticha et al., 2005; Tahara et al., 2005; Jones et al.,
2006 ). Al-induced callose formation in root tips is recognized as an excellent indicator of
Al sensibility (Horst et al., 1997; Massot et al., 1999; Meriga et al., 2003; Bhuja et al.,
2004; Hirano et al., 2004; Tahara et al., 2005) and some works negatively correlated root
elongation with callose formation during Al exposure (e.g. Nagy et al., 2004; Tahara et al.,
2005). In maize roots, Jones et al (2006) found a close spatial and temporal coordination
between Al accumulation and callose production in roots. Still, Tahara et al (2005)
reported that, in some Myrtaceae species, induction of callose formation was not
accompanied by root growth inhibition and suggested that callose formation is a more
sensitive indicator to Al than root elongation.
Since Al induces a transient rise of cytosolic Ca2+, an increase of callose accumulation
under Al stress is not unexpected. Cytosolic Ca2+ is one of the prerequisites for the
induction of callose synthesis, but not the only factor modulating increases in callose
synthesis and deposition (Bhuja et al., 2004). Callose formation, as response to Al, is
described in sensitive and, to a lesser extent, in tolerant roots (Horst et al., 1997; Eticha et
al., 2005). In a less extent, callose deposition has been considered as a mechanism to
prevent Al from penetrating into the apoplast. Also, this accumulation is reported to inhibit
the symplastic transport and cell communication by blocking plasmadesmata, avoiding Al
induced lesions in the symplast (Sivaguru et al., 2000). However, callose deposition in
sensitive roots has also been shown to lead to uncontrolled rigidity of cell walls (Jones et
al., 2006) leading ultimately to protoplast degradation.
I.2.6 Others
Al-induced effects/damages are first detected in the root system (Barceló and
Poschenrieder, 2002; Doncheva et al., 2005). Changes in the root system, may affect
12
Aluminium toxicity in wheat and rye
nutrient uptake, which can lead to nutritional deficiencies in shoots and leaves (Moustakas
et al., 1996). Except for Al-accumulator plants, Al accumulates more in roots than in
leaves (Konarska, 2010). In some species, Al-induced alterations in leaves were considered
indirect, since Al accumulation was not detected in leaves (Moustakas et al., 1996).
Nevertheless, alterations in leaves induced by Al-exposure were reported for many species.
Several works reported leaves biomass reduction (Azmat and Hasan, 2008), thickness
(Konarska, 2010), lipid peroxidation (Zhang et al., 2007b), nutritional imbalances (Guo et
al., 2007), changes in the photosynthetic performance (Jiang et al., 2008), changes in
chlorophyll contents (Chen et al., 2005; Zhang et al., 2007; Azmat and Hasan, 2008; Jiang
et al., 2008), among others. Reductions in carbon dioxide (CO2) assimilation rate due to Al
toxicity are reported for several species (Moustakas et al., 1996; Oleksyn et al., 1996;
Lidon et al., 1999; Pereira et al., 2000; Peixoto et al., 2002; Chen et al., 2005; Jiang et al.,
2008) and some works indicated that Al-exposure induced damage of the photosystem II
(Zhang et al., 2007b; Reyes-Dias et al., 2010). Very few works focused on the
consequence of Al-treatment in the carbohydrate metabolism. The effects of Al exposure
on Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) content and activity is
still unclear and the few reports available were performed in citrus (Chen et al., 2005;
Jiang et al., 2008) and in wild rice (Cao et al., 2010).
I.3 Aluminium Resistance
Aluminium resistance is the property which allows a plant to grow with little or no injury
with elevates Al supplies. Resistance strategies can be separated into: a) exclusion of Al
from the root apex; b) Al-tolerance (Al binding and uptake without Al injury) (Panda et al.,
2009; for review see Horst et al., 2010).
I.3.1 External Detoxification
I.3.1.1 Organic Acids
There has been considerable evidence in the literature for an Al-exclusion mechanism
based on carboxylate exudation from the root apex (for review see Barceló and
13
Aluminium toxicity in wheat and rye
Poschenrieder, 2002; Kochian et al., 2005; Horst et al., 2010). The ability of organic acids
to chelate and render Al nonphytotoxic is well established. However, the exudation rate
and the form of organic acid anions exuded depend on plants species and Al-resistance. Al
induced exudation of several organic acids has been described from roots of several
species, as malate in wheat (Ryan et al., 1995; Zheng et al., 1998; Li et al., 2000), citrate in
maize (Pellet at al, 1995), citrate and malate in rye (Li et al., 2002), citrate in soybean
(Yang et al., 2001) and oxalate in buckwheat (Zheng et al., 1998).
In Al-resistant genotypes, the efflux of organic acids may present two patterns (Ma et al.,
2001): I) immediately upon Al exposure (e.g. malate and/or citrate efflux in wheat, bean,
soybean, maize, shrub bushclover); II) requires a lag-time of several hours (e.g. malate
exudation in rye, triticale, Cassia sp.). For pattern I, it seems that Al induce the opening of
anion efflux channels facilitating the organic acid efflux (Delhaize and Ryan, 1995; Ryan
et al., 1995), while it seems that there is some gene activation in the pattern II, with protein
synthesis that may be involved in organic acid metabolism or in the transport of organic
acid anions.
Since organic acids in the cytoplasm are largely deprotonated and exist as anions, the
equilibrium potencial of organic acid anions is much more positive than that of the resting
membrane potencial. So, activation of membrane anion channels will result in an anion (as
organic acids) efflux down the outward electrochemical gradient. Although many types of
organic acids are found in root cells, only one or two are secreted in response to Al
treatment for a given species. This suggests the existence of specific transport systems for
the organic acid anions. In protoplasts from wheat roots apex, Ryan et al (1997) reported
that Al activated an ion channel that allows anion efflux. Later, in maize protoplasts
derived from cortical cells of the DTZ (Kollmeier et al., 2001) and in protoplasts from the
maize hybrid South American 3 (Piñeros et al., 2001), was found that Al activated anion
channel permeable to malate and citrate anions and to citrate, respectively. Also in maize,
Piñeros et al (2002) found Al-induced root citrate release (as far as 5 cm beyond root cap)
within 30 min, where the rate of exudation was positively correlated with the level of Al in
the nutrient solution, and Al-induced plasma membrane anion channel in protoplasts
isolated from stellar or cortical tissues. Later, Yang et al (2006) used a nonspecific proteinsynthesis inhibitor (CHM) to investigate the relationship between Al stress and organic
acid secretion in buckwheat, which presents a typically pattern I behavior. They
demonstrated that the induction of oxalate secretion was rapid, and CHM did not affect the
14
Aluminium toxicity in wheat and rye
process, showing that all the necessary metabolic “machinery” was constitutively
expressed in buckwheat roots, and that oxalate secretion was simply triggered by the Al
stimulus, no induction of a novel protein is required.
The Al response in some species, mostly those that present a lag-time in organic acids
exudation, is accompanied by enhancements of key enzymes in organic acids synthesis, as
malate dehydrogenase (MDH), citrate synthase (CS) (Li et al., 2000; Yang et al., 2001),
and phosphoenolpyruvate carboxylase (PEPC) (Gaume et al., 2001). Furthermore, some
works have shown, that genetic transformation of plants to overexpress enzymes involved
in organic acid synthesis, lead to increased efflux of organic acid and Al resistance (de la
Fuente et al., 1997; Tesfaye et al., 2001; Anoop et al., 2003). However, several studies
reported that the activity of enzymes related to organic acid synthesis, was not enhanced
under Al exposure, even when organic acids efflux occurred. For example, in barley, Zhao
et al (2003) reported an increase in citrate secretion, which was positively correlated with
Al resistance and negatively correlated with Al root apices content. However, the activity
of CS was unaffected by Al in both Al-resistant and Al-sensitive barley varieties. Also, in
triticale were not detected differences in the activity of CS, MDH and PEPC in both
tolerant and sensitive lines (Hayes and Ma, 2003). Furthermore, those authors concluded
that the release of organic acids does not appear to be regulated by their levels in the roots
or by the capacity of the root tissues to synthesize them. Yang et al (2006) used Cassia
tora L., which belongs to pattern II, and observed that CHM inhibited the citrate efflux, de
novo protein synthesis and the CS activity, but Al treatment alone induced significant
exudation of citrate and was not able to affect CS activity. So, these authors suggested that
the enhancement of citrate efflux was mainly caused by the de novo synthesis of anion
channels.
Among the organic acid anions, citrate forms the most stable complexes with Al. The Alcitrate 1:1 complex is not phytotoxic and its transport through the plasmalemma seems to
be very slow (Kochian, 1995). At a 1:1 ratio the Al-oxalate complex also had little toxic
effects in Al-sensitive wheat and the complex prevented Al root accumulation (Ma et al.,
2001). Malate efflux is specifically triggered by Al3+, whereas it is apparently the less
effective mechanism in preventing Al uptake.
The major Al resistance gene in wheat (ALMT1-aluminium-activated malate transporter)
encodes an Al activated malate transporter protein located in the plasma membrane of root
cells, which contains six transmembrane domains with the amino and carboxyl termini
15
Aluminium toxicity in wheat and rye
located on the extracellular side of the plasma membrane (Motoda et al., 2007). ALMT1
confers an Al activated efflux of malate from root apices (Raman et al., 2005). Expression
of ALMT1 of in vitro tobacco cells (Sasaki et al., 2004), Xenopus oocytes (Sasaki et al.,
2004), rice (Sasaki et al., 2004), and transgenic barley (Delhaize et al., 2004), conferred an
Al-activated efflux of malate and in some of them increased resistance to Al. Recently,
transgenic wheat lines with over-expression of the TaALMT1 gene showed increases in
malate efflux and in Al resistance (Pereira et al., 2010). Also, Hoekenga et al (2006)
identified in Arabidopsis a homolog of the ALMT1 (AtALMT1) as an essential factor for Al
resistance. Beyond ALMT1 genes, Al-resistance in some crop species such sorghum
(SbMATE) and barley (HvAACT1), was related to the expression of some genes of the
MATE (multidrug and toxic compound exudation) family based on Al-activated citrate
exudation (Magalhaes, 2010). Based on the homology to these genes (ALMT1 and MATE),
others resistance genes were isolated in others species, such as rye (Collins et al, 2008;
Yokosho et al, 2010), maize (Maron et al, 2010) and common bean (Eticha et al, 2010). It
is clear that Al-resistance mechanisms are controlled by numerous genes, but their
physiology and molecular biology is not well understood (Ryan et al, 2010).
I.3.1.2 Phenolic Compounds
Some studies did not find a correlation between Al resistance and the amount of organic
acid efflux (Piñeros et al., 2005). This indicates that exudation of organic acids may not be
the only mechanism of Al exclusion. Other ligands and mechanisms may also play a role in
this process (Barceló and Poschenrieder, 2002). According to stability constants of metalligand complexes, citrate, oxalate, and malate are more suited for binding Al in acid soil
solutions than simple phenolics, because of the high competition of protons for binding.
However, in flavonoid-type phenolic, the competitive effect of protons is substantially
lower (Barceló and Poschenrieder, 2002). Under the less acidic conditions in the apoplast
or inside plant cells, Al-binding by phenolics may even be more relevant (Tolrà et al.,
2005). Furthermore, the phenolic compounds in presence of carboxylic groups from
organic acids can, by a deprotonation reaction, strengthen the interaction between Al3+ and
the organic acid ligand (Barceló and Poschenrieder, 2002).
Enhancements of phenolic compounds contents were already reported under certain
conditions. It was also detected, by fluorescence spectroscopy, the complexation of Al by
16
Aluminium toxicity in wheat and rye
phelonics released by the roots of Norway spruce (Heim et al., 1999). In maize, Si induced
amelioration of Al toxicity enhanced root exudation of flavonoid-type phenolics (Kidd et
al., 2001; Barceló and Poschenrieder, 2002). Also, in Rumex acetosa L., Tolrà et al (2005)
reported that Al induced high levels of anthraquinone in roots and of catechol, catechin and
rutin in shoots and concluded that phenolics, rather than organic acids, are involved in
detoxification of Al in leaves. Recently, in Matricaria chamomilla plants phenolics
increased in the shoots of plants exposed to Al (Kovacik et al., 2010).
I.3.1.3 Root Mucilage
Rhizodepositions in the form of mucilage has also been implied in Al resistance
mechanisms (Horst, 1995). Root mucilage consists mainly of polysaccharides, and is
exuded from the outer layers of the root cap. It was proposed that one of the roles of
mucilage is the detoxification of toxic metal cations, as Al3+ (Horst et al., 1982). That
ability is due to the uronic acids, in its structural polysaccharides, which adsorb metal
cations. Archambault et al (1996) found that Al bound to the mucilage accounted
approximately 25-35% of nonexchangeable Al, representing a significant pool of
apoplastic Al in wheat. In maize, Li et al (2000) reported that mucilage strongly binds to
Al, and Al bound to mucilage is not phytotoxic. Higher mucilage production was found in
the Al tolerant wheat cultivar (Atlas 66) than in the sensitive (Puthota et al., 1991).
However, the mucilage contribution to Al tolerance is not still understood and is under
debate. Li et al (2000) reported that inhibition of root elongation by Al, was independent of
the presence or absence of mucilage prior to the Al treatment, and concluded that although
mucilage affects the accumulation of Al by roots, it does not confer Al resistance to Z.
mays root apices. Furthermore, in Melastoma malabathricum, an Al accumulator, the
removal of root mucilage reduced the Al concentration in shoots (Watanabe et al., 2008).
Those authors also reported that binding strength between mucilage and Al was very weak,
and that the characteristics of mucilage in adsorption of cations differ between M.
malabathricum and Z. mays. M. malabathricum mucilage has higher adsorption affinities
for trivalent cations, whereas Z. mays mucilage has a higher affinity for divalent cations
(Watanabe et al., 2008). All this together, shows that the rule of root mucilage in Al
detoxification is still unknown.
17
Aluminium toxicity in wheat and rye
I.3.2 Internal Detoxification
Some species have the ability to accumulate high Al concentrations without showing
symptoms of Al toxicity. Al accumulator plants have been defined as those that have
≥1000 mg kg-1 DM Al in aerial tissues, as more recently suggested by Jansen et al (2002)
in agreement with Chenery (1948). However, other authors proposed, in addition to the
refered criterion, the use of Al/Ca ratio to define Al accumulators, principly when Al
concentration is higher than 3000 mg kg-1 DM (Masunaga et al., 1998). Accumulation of
Al in leaves may occur in cell walls or in the leaf vacuoles (Poschenrieder et al., 2008).
One of Al accumulator species is M. malabathricum which growth is even stimulated in
the presence of Al (Watanabe et al., 2008). Also, tea and hydrangea plants are well known
Al accumulators. The concentration of free Al3+ at the pH (app. 7.4) of the symplasm, is
<10–11M because of the pH-dependent hydrolysis of Al in solution and the formation of
insoluble Al(OH)3 (Martin, 1986). Despite low concentration, Al can still be phytotoxic
because of the strong affinity for oxygen ligands (Martin, 1986). This suggests that Al
accumulating plants must have Al3+ internal detoxification.
A number of low-molecular-weight di- and tri-carboxylic acids form strong complexes
with Al3+, which are less phytotoxic than free Al3+ions. Among the ligands that form stable
complexes with Al, organic acids, phenolics and silicon may be implied in Al internal
detoxification (Wenzl et al., 2002; Tolrà et al., 2005; Kovacik et al., 2010; for review see
Ma et al., 2001 and Barceló and Poschenrieder, 2002). For example, considerable fraction
of the total Al accumulation in roots of both Brachiaria decumbens and Brachiaria
ruziziensis was complexed by soluble low-molecular-weight ligands (Wenzl et al., 2002).
In B. decumbens, 90% of organic acids were retained and accumulated within root apices,
whereas in mature root zones were secreted into the growth medium (Wenzl et al., 2002).
This pattern shows that Brachiaria species employ organic acids (citrate and transaconitate) for Al internal detoxification. In buckwheat, Al, once taken up, is chelated
internally by oxalate (forming 1:3 complex) in root cells and when is loaded to the xylem,
ligand exchange from oxalate to citrate (Ma and Hiradate, 2000). When Al is unloaded
from the xylem to the cells of the leaf, citrate ligand exchange to form again Al-oxalate
complex and is stored in the vacuole (Ma et al., 2001).
18
Aluminium toxicity in wheat and rye
I.4 Model species
Although some crops (e.g. pineapple, tea) are considered tolerant to high levels of
exchangeable of Al, for most crops it is a serious constraint. For most crops, fertilization
and attempts of soil correction (e.g. liming) may not be enough per se to reduce Al toxicity
(e.g. as the soil reaction remains strongly acid), and in most target countries these
strategies may also be jeopardized by economical constrains (Marschner et al., 1995).
Therefore, is imperative to fully understand the mechanisms that are used by the Altolerant species to cope Al toxicity, as well which genotypes, within the most
resistant/tolerant cereal species, are more suitable to grow in acidic soils in order to
increase world cereal production. Furthermore, the development of new cultivars (or the
reinvestment in ancient genotypes from Al rich regions) with increased Al-tolerance is
fundamental and economic solution to increase world food productionnn. It is widely
known that species and genotypes within species greatly differ in their tolerance to Al.
Among the cereal species, rye (Secale cereale L.) is one of the most Al-tolerant and wheat
(Triticum aestivum L.) is less tolerant (Aniol and Gustafson, 1984; Pinto-Carnide and
Guedes-Pinto, 2000; Kim et al., 2001). Within rye genotypes, the Portuguese regional
populations showed higher Al-tolerance than others European cultivars/populations, since
they show a great intraspecific variability and a higher Al-tolerance than others European
cultivars (Pinto-Carnide and Guedes-Pinto, 2000). One of them is the Montalegre
population from Trás-os-Montes regions, which is considered an Al-tolerant genotype
(Pinto-Carnide and Guedes-Pinto, 2000). Furthermore, within this population, two lines
were selected based on their Al-tolerance during six generations, at 30 ppm Al
concentration: AS6 (sensitive: without root regrowth) and AT6 (tolerante: with root
regrowth). Other rye genotype is ‘Riodeva’, which is a sensitive inbred line at 150 µM Al
(4 ppm) (Gallego and Benito, 1997). ‘Dankowski Zlote’, a rye Polish cultivar has being
used as tolerant tester (Pinto-Carnide and Guedes-Pinto, 1999; Kim et al., 2001).
Relatively to wheat genotypes, again Portuguese landraces are very tolerant to Al.
‘Barbela’, a very old Portuguese landrace, revealed high Al-tolerance (Pinto-Carnide and
Guedes-Pinto, 1999; Martins-Lopes et al., 2009) and a unique repository of genetic
variability (Guedes-Pinto et al., 1998). Line 7/72, selected from ‘Barbela’ landrace,
showed particular tolerance to Al (Martins-Lopes et al., 2009). On the other hand,
19
Aluminium toxicity in wheat and rye
‘Anahuac’, a Polish cultivar (IHAR-Radzików, Poland) has being used as non-tolerant
tester without root regrowth at 5 ppm Al (Pinto-Carnide and Guedes-Pinto, 1999).
Triticum aestivum L. (common wheat) and Secale cereale (rye), grain cereals, belong to
the Triticeae tribe. Bread wheat is the most widely grown food crop in the world and it
primary use is for bread manufacture. Furthermore, wheat is also used for flour, animal
feed, conversion of wheat starch to ethanol, for wheat beer, biofuel, among others. World
wheat production is currently forecast to reach near 650 million tones (2010), having been
produced 611 and 685 million tones in 2007/08 and in 2008/09, respectively
(http://www.fao.org/docrep/012/ak354e/ak354e00.pdf). According to the Food and
Agriculture Organization (FAO), Europe produced in 2009 more than 228 million tones of
rye, which more than 61 million tones were produced in the Russian Federation. In
Portugal, the production was about 110200 tones. The area cultivated with wheat in 2009
in the world was about 225.5 millions ha, which more than 61 millions ha are located in
Europe (see details in http://faostat.fao.org).
Rey grain is used for flour, bread, beer, some whiskies, vodkas and animal fodder.
According to the same international organization FAO, World rye production was in 2009
approximately 18 million tones, which near 16.5 million tones were produced in Europe. In
Europe, rye production is mainly located in the Eastern region with about 10.5 million
tones produced in 2009. Russian Federation is on the top of the table with about 4.3 million
tones produced. In Portugal, rye production in 2009 was 18300 tones. The world area
cultivated with rye in 2009 was about 6.5 millions ha and in Europe about 5.8 million ha
(see http://faostat.fao.org).
As conclusion, many regions where wheat is grown have acidic problems. This implies
that wheat production has to be improved or alternative crops should be searched. On other
hand, although rye production diminished during the past decades and is not a major crop,
it continues a good food source. Furthermore, rye can resist in extreme conditions, as high
Al external levels, representing an important crop for those critical regions. Here we
highlight the need and urgency to study the Al-tolerance physiology of these related crops
and contribute to their improvement and incorporation in plant breeding strategies.
Most of the studies on Al tolerance using cereal focus rice, barley, maize and wheat
species (e.g. Samac and Tesfaye, 2003). However, most studies are performed with
different media composition (as simple CaCl2 solution (Liu et al., 2008a; Panda et al.,
2010), Hoaglands nutritive solution (Azmat and Hasan, 2008; Giannkoula et al., 2008),
20
Aluminium toxicity in wheat and rye
variations of Hoaglands (Darkó et al., 2004; Guo et al., 2007) among others (Stass et al.,
2008)), Al concentration and period of exposure. This battery of non harmonized
experimental data needs caution during interpretation, mostly concerning generalizations
of functional models. A particular critical issue is the period of Al-exposure, which vary
between some minutes to several weeks. In this thesis, was considered as a short-term
exposure the experiments that last less than one week period and as long-term exposure the
experiments that last more than one week. Comparing, for the same species, short and
longer time exposure would represent an important insight to harmonize some of the
previous data and also clarify different mechanisms involved in the different periods.
I.5 Aims
Considering the above mentioned and the importance of both rye and wheat species, as
well as the unique patrimony of some regional genotypes (e.g. Montalegre rye and
‘Barbela’ wheat), and also considering the high levels of Al present in the most part of the
soils, namely in Portugal, the main aim of this thesis was to study functional aspects
related with Al toxicity (e.g. root growth related with cell cycle unbalances, nutrient
impairment, oxidative stress and carbon metabolism) in tolerant vs sensitive genotypes of
wheat and rye, using some regional genotypes among others.
Most functional aspects studied in this thesis were selected based on the lack of
information in literature (e.g. carbon metabolism), complexity and ambiguity of data
available (nutrient imbalances, cell cycle regulation and oxidative stress).
To achieve this main goal, several restrict hypothesis were designed, considering short
and/or long Al exposures:
a) Al toxicity induces damages in wheat and rye genotypes after short and long-term
exposure.
b) Al differently affects sensitive and tolerant genotypes.
c) Short-term Al exposure leads to nutritional imbalances, cell cycle alterations, root
structure modifications and/or callose deposition.
d) The tolerant genotypes are able to revert more efficiently the damages induced by
Al short-term exposure after Al removal.
e) Al exposure affects the plant oxidative status and the carbon metabolism.
21
Aluminium toxicity in wheat and rye
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Aluminium toxicity in wheat and rye
34
Aluminium toxicity in wheat and rye
CHAPTER II
SHORT-TERM ALUMINIUM EXPOSURE
35
Aluminium toxicity in wheat and rye
36
Aluminium toxicity
oxicity in wheat and rye
II 1: Differential aluminium changes on nutrient accumulation and root differentiation
differe
in
Al sensitive vs tolerant wheat
Chapter published as an original paper in a SCI journal:
Silva S, Pinto-Carnide
Carnide O, Martins
Martins-Lopes P, Matos M, Guedes-Pinto
Pinto H, Santos C. 2010.
20
Differential aluminium changes on nutrient accumulation and root dif
differ
ferentiation in an Al
sensitive vs tolerant wheat. Environmental and Experimental Botany 68, 91-98.
91
37
Aluminium toxicity in wheat and rye
38
Aluminium toxicity in wheat and rye
Abstract
Aluminium (Al) toxicity is particularly evident under acid soil conditions limiting crop
production, namely in wheat (Triticum aestivum). Although largely studied, the effects of
Al on nutrition imbalances and putative correlations with Al tolerance degrees still remain
inconclusive. We investigated the uptake of Al and transport to shoots in one wheat line
and one cultivar differing in their tolerance to Al: ‘Barbela 7/72’ (Al-tolerant) and
‘Anahuac’ (Al-sensitive). Wheat seedlings were exposed for a short period to 185 µM
AlCl3 and then grown in the absence of the metal. In general, Al inhibited root growth,
mostly in the sensitive cultivar ‘Anahuac’, while water content decreased and organic
matter increased in ‘Barbela 7/72’ line. Similar but less evident effects were found in
leaves of both genotypes. The profiles of macro- (Ca, K, P, Mg) and micronutrients (Mn,
Fe, Zn, B, Cu) accumulation in response to aluminium were analyzed in both genotypes:
Al levels increased, mostly in ‘Anahuac’ cultivar. Overall, P, Mg and K levels decreased
and Si and Ca increased in roots of both genotypes, while other nutrients had a more
inconsistent behaviour. Histological data showed that ‘Barbela 7/72’ initiated endodermis
differentiation more efficiently than ‘Anahuac’, at the hairy root zone. These data show
that: (a) Al negatively interferes with P, Mg and K in wheat and (b) Al interactions with
some nutrients depend on the level of plant tolerance. Finally, data also provided support to
the hypothesis that endodermis differentiation may be stimulated limiting Al
accumulation/allocation in roots of the tolerant wheat landrace.
39
Aluminium toxicity in wheat and rye
40
Aluminium toxicity in wheat and rye
Introduction
Aluminium (Al) toxicity is a major agricultural problem in acid soils (pH below 5.5) due to
Al solubility at low pH. Therefore, crops grown in acid soils conditions often show Alphytotoxic symptoms, such as undeveloped root system and/or nutrient imbalances (e.g.
Pietraszewska, 2001) and, consequently, lower production. Aluminium mechanisms of
phytotoxicity are complex and models to explain it, still remain unclear (e.g. Zheng and
Yang, 2005; Ma, 2007; Zheng et al., 2007). Furthermore, Al may be present in several
chemical forms, with different toxicities, and soil acidification may change soil properties,
which in turn affect plant growth and development increasing the complexity of the
problem (e.g. Pietraszewska, 2001). Several mechanisms for Al plant exclusion have been
proposed including, Al efflux, selective permeability, plant induced changes in pH
rhizosphere or root apoplast, and exudation of chelate ligands (Hossain et al., 2006;
Piñeros et al., 2008). Al interferes with uptake or transport of nutrients such as Ca, Mn, P,
Mg, B, Fe, Cu or K (e.g. Keltjens and Tan, 1993; Keltjens, 1995; Lukaszewski and
Blevins, 1996; Slaski et al., 1996; Taylor et al., 1998; Lidon et al., 2000; Guo et al., 2003,
2007; Olivares et al., 2009). Furthermore, Al tolerance in some species was stated to be
due to their ability to maintain in their roots and/or shoots adequate levels of some macroand micronutrients (e.g. Tan et al., 1993). Sivaguru and Paliwal (1993) and Giannakoula et
al (2007) reported that Al-sensitive maize and rice lines accumulate more Al than the
tolerant one, which retained higher concentrations of Ca2+, Mg2+ and K+. Some tolerant
rice cultivars always presented higher levels of P (Jan, 1991) and Ca under Al stress (e.g.
Sivaguru and Paliwal, 1993). This correlation may be due to direct effects of Al on the
absorption of those nutrients and/or to higher root growth rates and deeper root system of
the tolerant genotypes that, therefore, are able to explore a larger volume of soil. Due to the
reported lesions of Al in the meristematic zone, most studies on Al effects in roots are
particularly focused in this region (e.g. Sivaguru and Horst, 1998), in detriment to other
more differentiated regions (e.g. hairy root zone). Ryan et al (1993) demonstrated that 15
mm above the root apex, Al had no deleterious effects in maize. However, the interference
of Al, with other nutrients accumulations claims better studies on Al-induced effects in the
hairy root zone histoanatomy (the most specialized region for nutrient absorption) together
with its effects in nutrient absorption and allocation. Cereal crops exhibit variation in Al
tolerance and wheat is considered to be sensitive to Al (e.g. Barabasz et al., 2002),
41
Aluminium toxicity in wheat and rye
followed by triticale and rye. However, in some regions of Northern Portugal Al
availability in soils is very high, due to specific geoedaphic characteristics and to soils’ low
pH levels. ‘Barbela 7/72’ wheat landrace was widely grown in this region and showed to
be highly tolerant to Al when comparing with other varieties, being selected and
genetically characterized (Guedes-Pinto et al., 1998; Pinto-Carnide and Guedes-Pinto,
1999). Contrarily, ‘Anahuac’, a Polish cultivar, is considered non-tolerant when compared
with Portuguese wheat genotypes (Pinto-Carnide and Guedes-Pinto, 1999). Regional
germplasm of ancient landraces, expressing different degrees of tolerance to Al, provide
unique sources of genotypes for better understanding Al tolerance mechanisms. Based on
these two ancient genotypes behaviour, with ‘Barbela 7/72’ (tolerant) showing root
regrowth after Al exposure, we hypothesize that tolerant genotypes differ from sensitive
ones by developing protective mechanisms for controlling Al uptake and/or partition,
preventing Al-induced damage effects (e.g. histological, nutrition) and allowing root
growth. In this work we evaluated the behavior of Al sensitive and tolerant genotypes with
regard to: (a) Al accumulation and allocation; (b) interference of Al with macro- and
micronutrients; (c) root differentiation, in particular endodermis, under Al treatment; and
(d) recovery ability after Al removal. These data will contribute to future design general
physiological models of Al-nutrient interactions in wheat.
Material and Methods
Plant material selection, growth and exposure to aluminium
Two Triticum aestivum L. genotypes, ‘Barbela 7/72’ (tolerant) line, selected from the
‘Barbela’ landrace, and ‘Anahuac’ cultivar (sensitive), were used. Seeds were disinfected,
rinsed in distilled water and germinated (in the dark at 24 ºC) in Petri dishes with
moistened filter paper until roots reached 0.5 cm long. Afterwards, plants were transferred,
for two days, to a nutritive solution as described by Polle et al (1978). Plants were then
exposed to the same nutritive solution containing 185 µM AlCl3·6H2O corresponding to
4.81 µM Al activity, as estimated by Geochem-EZ.
To compare sensitive vs tolerant genotypes response to continuous exposure to Al and if
the genotypes are able to recover after Al exposure, two experimental conditions were
42
Aluminium toxicity in wheat and rye
conducted: (A) plants were continuously exposed to Al for 72 h. (B) Plants exposed to Al
for 24 h, were transferred to the same solution without Al during 48 h for recovery
assessment (total culture period was 72 h). As control, a third group of plants was grown
for 72 h on the same nutritive solution without Al. The pH was maintained at 4.0
throughout the assay.
Aluminium and nutrients partition, water content and organic matter determinations
For aluminium and nutritional analyses, plants were collected at the following times: 24
and 72 h of exposure and 48 h after recover. Fresh weight of leaves and roots was
measured (Brito et al., 2003). After washing, samples were dried at 60 ºC until constant
weigh and dried weight assessed. Subsequently, samples were incinerated (Azevedo et al.,
2005) and for elemental quantification of Ca, Si, Al, P, K, Mg,Mn, Zn, Cu, B and Fe ashes
were treated whit HCl and elements were determined by inductively coupled plasma
spectroscopy (ICPS, Jobin Ivon, JY70 Plus, Longjumeau Cedex, France).
For water content and organic matter determinations, the following formulas were used:
water content = (fresh weight−dry weight/fresh weight)×100; organic matter = (dry
weight−ashes weight/dry weight)×100.
Histological analyses
For histocytological analyses, sections from the elongation (10–15mm above apices) (EZ)
and hairy root zones (more than 30mm above apices) (HZ) were analyzed during exposure
(24 and 72 h) and recovery (after 48 h). Briefly, fresh samples were fixed in 2.5%
glutaraldehyde in 1.25 % (w/v) piperazine-N,N’-bis-2-ethanesulfonic acid (PIPES) buffer
(pH 7.4) overnight at 4 ºC and then washed in PIPES. Tissues were transferred to 1.0 %
(w/v) osmium tetroxide in PIPES buffer and dehydrated using increasingly concentrated
acetone solutions (30 – 100 %, (v/v)). Samples were embedded in an epoxy resin (Embed812). Semi-thin sections (app.1µm) were stained with periodic acid-Schiff reaction (PAS)
or toluidine blue 1 % (Pinto et al., 2008). Samples were analyzed with a Nikon Eclipse 80i
light microscope (Nikon Co., Kanagawa, Japan) and photographs were taken using a Leica
DC 200 digital camera (Leica Microsystems AG, Germany). Histological images were
analyzed using the image analyzing program UTHSCSA Image Tool, version 3.00
43
Aluminium toxicity in wheat and rye
(University of Texas Health Science Center, USA). The percentage of differentiated
endodermis cells was determined.
Statistical analysis
Values are given as mean ± SD as calculated from data from independent experiences. In
each experience three plants were used. The comparison between exposure vs. control and
recovery vs. control was made using a Student’s t-test or one-way ANOVA test to our
endpoints data, followed by a Holms Sidak Test when data was statistically different
(p<0.05). Comparisons of treatment vs genotype were made by two-way ANOVA.
Correlations among nutrients and Al accumulations were tested by Pearson’s analysis.
Results
Growth, water content and organic matter
In the sensitive ‘Anahuac’ plants, root elongation decreased in all conditions (Fig. 1A),
with first symptoms being evident after 6 h of Al exposure. Contrarily, root growth
reduction was evident only after 24 h in tolerant plants (‘Barbela 7/72’). Moreover, after 48
h in recovery medium, ‘Barbela 7/72’ roots restarted growth (p < 0.05) while ‘Anahuac’
roots did not show any root regrowth (Fig. 1A).
Concerning shoots length (Fig. 1D), those of ‘Anahuac’ slightly increased (p < 0.05) after
72 h of exposure. When Al-treated ‘Anahuac’ plants were transferred to recovery
conditions, shoot elongation increased after 48 h (p < 0.05) (Fig. 1D). Contrarily, ‘Barbela
7/72’ shoots were, in general, not affected by Al exposure (p > 0.05).
After 48 h of recovering, water content slightly decreased (−1.46 %) (p < 0.05) while
organic matter increased (+3.84 %) (p < 0.05) in ‘Barbela 7/72’ roots (Fig. 1B and C). On
the contrary, these parameters (Fig. 1B and C) were not affected (p > 0.05) in ‘Anahuac’.
Different behaviours were also observed, in both genotypes after 72 h exposure to Al (p <
0.05). Concerning shoots (Fig. 1E and F), while those of ‘Barbela7/72’ increased organic
matter (+6.34%) (p < 0.05), in ‘Anahuac’ no changes (p > 0.05) in water or organic matter
were found.
44
Aluminium toxicity in wheat and rye
Fig. 1. Variation in growth (A and D), organic matter (B and E) and water content (C and F) in roots and shoots of
‘Barbela 7/72’ and ‘Anahuac’ plants exposed to 185 µM Al and after 48 h in recovery. Data followed by * is significantly
different at p < 0.05 with respect to correspondingly control.
Al and nutrient accumulation
The average levels of macro- and micronutrients in control and in Al-treated plants are
expressed in Tables 1 and 2 and the main trend is presented in Fig. 2. ‘Barbela 7/72’ and
‘Riodeva’ roots differed with respect to nutrients contents. In particular, after 24 h, control
roots of both genotypes, differed significantly in all macronutrients contents. With
exposure, some nutrient content differences disappear (P and K), as result of different
45
Aluminium toxicity in wheat and rye
behaviour of these genotypes. For Al content, plants of both genotypes presented similar
residual levels (p > 0.05), but significant differences were found during exposure as well as
after recovery period (p < 0.05).
Table 1
Nutrient (Ca, P, K, Mg, Al, Mn, Fe, Zn, Si and Cu) quantification in roots and shoots of ‘Barbela 7/72’ plants exposed to
185 µM Al for 72 h and after 48 h in recovery. Values are given per dry weight (DW).
C a (µg g -1 DW)
P (µg g -1 DW
290.4 ± 11.35
737.3 ± 53.92
4555.1 ± 363.65
157.4 ± 7.94
3.6 ± 0.30
300.1 ± 43.71
727.2 ± 61.82
3366.4 ± 256.48*
153.2 ± 9.39
9.3 ± 2.60*
0µM
280.6 ± 31.00
640.0 ± 90.48
4551.7 ± 491.40
163.1 ± 23.39
1.6 ± 0.30
185µM
261.5 ± 22.61
660.5 ± 52.93
3047.5 ± 316.61*
162.1 ± 12.54
2.9 ± 0.10*
0µM
280.6 ± 31.00
640.0 ± 90.48
4551.7 ± 491.40
163.1 ± 23.39
1.6 ± 0.30
185µM
269.5 ± 27.35
566.8 ± 56.72
4233.9 ± 454.70
149.8 ± 15.84
2.0 ± 0.20
M n (µg g -1 DW)
F e (µg g -1 DW)
S ho o ts
K (µg g -1 DW)
M g (µg g -1 DW)
Al (µg g -1 DW)
24h e xp
0µM
185µM
72h e xp
48h re c
Zn (µg g -1 DW)
S i (µg g -1 DW)
C u (µg g -1 DW)
24h e xp
0µM
2.1 ± 0.10
14.6 ± 0.80
7.0 ± 0.30
8.8 ± 0.50
4.5 ± 0.50
185µM
2.3 ± 0.30
27.0 ± 6.30
6.5 ± 0.80
11.0 ± 3.10
6.4 ± 1.30*
72h e xp
0µM
4.9 ± 3.40
68.9 ± 50.80
6.5 ± 1.20
8.1 ± 0.90
4.8 ± 0.90
185µM
1.9 ± 0.20
52.9 ± 6.20
6.1 ± 0.40
8.5 ± 0.30
6.4 ± 0.60*
0µM
4.9 ± 3.40
68.9 ± 50.80
6.5 ± 1.20
8.1 ± 0.90
4.8 ± 0.90
185µM
2.0 ± 0.40
39.9 ± 7.60
5.6 ± 0.60
8.0 ± 0.80
4.5 ± 0.70
P (µg g -1 DW)
K (µg g -1 DW)
M g (µg g -1 DW)
48h re c
C a (µg g -1 DW)
Ro o t
Al (µg g -1 DW)
24h e xp
0µM
3930.4 ± 139.64
5413.2 ± 300.91
38796.5 ± 851.25
185µM
3734.8 ± 306.85
3907.1 ± 490.14*
21955.5 ± 2448.39*
993.8 ± 3609.46*
0µM
4267.0 ± 899.31
4852.3 ± 393.82
44393.6 ± 1802.78
1619.2 ± 23135.12
368.6 ± 209.40
185µM
6877.0 ± 1186.13*
3867.1 ± 76.65*
17236.7 ± 2872.24*
828.8 ± 1878.49*
1133.0 ± 116.80*
4267.0 ± 899.31
4852.3 ± 393.82
44393.6 ± 1802.78
1619.2 ± 23135.12
368.6 ± 209.40
7373.2 ± 1775.77*
3784.4 ± 87.37*
23247.4 ± 7025.24*
1121.6 ± 5306.39*
F e (µg g -1 DW)
Zn (µg g -1 DW)
2098.0 ± 610.74
168.6 ± 73.00
577.9 ± 70.80*
72h e xp
48h re c
0µM
185µM
M n (µg g -1 DW)
S i (µg g -1 DW)
511.0 ± 103.70*
C u (µg g -1 DW)
24h e xp
0µM
21.2 ± 0.60
350.6 ± 27.50
219.4 ± 4.80
268.6 ± 59.50
94.2 ± 8.60
185µM
19.9 ± 3.60
697.6 ± 351.50
146.8 ± 5.20*
241.3 ± 44.20
93.4 ± 1.70
0µM
37.2 ± 23.10
1469.0 ± 1149.80
236.7 ± 12.50
309.2 ± 50.50
187.1 ± 48.20
185µM
19.1 ± 1.90*
1210.3 ± 114.80
224.0 ± 61.00
437.8 ± 63.30*
173.1 ± 41.60
0µM
37.2 ± 23.10
1469.0 ± 1149.80
236.7 ± 12.40
185µM
15.7 ± 5.30*
520.1 ± 16.40*
72h e xp
48h re c
170.1 ± 37.20
309.2 ± 50.50
187.1 ± 48.20
393.4 ± 72.70*
281.7 ± 78.70
* Data are significantly different at p < 0.05 with respect to correspondingly control.
In the ‘Barbela 7/72’ roots, 24 h after exposure to Al, the levels of P, Zn, Mg and K
decreased (p < 0.05), while Al content increased (+242.87 %) (p < 0.05). In the longer Alexposures (72 h), besides those nutrients (except Zn), also Mn contents decreased (−48.58
%) (p < 0.05), while Si, Ca and mostly Al increased (Al increased approximately four fold
46
Aluminium toxicity in wheat and rye
compared to control; p < 0.05). In the roots of the recovery group (48 h of recovery) the
pattern was similar to the one of plant exposed for 72 h (p < 0.05), but the levels of some
nutrients (e.g. K) were closer to those of the controls and, contrarily for 72 h exposure a
decrease in Fe levels was detected. Also Al content was lower than its content in 72 h
exposed roots.
Table 2
Nutrient (Ca, P, K, Mg, Al, Mn, Fe, Zn, Si and Cu) quantification in roots and shoots of ‘Anahuac’ plants exposed to 185
µM Al for 72 h and after 48 h in recovery. Values are given per dry weight (DW).
C a (µg g -1 DW)
S ho o ts
P (µg g -1 DW
K (µg g -1 DW)
M g (µg g -1 DW)
Al (µg g -1 DW)
24h e xp
0µM
324.3 ± 16.53
702.8 ± 46.20
3882.4 ± 294.84
156.1 ± 12.79
9.6 ± 4.20
185µM
318.6 ± 93.42
723.5 ± 294.50
2972.1 ± 1291.53
151.6 ± 68.01
17.7 ± 14.50
72h e xp
0µM
185µM
337.1 ± 13.71
659.5 ± 32.78
4833.5 ± 458.11
175.0 ± 8.56
3.0 ± 1.10
236.6 ± 23.24*
698.7 ± 67.72
3190.3 ± 352.82*
152.7 ± 13.57
3.0 ± 0.90
337.1 ± 13.71
659.5 ± 32.78
4833.5 ± 458.11
175.0 ± 8.56
3.0 ± 1.10
240.1 ± 32.36*
536.3 ± 183.15
2759.8 ± 981.95*
132.6 ± 45.61
6.2 ± 3.30
48h re c
0µM
185µM
M n (µg g -1 DW)
F e (µg g -1 DW)
Zn (µg g -1 DW)
S i (µg g -1 DW)
C u (µg g -1 DW)
24h e xp
0µM
2.8 ± 0.10
28.9 ± 3.70
9.5 ± 0.70
12.9 ± 2.00
4.2 ± 0.60
185µM
3.2 ± 1.00
52.8 ± 12.00*
10.6 ± 4.50
13.5 ± 4.10*
5.8 ± 2.90
10.5 ± 0.80
9.8 ± 0.80
6.7 ± 1.60
9.8 ± 0.10
8.3 ± 1.10*
4.6 ± 0.40
72h e xp
0µM
2.8 ± 0.10
28.8 ± 2.20
185µM
3.7 ± 0.40
53.3 ± 8.60*
48h re c
0µM
2.8 ± 0.10
28.8 ± 2.20
10.5 ± 0.80
9.8 ± 0.80
6.7 ± 1.60
185µM
2.6 ± 0.70
25.7 ± 2.70
7.5 ± 2.00
10.2 ± 3.20*
9.0 ± 1.30
C a (µg g -1 DW)
Ro o t
P (µg g -1 DW
K (µg g -1 DW)
M g (µg g -1 DW)
Al (µg g -1 DW)
24h e xp
0µM
4899.5 ± 772.26
4132.0 ± 220.49
32784.3 ± 4552.71
1279.9 ± 32.30
185µM
5746.4 ± 319.94
3578.2 ± 493.39* 25063.9 ± 553.61*
671.0 ± 7.20*
4041.8 ± 360.36
3424.6 ± 298.61
27152.7 ± 1229.26
1135.9 ± 106.91
7178.7 ± 1375.82*
3777.0 ± 92.48
15844.2 ± 1438.79*
603.2 ± 46.13*
0µM
4041.8 ± 360.36
3424.6 ± 298.61
27152.7 ± 1229.26
1135.9 ± 106.91
301.7 ± 86.60
185µM
7032.3 ± 1102.62*
3830.0 ± 313.66
21588.4 ± 3076.51*
677.4 ± 19.41*
850.7 ± 37.90*
F e (µg g -1 DW)
Zn (µg g -1 DW)
256.8 ± 105.00
896.2 ± 119.90*
72h e xp
0µM
185µM
301.7 ± 86.60
1613.6 ± 160.30*
48h re c
M n (µg g -1 DW)
S i (µg g -1 DW)
C u (µg g -1 DW)
24h e xp
0µM
57.9 ± 14.40
1695.9 ± 225.40
284.6 ± 148.90
259.3 ± 24.10
91.9 ± 8.00
185µM
33.5 ± 6.80
1363.4 ± 597.40
254.8 ± 103.30
365.9 ± 70.8*
124.5 ± 0.70*
72h e xp
0µM
9.6 ± 2.00
185µM
21.5 ± 6.90
387.0 ± 9.80
112.2 ± 15.00
259.1 ± 21.20
1291.1 ± 353.1*
177.4 ± 22.90
463.5 ± 52*
114.1 ± 3.00
139.8 ± 5.30*
48h re c
0µM
9.6 ± 2.00
387.0 ± 9.80
112.2 ± 15.00
259.1 ± 21.20
114.1 ± 3.00
185µM
9.9 ± 1.00
1015.2 ± 75.6*
136.1 ± 20.80
426.3 ± 43.7*
121.5 ± 16.20
* Data are significantly different at p < 0.05 with respect to correspondingly control.
In ‘Barbela 7/72’ leaves, after 24 h exposure, only K levels decreased (p < 0.05), while Cu
and mostly Al (+158.26 %) levels increased (p < 0.05, Table 1). This pattern was
47
Aluminium toxicity in wheat and rye
maintained in leaves of plants exposed to aluminium for 72 h. In the group transferred to
recovery medium (48 h), K, Cu and Al contents levels reached those of the control,
suggesting a recovery ability in this genotype to regulate the nutrient imbalances induced
by Al exposure in the shoot.
The levels of P, Mg and K decreased (p < 0.05) in ‘Anahuac’ roots, 24 h after exposure.
Contrarily, Si, Al (+249.00 %) and Cu levels increased (p < 0.05) (Table 2). After 72 h in
Al, mostly Mg and K levels decreased, while Fe, Si, Cu, Ca and, mostly, Al levels
increased (+434.87 %; p < 0.05). After recovering for 48 h, roots continued to present
lower K and Mg levels (p < 0.05) though higher Al, Ca, Si and Fe levels (p < 0.05) than
control. Concerning leaves, Fe and Si levels increased (p < 0.05) in ‘Anahuac’ leaves after
24 and 72 h exposure, while Ca and K levels decreased in longer exposures (p < 0.05).
Contrarily to ‘Barbela 7/72’ leaves, no differences (p > 0.05) were found in Al
accumulation of ‘Anahuac’ leaves among the different treatments.
Fig. 2. Representation of nutrients variations in roots and shoots after 72 h exposure and 48 h recovery. Arrows
represents an increase (up arrow) or decrease (down arrow) in nutrient values.
Root anatomy and endodermis differentiation
Aluminium exposure affected endodermis differentiation in the hairy root region of
‘Barbela 7/72’ roots (Table 3). After 24 h exposure, ‘Barbela 7/72’ Al-treated plants had
100.00 ± 0.000% of thickened cells in the hairy root zone, while in the control this value
was only 27.03 ± 18.510. This stimulation of differentiation was also observed in roots
48
Aluminium toxicity in wheat and rye
exposed for longer periods or transferred to recovery medium (p < 0.05), although
difference was lower after recover period.
For ‘Anahuac’, the major Al effect on endodermis differentiation was observed in the
elongation zone (Table 3; Fig. 3). Al-treated roots for 24 h and those on recovery medium
showed higher endodermis differentiation than control ones in the EZ, but with exposure,
differences in this zone were no longer detected (Table 3).
Table 3
Percentage of endodermis cells with cell wall thickened for ‘Barbela 7/72’ and ‘Anahuac’. Plants were exposed to Al for
24 h and 72 h and 48 h in recover.
Percentage of endodermis cells with cell wall thickened
Barbela 7/72
Anahuac
Elongation zone
Hairy root zone
Elongation zone
Hairy root zone
24h exp
0 µM
185µM
0.00 ± 0.000
3.05 ± 2.675
27.03 ± 18.510
100.00 ± 0.000*
0.00 ± 0.000
87.12 ± 1.553*
100.00 ± 0.000
100.00 ± 0.000
72h exp
0 µM
185µM
28.33 ± 38.046
10.96 ± 3.134
71.33 ± 23.214
100.00 ± 0.000*
0.00 ± 0.000
16.57 ± 9.160
92.99 ± 7.246
100.00 ± 0.000*
48h rec
0 µM
185µM
28.33 ± 38.046
30.54 ± 27.615
71.33 ± 23.214
84.92 ± 1.089*
0.00 ± 0.000
87.82 ± 4.484*
92.99 ± 7.246
81.65 ± 4.760
* Data are significantly different at p < 0.05 with respect to correspondingly control.
Fig. 3. Root cross sections of ‘Anahuac’ plants showing endodermis thickness in exposed plants (bar = 50 µm). (A)
Control after 24 h in culture with no thickness; (B) exposed root after 24 h in Al showing thickness (arrow); (C) control
after 72 h in culture with no thickness; (D) root after 24 h in Al and 48 h in recover showing thickness (arrow).
49
Aluminium toxicity in wheat and rye
Discussion and Conclusions
Many reports concerning Al-phytotoxic effects describe inhibition of root growth (e.g.
Vázquez et al., 1999; Pietraszewska, 2001; Ahn et al., 2002; Ma et al., 2004; Guo et al.,
2007; Ali et al., 2008). Some phytotoxic effects on root growth were detected after very
short periods in Al, such as after 30/60 min (e.g. Panda and Matsumoto, 2007; Rangel et
al., 2007). These phytotoxic effects suggest damages in the root histoanatomical system
(e.g. Oleksyn et al., 2006). Root elongation is a complex process involving histoanatomical
modifications, as well as cell division and expansion changes. Recently our group also
detected that Al affected wheat root cell cycle dynamics.
Besides, those putative impairments on histology and/or cell cycle, reducing therefore root
growth, we show that Al also affects nutrients accumulation/partition in both genotypes.
Moreover, nutrients accumulation/partition in the plant may be conditioned by root
histoanatomy.
Comparative elongation rates were already used to discriminate differences in Al response
in these sensitive (‘Anahuac’) and tolerant (‘Barbela 7/72’) wheat genotypes (e.g. PintoCarnide and Guedes-Pinto, 1999). Due to their different ability to tolerate aluminium they
became a powerful tool to better understand Al effects in wheat.
Some authors also reported that Al toxicity may decrease shoot growth (e.g. Ryan et al.,
1993; Ali et al., 2008). Shoot growth reduction may be a consequence of root damages or a
strategy to reduce transpiration rates. In sensitive ‘Anahuac’ plants, shoot growth increased
48 h after transfer to recovery medium (without Al), while no changes in water content
were found. Contrarily, the decrease of water content and increase of dry matter in
‘Barbela 7/72’ after the same period suggest a different strategy to control cell osmotic
potential, and reduction of the shoot growth rates in this genotype may represent a strategy
to reduce transpiration rates and restrict aluminium, instead of being a consequence of root
damage. Furthermore, as Al availability increases under acid soils, the accumulation of
other elements as Cu or Fe also may increase and eventually may reach toxic levels
(Schroth et al., 2003). We demonstrated here that Cu accumulation in sensitive wheat
plants is stimulated by Al. It is presently under study how the presence of Al stimulates the
accumulation of Cu mostly in the sensitive plants.
After 72 h of exposure, ‘Barbela 7/72’ roots accumulate less Al than ‘Anahuac’ roots,
while, some of this metal is translocated to leaves (at higher rate during the first hours of
50
Aluminium toxicity in wheat and rye
exposure), showing that this tolerant genotype has the ability to accumulate Al in the
shoots. Contrarily, the sensitive genotype, ‘Anahuac’ (after the treatment) remained with
larger amounts of Al in the root than ‘Barbela7/72’ but excluded Al from the shoots.
Giannakoula et al (2007) also reported that an Al-sensitive maize line accumulated more
Al than the tolerant one. Furthermore, in ‘Barbela 7/72’ recovering shoots, Al and Cu
contents had the ability to return to control values. These data support that the tolerant
genotype has an effective control of aluminium and nutrients accumulation and partition, a
characteristic not demonstrated by the sensitive one. We are presently evaluating the
mechanisms involved in these controls.
Al and/or low pH of the soils may also lead to deficiencies of P, Mg, and K (Poschenrieder
et al., 1995; Schroth et al., 2003). Poschenrieder et al (1995) reported for maize varieties
exposed to Al in acidic conditions, an inhibition of root elongation and of nutrient
absorption (B, Fe, Mg, Ca and P) related to low pH, while only B also seemed to be related
to Al exposure. Therefore, in wheat, the consistent decrease pattern of P (which increased
to control levels during recovery in ‘Anahuac’ roots), Mg and K in roots of both wheat
genotypes may be related to Al and/or to acid conditions, which synergically reduced the
absorption of these elements.
The inhibition of phosphorus accumulation in roots by aluminium was already described
(e.g. Pietraszewska, 2001). Once within the cell, Al may react with P compounds, and
upset the plant P metabolism. For the genotypes in study, P root accumulation is affected
in both genotypes, supporting previous studies on P accumulation decrease in Al exposed
plants (e.g. Howeler and Cadavid, 1976; Foy et al., 1978; Mugwira et al., 1980). Al
tolerance may be associated with an efficient balance of phosphorus such as adsorption–
precipitation reactions between Al and P or other cell reactions (Petrersson et al., 1988).
Also K and Mg were affected by Al exposure. K is extremely important for osmotic
balances, and therefore for cell extension. Despite a general decrease in both genotypes, K
levels recovered in tolerant plant shoots when Al was removed. The more drastic effects in
the sensitive ‘Anahuac’ plants suggest higher disorders in osmoregulation and ultimately in
cell extensibility in this genotype. It has been shown that even in severely Al-intoxicated
roots plasma membranes remain intact (Kinraide, 1988), and as already suggested K efflux
may accompany the extrusion of organic acids (Samac and Tesfaye, 2003; Gonçalves et
al., 2005). The decrease of K verified in this work may be a consequence of this extrusion
to contrabalance the extrusion of organic acids but further studies are needed. However,
51
Aluminium toxicity in wheat and rye
preliminary data show that short term exposure to Al seems not to induce malic extrusion
in these ‘Anahuac’ roots (data not shown), and therefore mechanisms involved in Al–K
interaction must be further explored. Al also affected Ca and, mostly, Mg accumulation in
both genotypes. Recently, Yang et al (2007) demonstrated that Mg can alleviate the Alinduced decrease root growth in rice and bean, and that this effect was probably due to
citrate efflux. These Al effects may be due to its interference on membrane and of cell wall
properties and architecture (e.g. Pietraszewska, 2001). Another hypothesis is that to
maintain the root surface pH above 5.0, roots have to efflux alkaline compounds, as Mg, K,
Na and Ca. So, the decrease of K and Mg levels may be due to reduce cation uptake in an
attempt to keep a greater anion uptake. The Ca accumulation in roots may be due to
negative effects of Al in xylemic sieve translocation but further studies are needed.
Aluminium first contact with the plant is with the apoplast/cell wall. Al effects may be at
the apoplast such as increasing cell wall rigidity (Mohanty et al., 2004) or affect cell wall
extensibility, as already described for wheat roots (Ma et al., 2004), or cell wall
polysaccharides composition (Yang et al., 2008). Other Al toxic effects are at the symplast
(e.g. Silva et al., 2000; Pietraszewska, 2001; Barceló and Poschenrieder, 2002; Samac and
Tesfaye, 2003; Kochian et al., 2002; Doncheva et al., 2005). Endodermis represents an
important barrier to nutrients (and metals) transport to shoots. For example, spruce
seedlings exposed to Al presented lower concentration of Ca inside endodermal layers
suggesting controlling mechanisms of endodermis (e.g. Godbold et al., 1988). Also, in
Picea abies exposed to aluminium, was almost entirely confined to the root cortical cell
walls, and was not detectable inside the endodermis (Hodson and Wilkins, 1991). In wheat,
Delhaize et al (1993) showed that Al entered root apices of Al-sensitive plants and
accumulated in the epidermal layer and in the cortical layer immediately below the
epidermis.
The two wheat genotypes studied here behaved differently concerning Al and nutrients
accumulation and partitioning in roots and shoots and also differed in their endodermis
differentiation in response to aluminium, with stimulation in the tolerant genotype in the
hairy root zone (HZ). This may represent a defense mechanism of shortening the zone of
unhindered apoplastic transport into the central cylinder. Furthermore, the differences
found for ‘Anahuac’ may merely reflect that the roots stopped growing. This fact is being
evaluated with restricted zonal cell cycle and histocytological analyses (data not shown).
52
Aluminium toxicity in wheat and rye
In conclusion, our comparative data on the Al-tolerant vs the Al-sensitive wheat plants
showed different patterns between the two genotypes dependent on: (a) time of exposure
and recovery (Al effects were better alleviated in the tolerant genotype after 48 h on
recover solution); (b) the organ (the root was more sensitive to nutrient and Al
accumulation and partition than leaves) and this was more evident in ‘Anahuac’ roots; (c)
the nutrient uptake (e.g. K followed by other elements, as Mg and P, were extremely
sensitive to Al exposure). Al also affected endodermis differentiation, at the hairy root
zone with higher differentiation in the tolerant plants, supporting that this better
differentiation may be involved in controlling mechanisms of Al accumulation/partition in
the tolerant genotype.
Acknowledgements
The
Portuguese
Foundation
for
Science
and
Technology
FCT/MCT
project
POCI/AGR/58174/2004 supported this project. The authors are grateful to Armando Costa
assistance. The Portuguese Foundation for Science and Technology (FCT) financed the work of
Sónia Silva (FCT/BD/32257/2006).
53
Aluminium toxicity in wheat and rye
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57
Aluminium toxicity in wheat and rye
58
Aluminium toxicity
oxicity in wheat and rye
II 2: Different structural and functional responses of sensitive vs tolerant wheat roots
during Al exposure andd recovery
Chapter submitted as an original paper in a SCI journal:
Silva S, Eleazar R, Pinto-Carnide
Carnide O, Martins
Martins-Lopes
Lopes P, Matos M, Guedes-Pinto
Guedes
H, Santos
C. 2010. Different structural and functional responses of sensitive vs tolerant wheat roots
during Al exposure and recovery
recovery. Environmental and Experimental Botany (submitted).
(submitted)
59
Aluminium toxicity in wheat and rye
60
Aluminium toxicity in wheat and rye
Abstract
Aluminium (Al) irreversibly inhibits root growth in sensitive but not in some tolerant
genotypes. To better understand tolerance mechanisms, seedlings from tolerant
(’Barbela7/72’ line) and sensitive (‘Anahuac’) Triticum aestivum L. genotypes were
exposed to 185 µM AlCl3 for: a) 24h followed by 48h without Al (recovery); b) 72h of
continuous exposure. Three root zones were analyzed (meristematic (MZ), elongation (EZ)
and hairy (HZ)) for callose deposition, reserves (starch and lipids) accumulation,
endodermis differentiation and tissue architecture. Putative Al-induced genotoxic or
cytostatic/mytogenic effects were assessed by flow cytometry in root apices.
Tolerant plants accumulated less Al, presented less root damage and a less generalized
callose distribution than sensitive ones. Starch and lipid reserves remained constant while
drastically decreased in sensitive root tips. Al induced different profiles of endodermis
differentiation: differentiation was promoted in EZ and HZ, respectively, in sensitive and
tolerant genotypes. No ploidy changes or clastogenicity were observed, however
differences in cell cycle blockage profiles were detected, being less severe in tolerant roots.
After Al removal, only ‘Barbela7/72’ line reversed Al-induced effects to values closer to
control, mostly concerning callose deposition and cell cycle progression. We demonstrate
for the first time that: a) cell cycle progression is differently regulated by Al tolerant and
sensitive genotypes; b) Al induces callose deposition > 3cm above root apex (in HZ); c)
callose deposition is a transient Al-induced effect in tolerant plants; c) in HZ, endodermis
differentiation is also stimulated only in tolerant plants, probably functioning in tolerant
genotypes as protective mechanism in complement to callose.
61
Aluminium toxicity in wheat and rye
62
Aluminium toxicity in wheat and rye
Introduction
Approximately half of the arable soils are acid (Echart et al., 2002, Kochian et al., 2004),
and Al phytotoxicity is a primary factor limiting crop productivity in those soils (e.g.
Maron et al., 2008). Root growth inhibition is often pointed out as the most evident
symptom of Al toxicity (e.g. Barceló and Poschenrieder, 2002) and, using sensitive
genotypes, it was demonstrated that Al inhibits both root cell division and elongation
(Mossor-Pietraszewska, 2001; Doncheva et al., 2005; Wang et al., 2006). However, the
identification of the root zones (e.g. meristematic, elongation or hairy root zones) that are
primarily affected by Al and local protective mechanisms remain to clarify. In maize Alsensitive cultivars it was shown that the root apex (including root cap, meristematic and
elongation zones) had more physiological damages and accumulated more Al than the
mature tissues (Delhaize and Ryan 1995). Ryan et al (1993) showed that the exposure of
meristematic zone (MZ, 2–3mm) to Al was a prerequisite for root growth inhibition.
Sivaguru and Horst (1998) demonstrated in Al-sensitive maize that callose deposition and
cytoskeleton structure in the distal transition zone (DTZ, 1-2mm from root apex) were
especially sensitive to Al. These studies however do not explore putative effects at other
regions such as at the hairy root zone (HZ), where nutrient absorption is intense.
Al-induced callose formation in root tips is recognised as an excellent indicator of Al
sensibility (Horst et al., 1997; Massot et al., 1999; Meriga et al., 2003; Bhuja et al., 2004;
Tahara et al., 2005) and some works negatively correlated root elongation with callose
formation during Al exposure (e.g. Tahara et al., 2005). Most studies on callose deposition
only covered up to 1cm of the root tip region. In this young region, callose accumulated
during Al exposure in maize (Eticha et al., 2005; Jones et al., 2006), Norway spruce (Nagy
et al., 2004), Melaleuca and Eucalyptus (5 mm back from the root tip) (Tahara et al.,
2005). In more differentiated root regions (as HZ) it is consensually accepted that Al does
not induce significant callose deposition (Wissemeier and Horst, 1995; Horst et al., 1997;
Budikova and Mistrik, 1999; Eticha et al., 2005).
Callose deposition, as response to Al, is described in sensitive and, to a lesser extent, in
tolerant roots (Horst et al., 1997; Eticha et al., 2005), but comparative studies are lacking.
This accumulation is reported to inhibit the symplastic transport and cell communication
by blocking plasmadesmata, avoiding Al induced lesions in the symplast (Sivaguru et al.,
2000). Al bound to cell walls affects cell extension and root growth (e.g. Rengel, 1996;
63
Aluminium toxicity in wheat and rye
Schmohl and Horst, 2000; Hossain et al., 2005), while Al bound to plasmalema
(Poschenrider et al., 2008) may change membrane properties and nutrient homeostasis (Ma
et al., 2002; Zheng et al., 2007, Silva et al., 2010). However, callose deposition in sensitive
roots has also been shown to lead to uncontrolled rigidity of cell walls (Jones et al., 2006)
leading to protoplast degradation.
At molecular level, Al affects several macromolecules, including DNA (Zheng and Yang,
2005). Few data report its induction of mutations, clastogenicity and inhibition of cell
cycle. It was demonstrated that Al highly accumulates in the nuclei of sensitive genotypes,
and presumably leads to inhibition of cell division (Silva et al., 2000). Rice roots exposed
to AlCl3 showed cytotoxic disorders such as chromosome stickiness, micronuclei and
multinucleated cells (Mohanty et al., 2004). In maize mutants and wheat plants exposed to
high Al levels, Rayburn and Wetzel (2002) demonstrated, by flow cytometry (FCM), that
increases of coefficient of variation of the G0/G1 peaks were correlated with increases of
chromosomal damages and clastogenicity.
Considering the Eulerian model for organ growth, the rate of cell production is determined
by the time it takes to progress through the cell cycle and the number of dividing cells,
being speculated that both parameters are controlled by cell cycle checkpoints (West et al.,
2004). The information provided by Doncheva et al (2005) on Al-induced root cell cycle
patterning, concerning mitotic blockage (at S phase) near the MZ of sensitive roots,
demonstrates that Al influences cell cycle progression. However, comparative studies on
the mechanisms by which Al may influence the cell cycle progression in sensitive versus
tolerant roots remain unknown.
The works comparing sensitive and tolerant genotypes are in general restricted to the
following endpoints: growth (Rangel et al., 2007; Stass et al., 2008), callose deposition
(Eticha et al., 2005; Jones et al., 2006; Stass et al., 2008), Ca and K imbalances (Babourina
et al., 2005), root exudates (Wenzel et al., 2002; Zhang et al., 2003; Tolra et al., 2005;
Stass et al., 2008). So, for a more complete functional understanding of genotypes
tolerance to Al, there is a need to use a large multiparametric evaluation, and also evaluate
at what extension the effects are reversible after Al removal. An ancient wheat landrace
‘Barbela’ was selected for its extreme tolerance to high levels of Al (Martins-Lopes et al.,
2009), while ‘Anahuac’ plants have an Al-sensitive phenotype (e.g. Silva et al., 2010).
These two genotypes are therefore valuable and comparable models for comparing
mechanisms of Al induced toxicity and tolerance strategies.
64
Aluminium toxicity in wheat and rye
In order to identify some mechanisms used by tolerant wheat genotypes, and at what
extension Al-induced injuries become irreversible in both sensitive and tolerant plants, we
compared apical/elongation and mature regions of sensitive and tolerant roots, during Al
exposure and after Al removal. We covered multiple endpoints in particular: callose
distribution and tissue architecture throughout the root, Al and reserves accumulation, and
putative Al genotoxic effect (ploidy changes, clastogenicity) and cytostatic/mitogenic
effects both in leaves and roots.
Material and Methods
Plant material selection, growth and exposure to aluminium
Two Triticum aestivum L. genotypes were used: ‘Barbela 7/72’ (tolerant) from Trás-osMontes, Portugal) and ‘Anahuac’ (sensitive) from Poland. Seeds were disinfected, rinsed
and germinated in petri dishes with moistened Whatman paper until roots reached about
0.5 cm long. Germination took place in the dark at 24 ºC. Plantlets were transferred, for
two days, to a nutritive solution described by Polle et al. (1978).
Plantlets were exposed to the nutritive solution containing 5 ppm (185 µM) AlCl3.6H2O
corresponding to 4.81 µM Al activity, as estimated by Geochem-EZ (Shaff et al., 2010).
To assess genotypes ability to recover from Al induced effects, one group of plants
exposed for 24 h to Al, was then grown for 48 h on nutritive solution without Al. Another
group was continuously exposed to Al (72 h). As control, a third group was maintained in
nutritive solution without Al (72 h). The pH was maintained at 4.0 throughout the assay.
Root growth was assessed after 6 h, 24 h, 48 h and 72 h in Al and after 24 h and 48 h in
recover.
Aluminium content determination
For metal content determination, roots and shoots were sampled at the following
conditions: 24 h and 72 h during Al exposure, 24 h and 48 h after transfer to recovery
solution. Samples were washed and treated according to Santos et al (2002). Aluminium
65
Aluminium toxicity in wheat and rye
content was determined by inductively coupled plasma spectroscopy (ICP-MS, Jobin Ivon,
JY70 Plus, Longjumeau Cedex, France).
Histological and morphometric analyses
Histocytological analyses were performed in roots after 24 h, 48 h and 72 h exposure, 24 h
and 48 h after recovery. Based on preliminary studies for histological and functional
characteristics performed in roots of these genotypes (data not shown), sections from three
regions were analyzed: the meristematic zone (MZ, up to 2.5 mm behind the root cap,
based on Ryan et al., 1993), the elongation zone (EZ, 10 to 15 mm behind the root cap) and
the differentiated hairy root zone (HZ, 30 to 50 mm behind root cap). Briefly, fresh
samples were fixed in 2.5% glutheraldehyde in 1.25% (w/v) piperazine-N,N’-bis-2ethanesulfonic acid (PIPES) buffer (pH 7.4). Tissues were transferred to 1.0% (w/v)
osmium tetroxide in PIPES buffer, dehydrated and embedded in an epoxi resin (Embed812). Semi-thin sections (app. 1µm) were stained with periodic acid-Schiff reaction (PAS)
for carbohydrate detection, with Sudan Black B for lipid staining, with Comassie Brilliant
Blue for proteins and with toluidine blue 1% (w/v) for general staining. Samples were
analyzed with a Nikon Eclipse 80i light microscope (Nikon Co, Kanagawa, Japan) and
photographs were taken using a Leica DC 200 digital camera (Leica Microsystems AG,
Germany). Histological images were analyzed using the image analyzing program
UTHSCSA Image Tool, version 3.00 (University of Texas Health Science Center, USA)
(Pinto et al., 2008). For morphometric studies, the following parameters were measured:
root cross section area, cortex area (for MZ corresponds to fundamental meristem), central
region area (central cylinder area and endodermis area) (for MZ correspond to
procambium), central lacunae area, pericycle cell area and endodermis cell area.
Measurements were based on at least three images from different pieces of each sample.
For pericycle and endodermis calculations of cell areas, five measurements were made in
each image (total of 30 measurements for each parameter). Comparative studies on
endodermis differentiation were based on the difference between the percentages of
differentiated cells in the control and the correspondent Al exposure. For this, percentage
of differentiated cells was calculated on the percentage of cells showing cell wall thickness
for each condition.
66
Aluminium toxicity in wheat and rye
Callose detection
Callose was detected in roots after 24 h, 48 h and 72 h exposure, 24 h and 48 h after
recovery. The same regions selected for histological analyses were chosen: MZ, EZ and
HZ. MZ was divided in three sections: 1 mm, 1-2 mm and 2-2.5 mm behind root cap. Root
tips sections were collected and stored in 100% ethanol until analysis. Samples were
stained for 1h with 0.1% aniline blue and then mounted in a drop of 50% glycerine (Castro
et al., 2008). Samples were observed with a Nikon Eclipse 80i epifluorescence microscope
equipped with a UV-2A filter cube (330–380 nm excitation; Nikon Instruments, Inc.,
Kanagawa, Japan)
Flow cytometric analysis
For FCM studies, nuclear suspensions of root apices and leaves (after 6 h, 24 h, 48 h and
72 h in Al and after 24 h and 48 h in recover) were obtained by chopping 10 mg of sample
tissue in WPB buffer and processed as described by Loureiro et al. (2007). For DNA
content estimation, 20 mg of Vicia faba L. leaf (with 2C = 26.90 pg DNA (Dolezel et al.,
1992)) was added as an internal standard. After a 5 min incubation period, samples were
analyzed in a Coulter EPICS-XL flow cytometer (Coulter Electronics, Hialeah, Florida,
USA), equipped with an air-cooled argon-ion laser (15 mW operating at 488nm). The
results were acquired using the SYSTEM II software (v. 3.0, Beckman Coulter®). Only
samples with at least 3,000 nuclei were considered for statistical analysis. The DNA ploidy
level of each sample was given as an index relative to the internal standard (V. faba), by
analyzing three replicates per condition.
The nuclear genome size of the samples was calculated according to the following
formula:
Wheat landraceG 0 / G1 peack mean
× 26 .90
G 0 / G1 peack mean
a
b
a
f
.
V
Sample 2C nuclear DNA content (pg)
Putative clastogenicity was analyzed according to Rayburn and Wetzel (2002). For cell
cycle analysis, flow cytometric files were transformed to single histogram files with
WinMDI ver. 2.9 software (freeware by Joe Trotter, Scripps Inst., La Jolla, USA) and then
analyzed using Cylchred software (freeware by Terry Hoy, Univ. Cardiff, UK).
67
Aluminium toxicity in wheat and rye
Statistical analysis
Values are given as mean ± standard deviation as calculated from data of independent
experiences. The comparison between Al exposure periods and the control was made using
a t-student test or One Way ANOVA test, followed by a Holmes-Sidak multiple
comparison procedure (p < 0.05).
Results
Root growth and Al content
Under control conditions, both Al-sensitive and tolerant wheat genotypes presented similar
growth rates, and roots were pearl colored. After only 6 h exposure to Al, ‘Anahuac’
presented a reduction of root growth (-24.61 %) that was not restored after placing the
plants for recovery. Contrarily, the Al tolerant ‘Barbela 7/72’ line only showed root growth
inhibition 24h after exposure to Al, and growth was always restored after transfer to
solution without the metal (Fig. 1).
16,0
Root length (cm)
14,0
12,0
*
*
10,0
*
8,0
*
*
*
*
6,0
4,0
2,0
Barbela7/72
Anahuac
Barbela7/72
24h exp
Anahuac
72h exp
Barbela7/72
Anahuac
24h rec
Barbela7/72
185µM
0µM
185µM
0µM
185µM
0µM
185µM
0µM
185µM
0µM
185µM
0µM
185µM
0µM
185µM
0µM
0,0
Anahuac
48h rec
Fig. 1. Root length (cm ± standard deviation) of ‘Anahuac’ and ‘Barbela 7/72’ plants exposed to 185 µM Al during 24 h
and 72 h and after a recover period of 24 h and 48 h. Control is presented as 0 µM Al. * Data are significantly different at
p < 0.05 with respect to correspondingly control.
68
Aluminium toxicity in wheat and rye
After 72 h of exposure, tolerant ‘Barbela 7/72’ plants accumulated less Al in both roots (p
< 0.05) (1133.0 ± 116.80 µg g-1DW) (Fig. 2) and shoots (p ˃ 0.05) (2.9 ± 0.10 µg g-1DW)
than the sensitive ones (1613.6 ± 160.30 µg g-1DW in roots and 3.0 ± 0.90 µg g-1DW in
shoots).
Root Al content ( µg mg-1 DW)
0,02
*
0,018
0,016
0,014
*
0,012
*
0,01
*
*
0,008
*
0,006
*
0,004
0,002
185µM
0µM
185µM
0µM
185µM
0µM
185µM
0µM
185µM
0µM
185µM
0µM
185µM
0µM
185µM
0µM
0
Barbela 7/72 Anahuac Barbela 7/72 Anahuac Barbela 7/72 Anahuac Barbela 7/72 Anahuac
24h exp
72h exp
24h rec
48h rec
Fig. 2. Al content (µg mg-1 of dry weight ± standard deviation) in roots of ‘Anahuac’ and ‘Barbela 7/72’ plants exposed
to Al (185 µM) during 24 h and 72 h and after 24 h and 48 h of recover. Control is presented as 0 µM. * Data are
significantly different at p < 0.05 with respect to correspondingly control.
Root histological analyses
In control meristematic zone (MZ), the central lacunae ranged from 0.002 ± 0.00003 mm2
to 0.004 ± 0.0002 mm2 (‘Barbela 7/72’ line) and 0.001 ± 0.0003mm2 (‘Anahuac’ cultivar).
The central region showed abundance of mitotically active cells, which presented lipid
accumulation (Fig. 3A). Forty eight hours after exposure to Al, ‘Anahuac’ outer cell layers
showed disaggregation, cell disruption and increasing intercellular spaces (Fig. 3B). After
72 h exposure, the inner cells layers were totally destroyed (Fig. 3C). Also, after this
period, Al induced root tip deformation and enlargement (p < 0.05) (Table 1, 2).
In MZ controls, outer and inner cells strongly stained for lipids, while lipid reserves
disappeared in Al treated sensitive roots (Fig. 3D-G). Carbohydrates accumulated in
control root cap cells and outer cells (Fig. 3H), but similarly to lipids, carbohydrate
reserves disappeared in sensitive roots after 48 h (Fig. 3I). Moreover, in this genotype,
carbohydrates were not detected after 24 h of recovery. For ‘Barbela 7/72’, Al exposure
did not affect neither lipid nor carbohydrate accumulation (Fig. 3J).
69
Aluminium toxicity in wheat and rye
A
D
H
B
C
F
E
I
G
J
Fig. 3. Root cross sections at meristematic zone stained with Sudan black (A, D-G) and with PAS (B, C, H-J). A) Control
root of ‘Barbela 7/72’ after 24 h on nutritive solution. B) ‘Anahuac’ roots after 48h Al exposure. C) ‘Anahuac’ roots after
72 h Al exposure showing damages in central region caused by Al exposure. D-G) ‘Anahuac’ roots after 24 h (D) on
nutritive solution, after 24 h Al exposure (E), after 48 h Al exposure (F) and after 72 h Al exposure (G) showing lipidic
accumulation in control and decrease with time exposure. H) ‘Anahuac’ control root after 72 h revealing carbohydrates
accumulation in root cap and cortex outer cells. I) ‘Anahuac’ roots after 48 h Al exposure showing the absence in
carbohydrates accumulation after exposure. J) ’Barbela 7/72’ roots after 72 h Al exposure showing
carbohydrates accumulation. Arrows: carbohydrates. Bar: 50 µm.
70
Aluminium toxicity in wheat and rye
Oppositely to MZ, both EZ (elongation zone) and HZ (hairy root zone) did not show lipid
or carbohydrate accumulation, independently of the treatment. In EZ, differences (p <
0.05) were seen between ‘Anahuac’ and ‘Barbela 7/72’: while in the sensitive genotype,
the root section area decreased after 48 h and 72 h exposure, in the Al tolerant roots it was
not affected (Table 1, 2). Furthermore, after the same period, the central lacunae decreased
in’Anahuac’. After recovery period, differences (p < 0.05) between the two genotypes
were also visible in the EZ. ‘Anahuac’ plants showed an increase in lacunae area and
endodermis and pericycle cells areas and an increase of outer layers cell wall loosening
(Table 2).
Table 1
‘Anahuac’ roots area measurements. Average measurements of root meristematic (MZ), elongation (EZ) and
differentiated (HZ) zones for Al-exposed plants during exposure and recover. For MZ, cortex corresponds to fundamental
meristem and central region to procambium. Control is presented as 0 µM.
2
Root Area (mm ) ± SD
Anahuac
24h exp
0 µM
2
Cortex Area (mm ) ± SD
2
2
Central region Area (mm ) ± SD Lacune Area (mm ) ± SD
2
Endodermis cell Area (mm ) ± SD
2
Pericycle cell Area (mm ) ± SD
MZ
EZ
HZ
0.12 ± 0.005
0.24 ± 0.068
0.14 ± 0.017
0.06 ± 0.005
0.15 ± 0.060
0.08 ± 0.029
0.02 ± 0.001
0.06 ± 0.005
0.03 ± 0.002
0.001 ± 0.00005
0.005 ± 0.0001
0.002 ± 0.0004
0.0005 ± 0.00013
0.0003 ± 0.00006
0.0003 ± 0.00006
0.0002 ± 0.00005
MZ
EZ
HZ
0.09 ± 0.011
0.31 ± 0.006
0.19 ± 0.003 *
0.04 ± 0.009
0.21 ± 0.004
0.13 ± 0.003 *
0.02 ± 0.000
0.05 ± 0.001
0.03 ± 0.000 *
0.001 ± 0.0001
0.004 ± 0.0001
0.003 ± 0.0001
0.0005 ± 0.00009
0.0003 ± 0.00007 *
0.0004 ± 0.00007 *
0.0002 ± 0.00004 *
MZ
EZ
HZ
0.15 ± 0.016
0.32 ± 0.116
0.19 ± 0.010
0.09 ± 0.020
0.19 ± 0.046
0.12 ± 0.005
0.02 ± 0.003
0.08 ± 0.051
0.04 ± 0.002
0.001 ± 0.0001
0.006 ± 0.0033
0.003 ± 0.0005
0.0005 ± 0.00024
0.0003 ± 0.00010
0.0004 ± 0.00018
0.0003 ± 0.00005
MZ
EZ
HZ
0.12 ± 0.086
0.17 ± 0.009 *
0.15 ± 0.012 *
0.08 ± 0.058
0.12 ± 0.009 *
0.10 ± 0.011 *
0.02 ± 0.016
0.03 ± 0.001
0.03 ± 0.000 *
0.001 ± 0.0003
0.002 ± 0.0003
0.002 ± 0.0004
0.0004 ± 0.00011
0.0004 ± 0.00009 *
0.0002 ± 0.00004 *
0.0002 ± 0.00004 *
MZ
EZ
HZ
0.12 ± 0.004
0.38 ± 0.153
0.32 ± 0.216
0.07 ± 0.017
0.27 ± 0.108
0.20 ± 0.135
0.02 ± 0.004
0.06 ± 0.032
0.08 ± 0.050
0.001 ± 0.0003
0.004 ± 0.0023
0.005 ± 0.0027
0.0006 ± 0.00034
0.0007 ± 0.00056
0.0005 ± 0.00025
0.0005 ± 0.00033
MZ
EZ
HZ
0.24 ± 0.017 *
0.16 ± 0.015 *
0.13 ± 0.005
0.20 ± 0.017 *
0.11 ± 0.013 *
0.08 ± 0.004
0.03 ± 0.001
0.03 ± 0.001 *
0.03 ± 0.005 *
0.003 ± 0.0003
0.003 ± 0.0003
0.002 ± 0.0010
0.0003 ± 0.00009 *
0.0003 ± 0.00009 *
0.0002 ± 0.00005 *
0.0002 ± 0.00004 *
MZ
EZ
HZ
0.15 ± 0.016
0.32 ± 0.116
0.19 ± 0.010
0.09 ± 0.020
0.19 ± 0.046
0.12 ± 0.005
0.02 ± 0.003
0.08 ± 0.051
0.04 ± 0.002
0.001 ± 0.0001
0.006 ± 0.0033
0.003 ± 0.0005
0.0005 ± 0.00024
0.0003 ± 0.00010
0.0004 ± 0.00018
0.0003 ± 0.00005
MZ
EZ
HZ
0.18 ± 0.007 *
0.31 ± 0.167
0.42 ± 0.323
0.12 ± 0.003 *
0.21 ± 0.102
0.26 ± 0.203
0.03 ± 0.002 *
0.05 ± 0.030
0.09 ± 0.065
0.001 ± 0.0004
0.004 ± 0.0014
0.007 ± 0.0053
0.0008 ± 0.00049 *
0.0007 ± 0.00042 *
0.0005 ± 0.00034
0.0006 ± 0.00043
MZ
EZ
HZ
0.12 ± 0.004
0.38 ± 0.153
0.32 ± 0.216
0.07 ± 0.017
0.27 ± 0.108
0.20 ± 0.135
0.02 ± 0.004
0.06 ± 0.032
0.08 ± 0.050
0.001 ± 0.0003
0.004 ± 0.0023
0.005 ± 0.0027
0.0006 ± 0.00034
0.0007 ± 0.00056
0.0005 ± 0.00025
0.0005 ± 0.00033
MZ
EZ
HZ
0.19 ± 0.017 *
0.45 ± 0.019
0.65 ± 0.021 *
0.12 ± 0.012 *
0.28 ± 0.012
0.41 ± 0.018 *
0.03 ± 0.004 *
0.10 ± 0.003
0.12 ± 0.004 *
0.001 ± 0.0001
0.008 ± 0.0005
0.008 ± 0.0002
0.0011 ± 0.00018 *
0.0010 ± 0.00013 *
0.0007 ± 0.00011 *
0.0009 ± 0.00014 *
185 µM
48h exp
0 µM
185 µM
*
72h exp
0 µM
185 µM
24h rec
0 µM
185 µM
*
48h rec
0 µM
185 µM
*
*
*
* Data are significantly different at p < 0.05 with respect to correspondingly control.
71
Aluminium toxicity in wheat and rye
Table 2
‘Barbela 7/72’ roots area measurements. Average measurements of root meristematic (MZ), elongation (EZ) and
differentiated (HZ) zone for Al-exposed plants during exposure and recover. For MZ, cortex corresponds to fundamental
meristem and central region to procambium. Control is presented as 0 µM.
2
Root Area (mm ) ± SD
Barbela 7/72
24h exp
0 µM
2
Cortex Area (mm ) ± SD
2
2
Central region Area (mm ) ± SD Lacune Area (mm ) ± SD
MZ
EZ
HZ
0.70 ± 0.021
0.25 ± 0.017
0.26 ± 0.022
0.37 ± 0.071
0.18 ± 0.016
0.16 ± 0.014
0.15 ± 0.006
0.04 ± 0.002
0.06 ± 0.005
MZ
EZ
HZ
0.10 ± 0.015 *
0.27 ± 0.001
0.12 ± 0.007 *
0.06 ± 0.012 *
0.15 ± 0.003
0.08 ± 0.006 *
0.02 ± 0.002 *
0.05 ± 0.000 *
0.02 ± 0.001 *
MZ
EZ
HZ
0.13 ± 0.003
0.28 ± 0.107
0.28 ± 0.264
0.08 ± 0.003
0.18 ± 0.059
0.24 ± 0.225
0.03 ± 0.001
0.05 ± 0.026
0.05 ± 0.046
MZ
EZ
HZ
0.16 ± 0.002 *
0.19 ± 0.016
0.21 ± 0.007
0.07 ± 0.055 *
0.12 ± 0.029
0.15 ± 0.006
MZ
EZ
HZ
0.17 ± 0.030
0.22 ± 0.150
0.46 ± 0.417
MZ
EZ
HZ
2
Endodermis cell Area (mm ) ± SD
0.004 ± 0.0002
0.002 ± 0.0004
0.004 ± 0.0004
2
Pericycle cell Area (mm ) ± SD
0.0005 ± 0.00014
0.0005 ± 0.00005
0.0003 ± 0.00011
0.0005 ± 0.00005
0.0005 ± 0.00014
0.0003 ± 0.00009 *
0.0005 ± 0.00013 *
0.0002 ± 0.00004 *
0.002 ± 0.0000
0.004 ± 0.0018
0.005 ± 0.0038
0.0006 ± 0.00028
0.0006 ± 0.00053
0.0006 ± 0.00063
0.0005 ± 0.00040
0.02 ± 0.000 *
0.03 ± 0.001
0.03 ± 0.001
0.001 ± 0.00004 *
0.002 ± 0.0002 *
0.002 ± 0.0002
0.0003 ± 0.00012 *
0.0003 ± 0.00006 *
0.0003 ± 0.00006 *
0.0002 ± 0.00004
0.10 ± 0.028
0.14 ± 0.095
0.21 ± 0.207
0.03 ± 0.006
0.05 ± 0.031
0.08 ± 0.067
0.002 ± 0.0005
0.005 ± 0.0037
0.007 ± 0.0052
0.0004 ± 0.00026
0.0007 ± 0.00060
0.0003 ± 0.00052
0.0002 ± 0.00041
0.23 ± 0.010 *
0.20 ± 0.023
0.26 ± 0.013
0.12 ± 0.008
0.14 ± 0.020
0.18 ± 0.011
0.03 ± 0.001
0.03 ± 0.002
0.04 ± 0.001
0.001 ± 0.0006
0.002 ± 0.0003
0.004 ± 0.0001
0.0003 ± 0.00007
0.0004 ± 0.00011
0.0003 ± 0.00004 *
0.0003 ± 0.00006
MZ
EZ
HZ
0.13 ± 0.003
0.28 ± 0.107
0.28 ± 0.264
0.08 ± 0.003
0.18 ± 0.059
0.24 ± 0.225
0.03 ± 0.001
0.05 ± 0.026
0.05 ± 0.046
0.002 ± 0.00003
0.004 ± 0.0018
0.005 ± 0.0038
0.0006 ± 0.00028
0.0006 ± 0.00053
0.0006 ± 0.00063
0.0005 ± 0.00040
MZ
EZ
HZ
0.21 ± 0.027 *
0.40 ± 0.149
0.68 ± 0.025
0.14 ± 0.017 *
0.25 ± 0.091
0.43 ± 0.017
0.03 ± 0.005
0.06 ± 0.023
0.12 ± 0.004
0.001 ± 0.0001
0.005 ± 0.0017
0.008 ± 0.0006
0.0005 ± 0.00024
0.0012 ± 0.00028 *
0.0004 ± 0.00018
0.0007 ± 0.00014 *
MZ
EZ
HZ
0.17 ± 0.030
0.22 ± 0.150
0.46 ± 0.417
0.10 ± 0.028
0.14 ± 0.095
0.21 ± 0.207
0.03 ± 0.006
0.05 ± 0.031
0.08 ± 0.067
0.002 ± 0.0005
0.005 ± 0.0037
0.007 ± 0.0052
0.0004 ± 0.00026
0.0007 ± 0.00060
0.0003 ± 0.00052
0.0002 ± 0.00041
MZ
EZ
HZ
0.23 ± 0.014 *
0.21 ± 0.008
0.56 ± 0.024
0.15 ± 0.021 *
0.14 ± 0.004
0.34 ± 0.012
0.03 ± 0.001
0.04 ± 0.001
0.14 ± 0.006
0.001 ± 0.0001
0.005 ± 0.0023
0.010 ± 0.0002
0.0009 ± 0.00033 *
0.0011 ± 0.00023 *
0.0005 ± 0.00016 *
0.0011 ± 0.00024 *
185 µM
0.0003 ± 0.00001
0.004 ± 0.0003 *
0.002 ± 0.0001 *
49h exp
0 µM
185 µM
72h exp
0 µM
185 µM
24h rec
0 µM
185 µM
*
48h rec
0 µM
185 µM
*
* Data are significantly different at p < 0.05 with respect to correspondingly control.
In the differentiated HZ, 72 h continuous exposure did not affect ‘Barbela 7/72’ tissue
structure. However, in the sensitive genotype and for the same exposure, the central region
area decreased as well as the average cell area of endodermis and pericycle (p<0.05). After
the recovery period, and for ‘Anahuac’ plants, all morpho-histological parameters
measured increased with respect to control (Table 1), while for ‘Barbela 7/72’, this was
only detected in the endodermis and pericycle cells areas (p < 0.05) (Table 2).
Together with alterations in endodermis cell area, higher endodermis differentiation in the
HZ of the tolerant genotype was observed (Table 3). Contrarily, the sensitive genotype
showed a stimulatory effect only at the EZ. During recovery, the main trend found in
exposed roots was maintained.
72
Aluminium toxicity in wheat and rye
Table 3
Differences between percentages of control endodermis thickened cells and its correspondent Al-exposure in elongation
(EZ) and hairy root (HZ) zones. Values below and above 0.00 correspond to thickening inhibition and stimulation,
correspondingly. Values equal to 0.00 represent no alteration in thickness when compared to the control ones.
Barbela 7/72
24h
48h
72h
24h
48h
exp
exp
exp
rec
rec
EZ
3.05
0.00
-17.38
0.00
2.21
HZ
72.97
34.47
28.67
25.02
13.59
Anahuac
EZ
87.12
44.40
16.57
64.54
87.82
HZ
0.00
0.00
7.01
-19.78
-11.34
Callose detection
Control roots (‘Anahuac’ and ‘Barbela 7/72’) presented a discrete localization of callose,
generally observed as spots. Some root cap cells showed small callose dots, while above
the root cap, callose deposition was mostly restricted to epidermis, mainly associated with
root hairs, and near periclinal walls of some cortical cells.
In the three MZ subregions, a general increase of callose deposition was observed in
‘Anahuac’ exposed to Al: a) in the root cap cells, callose deposition increased (Fig. 4A),
becoming uniformly distributed with time; b) 1-2 mm behind the root cap, callose
deposition reached uniform distribution after 24 h in the epidermal cells, and after 48h in
the 2-2.5 mm region. In these two subregions, the outer cortical cell layer was the first
presenting callose (as largely distributed spots), which, with time, also accumulated in the
inner layers. After 72 h, the inner cell layer showed highest callose staining. At EZ, spots
of callose accumulation were visible in epidermis and cortex layers after 24h exposure,
increasing to homogeneous distribution with exposure (Fig. 4B). Still in this sensitive
genotype, at HZ, callose deposition was mostly restricted to cortex (mostly inner layers),
while its occurrence in epidermis was restricted to few spots independently of the exposure
period. After 72h exposure callose was also detected in endodermic cell (Fig. 4C).
In ‘Barbela 7/72’ MZ subregions, callose deposition in the root cap cells increased with
exposure (Fig. 4D). In the other two subregions (1-2 mm and 2-2.5 mm behind root cap),
callose deposition initially appeared as spots in epiderm, increasing with time. Cortical
cells presented few callose spots, particularly near periclinal cell walls of inner layers. At
EZ, callose first accumulated in the epidermis and outer cortical layers and with time, spot
deposition extended also to inner cell layers (Fig. 4E). At HZ and since 24 h exposure
73
Aluminium toxicity in wheat and rye
callose spots were detected mostly in cortex (with inner cell layers presenting higher
fluorescence) (Fig. 4F).
A
D
B
E
C
F
G
H
Fig. 4. Spatial distribution of callose accumulation induced by aluminium (Al) exposure in longitudinal and transversal
root sections stained by aniline blue. ‘Anahuac’ roots after 72h Al-exposure (A-C) and 48h after Al removal (G);
‘Barbela 7/72’ roots after 72h Al-exposure (D-F) and 48h after Al removal (H). A, D) root cap; B, E) EZ: 10-15mm
behind root cap; C, F) HZ: 30-50mm behind root cap; G, H) EZ: 10-15mm behind root cap.
After recovery time, callose deposition was not reverted in ‘Anahuac’ roots. These roots
maintained the same callose accumulation and distribution profile that was shown after Al
exposure (Fig. 4G). Contrarily, in ‘Barbela 7/72’ roots, callose deposition was reversible
(Fig. 4H): these roots presented a clearly reduction in callose deposition in all root zones
except in HZ that maintained as after exposure.
74
Aluminium toxicity in wheat and rye
Flow cytometric analyses
Regarding the nuclei ability to disperse light, FCM analyses give estimations on both
nuclei granularity and size. For these parameters, no significant differences (p>0.05) were
obtained among all conditions.
The nDNA of control plants ranged between 2 C = 36.19 ± 0.253 pg, for ‘Barbela 7/72’
and 2 C = 35.19 ± 0.295 pg for ‘Anahuac’. Al exposure did not induce major DNA ploidy
changes and the small variations found in DNA content are within the range of normal
intraspecific variation. To score for possible clastogenic damage the full peak coefficient
of variation (FPCV) of the G0/G1 peaks were analyzed.
Table 4
Nuclei’s full peak coefficient of variation (FPCV) for ‘Anahuac’ and ‘Barbela 7/72’. Values are given as mean ± standard
deviation for each condition.
Genotype
Root
FPCV ± SD
Leaf
FPCV ± SD
7.20± 0.432
7.13 ± 0.464
11.50 ± 0.250
10.55 ± 0.499
7.53 ± 0.573
7.83 ± 0.287
10.17 ± 0.275
9.37 ± 0.368
8.23 ± 0.759
8.80 ± 1.337
9.53 ± 0.929
11.73 ± 1.841 *
5.97 ± 0.499
8.63 ± 0.531 *
9.33 ± 0.419
11.55 ± 0.589 *
8.23 ± 0.759
7.10 ± 0.668 *
9.53 ± 0.929
8.80 ± 0.216
5.97 ± 0.499
8.00 ± 1.759 *
9.33 ± 0.419
10.13 ± 0.262
6.80 ± 0.726
8.43 ± 0.478
10.55 ± 0.531
13.60 ± 1.878
7.77 ± 0.776
9.87 ± 0.818 *
12.20 ± 1.268
12.23 ± 1.223
7.63 ± 0.340
10.27 ± 1.337 *
13.37 ± 0.633
11.35 ± 0.776
9.23 ± 1.759
12.30 ± 0.589 *
11.70 ± 0.400
13.80 ± 0.650
7.63 ± 0.818
9.85 ± 0.204
13.37 ± 0.953
9.47 ± 2.223 *
9.23 ± 1.759
12.07 ± 1.551 *
11.70 ± 0.653
10.97 ± 1.841
6h exp
Barbela 7/72
Anahuac
0 µM
185 µM
24h exp
0 µM
185 µM
48h exp
0 µM
185 µM
72h exp
0 µM
185 µM
24h rec
0 µM
185 µM
48h rec
0 µM
185 µM
6h exp
0 µM
185 µM
24h exp
0 µM
185 µM
48h exp
0 µM
185 µM
72h exp
0 µM
185 µM
24h rec
0 µM
185 µM
48h rec
0 µM
185 µM
* Data are significantly different at p < 0.05 with respect to correspondingly control.
75
Aluminium toxicity in wheat and rye
In ‘Anahuac’ roots, Al exposure generally increased FPCV values (Table 4) while in
‘Barbela 7/72’, a somewhat heterogeneous response was found at 48 h recovery (increase)
and 72 h exposure (decrease).
A
1
Pea k
1
2
% eve nts M ean
57. 6
20 1.3
18. 8
28 6.9
CV (%)
2.02
1.83
B
1
1
Pea k
1
2
% eve nts
Mean
CV (%)
1
2
57.4
15.6
200.6
382.8
2 .8 4
1 .2 2
2
2
C
Pea k
% eve nts M ean
56. 0
19 5.2
16. 0
38 0.7
CV ( %)
1.79
1.48
D
1
Peak
1
% eve nts Mean
48.6
193.5
CV ( %)
2 .5 9
2
20.9
374.0
2 .1 4
Pe ak
% ev ents
Mea n
CV (%)
1
2
74.1
7. 5
201. 3
395. 0
2. 57
0. 68
2
2
E
1
Peak
1
2
% ev ents
55.4
7 .8
Mean
200.2
398.6
CV (%)
3. 15
0. 87
F
1
2
a
G
1
2
Peak
1
2
% ev ents
45.9
11.9
Mean
195.8
390.8
2
CV (%)
2. 25
0. 53
H
1
Peak
1
2
% eve nts Mean
65.9
192.6
8.5
381.4
CV ( %)
2 .3 6
0 .5 7
2
Fig. 5. Histograms of relative fluorescence intensity (FL) for ‘Anahuac’ (A, C, E, G) and ‘Barbela 7/72’ (B, D, F, H).
Plants were either exposed to Al (C, D, G, H) or controls (A, B). Histograms presented are representative to 6 h (A, B, C,
D) and 72 h (E, F, G, H) conditions. The values given as the percentage of nuclei, the mean position of the peak (mean)
and the coefficient of variation (CV%) of the G0/G1 and G2 peaks (1 and 2 in the histograms, respectively). Images are
representative of at least four replicate roots.
76
Aluminium toxicity in wheat and rye
FCM cell cycle dynamic analysis was performed by quantifying the proportion of cells in
the different cell cycle phases (G0/G1: S: G2) for each tissue analyzed (Fig. 5, 6). Control
plants, along the conditions tested showed a gradual increase in the proportion of G0/G1
cells at the expense of S and G2. Al-treated ‘Barbela 7/72’ roots presented a decrease of
cells in G2 phase after 6 h and 48 h exposure (p < 0.05), together with an increase in
G0/G1after 48 h (p < 0.05) (Fig. 6). Simultaneously, a trend of decreasing cells in S phase
was observed (mostly after 24 h, p < 0.05). Contrarily, in the sensitive genotype
‘Anahuac’, an increase of cells in S phase was observed after 72 h exposure (p < 0.05).
Concerning the recovery period, only ‘Anahuac’ roots presented significant changes: after
48 h of recovery, it was observed a blockage at S phase at the expenses of decreases in
G0/G1 and G2 phases (Fig. 6).
Barbela 7/72
A
4,9
12,8
14,7
19,0
20,0
11,8
13,0
11,5
4,8
11,8
13,0
6,9
15,7
16,0
10,6
15,4
5,4
13,6
6,3
6,0
9,9
9,3
6,0
7,3
10,5
10,4
4,0
6,4
13,3
12,7
7,3
6,8
6,4
5,2
10,4
9,9
12,7
13,5
10,6
15,2
22,9
2,5
11,5
19,0
17,7
19,0
28,3
28,1
Nuclei cells (%)
G1
S
69,9
64,3
73,0
68,0
79,1
77,9
79,5
77,9
87,7
83,9
84,7
83,9
83,6
82,3
82,8
80,9
83,3
82,3
81,3
80,9
G2
70,5
68,0
52,7
51,9
B 0µM
185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM
0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM
Anahuac
root
root
15,7
18,76h exp
root
12,5
19,5
24h exp
11,9
16,0
48h exp
9,1
root
root
10,9
72h exp
11,0
16,0
24h rec
9,1
root
8,2
48h rec
4,1
leaf 5,2
10,26h exp
17,1
15,3
19,4
21,6
28,1
17,7
15,3
26,6
30,2
17,7
28,8
6,2
leaf 4,5
4,7
leaf
10,3
24h exp
16,5
11,1
48h exp
18,4
14,5
7,2
leaf 7,8
72h exp
18,0
4,7
leaf 6,6
11,1
24h rec
11,4
7,2
leaf 3,6
48h rec
18,9
18,0
19,4
36,3
Nuclei cells (%)
20,9
G1
S
85,8
75,6
66,3
65,0
59,7
59,6
77,9
75,6
84,2
79,0
75,4
75,2
74,8
82,1
77,5
74,8
72,7
G2
66,3
61,6
59,4
84,2
60,3
58,9
55,6
0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM 0µM 185µM
root
root
root
root
root
root
leaf
leaf
leaf
leaf
leaf
leaf
6h exp
24h exp
48h exp
72h exp
24h rec
48h rec
6h exp
24h exp
48h exp
72h exp
24h rec
48h rec
Fig. 6. Cell cycle dynamics of roots and leaves of ‘Barbela 7/72’ (A) and ‘Anahuac’ (B) exposed to control (0 µM) or Al
(185 µM) containing nutritive solution, after 6 h, 24 h, 48 h and 72 h Al exposure (exp) or 24 h and 48 h with recovery
(rec). The values given are the means of each population of cells in each of the cell cycle stages (G1, S and G2).
77
Aluminium toxicity in wheat and rye
Discussion and Conclusions
Root growth inhibition is a primary symptom of Al sensitivity, resulting in a reduced and
damaged root system (Kochian et al., 2004). Data concerning root regrowth after Alexposure is generally used to classify genotypes as Al-sensitive or Al-tolerant (e.g. Aniol
and Gustafson, 1984; Pinto-Carnide and Guedes-Pinto, 1999; Aniol, 1984). Pinto-Carnide
and Guedes-Pinto (1999) characterized ‘Anahuac’ and ‘Barbela 7/72’ genotypes using this
end-point, as Al-sensitive and Al-tolerant genotypes, respectively.
Due to their different tolerance, these genotypes are being used to study induced Al
toxicity and tolerance strategies at physiological levels (e.g. Silva et al., 2010). The
physiological mechanisms involved in root response to Al are still under discussion and
usually are classified as those preventing Al uptake/accumulation by roots and those
involved in detoxification once Al is inside the cell (e.g. Silva et al., 2000; Kochian et al.,
2004). Our data show that Al accumulated at higher levels and severely inhibited root
growth and regrowth in ‘Anahuac’ when compared to the tolerant genotype, supporting the
previous classification of Pinto-Carnide and Guedes-Pinto (1999). In sensitive roots,
histological analyses showed collapsed cell structure and perturbation in tissue
architecture, namely in MZ. Similar lesions at the MZ were detected in sensitive Norway
spruce seedlings (Nagy et al., 2004) exposed to Al. The same authors also related the
increase of cell damages/death in MZ with the disappearance of carbohydrates. In the MZ
of the sensitive ‘Anahuac’ roots, carbohydrates and lipid accumulation also decreased,
confirming a clear cellular metabolism disturbance. Contrarily,’Barbela 7/72’ that is less
affected in growth by Al, showed no changes in carbohydrates and lipids accumulation
profiles between exposed and not exposed roots. Moreover, ‘Anahuac’ sensitivity was
even more evident by roots inability to revert these structural and functional damages after
Al removal.
The studies conducted on maize root growth inhibition by Ryan et al (1993) and Sivaguru
and Horst (1998) demonstrated that MZ is the most Al-sensitive site. Those studies,
however, only focused on the apical region, not covering more differentiated regions such
as the hairy root zone (HZ), a crucial region for absorption. Considering the HZ role in
water and nutrients absorption, it should be expected that, besides the described sensitivity
of MZ, HZ should also present sensitivity to Al exposure. In this zone, callose gradually
accumulated in epidermis and outer cortical layer (started with patches or spots and
78
Aluminium toxicity in wheat and rye
evolved to uniformly distribution in the cell wall surface) and gradually accumulated in
inner cortical layers and even in endodermis (in the sensitive genotype). We detected that
in the sensitive genotype, a uniform accumulation of callose (epidermis and outer cortical
layer) coincided with cell wall disruption. In maize it was demonstrated that Al initially
accumulates in epidermal and outer cortical cell layers and only later it enters inner layers
of root apex (Jones et al., 2006). These data support our findings that Al first affects the
epidermis cell structure and outer cortical layers architecture. We found a similar pattern of
callose deposition in these cell layers, suggesting that uniform deposition contributes to the
collapse of cell structure. Previously, Horst et al (1997) extracted and quantified, by
fluorimetry, callose in several Al-exposed root zones of maize, but the results above 1 cm
were inconsistent as they found, mostly, non specific fluorescence, hampering a clear
interpretation of those data. Alternatively, our results show that by microscopy, we were
able to clearly confirm that callose in response to Al accumulates (and how it is distributed
within the cell layers) in MZ, EZ and HZ.
Plants face a dilemma by accumulating callose when exposed to Al. On one hand callose
may act as a barrier to Al influx, being a protective strategy of blocking plasmodesmata,
inhibiting symplastic transfer of solutes (Sivaguru et al., 2000). On other hand, callose is
speculated to also inhibit root growth (Tahara et al., 2005) due to cell wall rigidification
and inhibition of the symplastic carbon supply required to fuel root growth (Jones et al.,
2006). Moreover, Jones et al (2006) postulated that callose response may be mediated at a
cell-specific level rather than at a systemic level. In this complex process, the nutritional
cell balance is mandatory. For example, elevated cytosolic Ca2+ activity is a prerequisite
and acts as a trigger for callose synthesis (Horst et al., 2010). Rengel and Zhang (2003)
also hypothesized that cells have a finite capacity to accumulate callose and that a decrease
in cytosolic Ca2+ activity would prevent callose accumulation.
The higher and more uniform accumulation of callose in the sensitive genotype compared
to the tolerant one is in accordance with findings in other sensitive species/genotypes (e.g.
Eticha et al., 2005; Tahara et al., 2005). Moreover, the sensitive genotype was unable to
reduce callose accumulation and Al contents, contrarily to the tolerant one. All this
together suggest that: a) the protective machinery against Al entrance/exclusion and
toxicity in ‘Anahuac’ isn’t efficient; b) protective measure of callose formation, being
higher and uniformly distributed in the sensitive genotype may jeopardize nutrient
absorption and increase rigidification, promoting cell disruption; c) Al induced signal to
79
Aluminium toxicity in wheat and rye
callose deposition remains after Al removal, suggesting that the machinery involved in
callose degradation is deregulated in the sensitive genotype.
Some works reported that callose production was detected only in actively growing root
zones (Jones et al., 2006). However, in our findings, callose production was also detected
in mature root regions (HZ), proving that Al exposure caused injuries in that zone.
Furthermore, after Al removal and in ‘Barbela 7/72’, callose was not degraded in HZ,
contrarily to other more young regions. This suggests that, concerning callose deposition,
Al affected more severely the HZ then the actively growing root zones (MZ and EZ).
Using these tolerant and sensitive wheat genotypes we have demonstrated for the first time
that genotypes differing in Al tolerance also show different endodermis differentiation
profiles in response to Al. Once the HZ is a specialized zone for absorption, the tolerant
‘Barbela 7/72’ seems to have stimulated the differentiation of endodermis as a
complementary barrier to Al entrance in this HZ. Contrarily, ‘Anahuac’ apparently does
not have this protective mechanism in HZ, and uses callose as the only barrier with the
consequent cell wall rigification. The different patterns of endodermis cell wall thickening,
at the sites of major entrance, between both wheat genotypes sustain that, differences in
cell wall chemistry and in cell signalling regulating endodermis thickening influence roots
tolerance to Al.
Root growth inhibition may be due to Al-effects in both apoplast and symplast, most
probably acting in combination. A first analysis of Al-treated wheat roots by FCM did not
reveal any significant effect induced by Al in the nuclei size and granularity. The nDNA
content found for both genotypes are within the range values described for wheat from
33.10 to 38.90 pg/2C (Bennett and Leitch, 2005) considering the large number of cultivars
of this species. The histograms for both genotype roots under Al-stress, revealed little
variation of DNA content (hence DNA-ploidy level), showing that the Al concentration
and/or the period of exposure used were not sufficient to induce significant DNA content
changes.
Rayburn and Wetzel (2002) showed in wheat and maize, that FCM was sensitive enough to
detect the intraplant nuclear DNA variation associated with sticky chromosomes and
abnormal mitosis. In Al-exposed wheat roots, the CV of G0/G1 peaks increased more
significantly in Al-exposed sensitive plants than in tolerant ones. These data support the
occurrence of Al-induced clastogenic effects, mostly in the sensitive genotype. However, a
higher variation in cellular compounds, which interfere with PI-DNA intercalation
80
Aluminium toxicity in wheat and rye
(Loureiro et al., 2006), may also be at the basis of the higher CV observed in the sensitive
cultivar compared to the tolerant one. Matsumoto (1991) demonstrated that Al can
adversely affect chromatin structure and template activity, to which PI intercalation is also
sensitive (Loureiro et al., 2007). In summary, our data demonstrate that Al exposure leads
to an overall increase of the G0/G1 peak CV with time, mostly in roots, and that this effect
is more drastic in the sensitive genotype. All this together point to a cause-effect scenario,
in which Al exposure leads to an effective DNA clastogenic damage alone, and/or
combined with plant strategies to cope with increasing exposure to Al.
Changes in the cell cycle patterning were described as an effect of Al-toxicity in the
symplast, which is mostly evident as a blockage of cells at the S phase in the Al-sensitive
maize (Doncheva et al., 2005). Our data confirms that Al interferes with cell cycle
patterning (G0/G1: S: G2) but the effect varies according to the tolerance degree of the plant
genotype. Similarly to Doncheva et al (2005) reports, we also found a blockage at the S
phase in the sensitive wheat genotype ‘Anahuac’, when exposed to Al. These exposed
roots had a cell cycle G0/G1: S: G2 patterning quite different from the one of controls.
Contrarily, the cell cycle dynamics of Al-exposed ‘Barbela 7/72’ roots followed a trend
close to the one of control roots, but the observed blockage is at the G0/G1 phase, while the
percentage of cells in S and G2 decreased. Data strongly suggests that Al affects either
G0/G1-S transition checkpoint or the S-G2 transition checkpoint, depending on the
tolerance degree of the genotype.
Stress induced cell cycle effects, were already described in salt stressed Arabidopsis roots
(West et al., 2004) that showed an arrest at the G2/M transition, justified by a decrease in
both meristem size and the proportion of cells showing CYCB1;2 promoter (mitotic cyclin)
activity. Besides, Bracale et al (1997) and Schuppler et al (1998) also found evidences for
a G2 blockage in water stressed pea root tips and wheat leaves, respectively. The arrest in
G2 was explained as a protective strategy, preventing stressed cells to enter into a stage (M)
where they might become highly fragile.
Though the regulators accountable for this Al-induced response remain unknown, Al
seems to act in different pathways of those regulated by salt and water stresses. In Al
sensitive roots, our data and the one of Doncheva (2005) suggest that the S to G2 transition
is the most sensitive checkpoint, while in the tolerant one, the blockage is mostly exerted
in the G1 to S transition. Checkpoints include monitoring the cell size and environment
prior proceeding from G1 to S, that all DNA has been synthesized before moving from S to
81
Aluminium toxicity in wheat and rye
G2 (Gahan, 2007). Therefore the arrest in the S phase might be due to damages of a (pos)
replication process. Our hypothesis is in agreement with the data found by Callegari and
Kelly (2006; 2007) in mutant yeasts exposed to UV radiation, which showed a delay in cell
cycle (mostly a blockage at the S phase) due to damages in a postreplication (for review
see Andersen et al., 2008).
Al effects on cell cycle and/or on the peak CV values occur even after removing Al, which
may be due to the remaining metal accumulated inside the root cells or that effects caused
are not reverted within the recovery period tested.
In conclusion, we demonstrate here that compared to tolerant roots, sensitive ones showed
an inhibition of root regrowth, and more differentiated cells closer to the root tip together
with a higher destruction of inner MZ cells. Moreover, both tolerant and sensitive
genotypes differ in both callose deposition/allocation and endodermis differentiation in
response to Al exposure. We therefore suggest that callose and endodermis play
complementary roles to prevent Al entrance in the symplast, with different contributions in
the different root regions, and that the tolerant genotype has more efficient mechanisms for
coping with Al entrance (e.g. at the HZ). These facts, combined with the different effects
of Al in cell cycle partitioning between the two genotypes contribute to better
understanding the tolerance mechanisms developed by tolerant genotypes under Al stress,
and that may contribute to the ability of root regrowth during recovery in these genotypes.
Acknowledgements
FCT/MCT supported this work (POCI /AGR/ 58174/2004), and S. Silva (FCT⁄BD⁄ 32257⁄2006)
and E. Rodriguez (FCT/BD/27467/06) grants. Thanks are due to Armando Costa for technical
support.
82
Aluminium toxicity in wheat and rye
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88
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oxicity in wheat and rye
II 3: Al toxicity mechanisms in tolerant and sensitive rye
ye genotypes: a comprehensive
physiological and histological study
Chapter accepted as an original paper in a SCI journal:
Silva S, Santos C, Matos M, Pinto
Pinto-Carnide O. 2011. Al toxicity mechanism in tolerant and
sensitive rye genotypes: a comprehensive physiological and histological study.
Environmental and Experimental Botany (accepted).
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90
Aluminium toxicity in wheat and rye
Abstract
Rye (Secale cereale L) plants have high tolerance to Al, and become unique tools to study
Al-toxicity and tolerance mechanisms. To better understand if and how Al-induced root
growth inhibition is related with root histological differentiation, nutrient balances and cell
cycle dynamics, tolerant (AT6) and sensitive (AS6) rye lines were exposed for 24 h to 1.1
mM Al. After that period, half of plants were transferred to medium without Al, while the
other group was maintained with Al for more 48 h. AS6 plants showed evident and
irreversible root growth inhibition, decreases in water content, organic matter and
carbohydrate reserves. Also, this line showed greater nutrient imbalances (e.g. an increase
of root Ca levels) and changes in root anatomy (e.g. thickness). Data revealed that AS6
plants accumulate more Al in roots and leaves, suggesting more Al translocation to the
shoots. The two lines responded differently to Al exposure in what concerns endodermis
differentiation and cell cycle profile. The tolerant line shows a tendency to arrest in G0/G1
phase whereas the sensitive have a tendency to arrest in S phase. These data point to an
arrest of cells in specific cell cycle phases, leading to eventual Al-induced cell cycle delay.
Taken all together, data show that even short-term exposure to Al leads to root anatomical
modifications that in combination with the cell cycle dynamic changes, may represent a
plant strategy to cope with this stress.
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Aluminium toxicity in wheat and rye
92
Aluminium toxicity in wheat and rye
Introduction
Aluminium (Al) is an abundant metal in the earth’s crust and becomes available in a
phytotoxic form to plants in acidic soils (pH below 5.5). It is estimated that over 40-50 %
of world’s arable non-irrigated land is acidic (e.g. Jardim, 2007; Panda et al., 2009), and in
those soils, Al phytotoxicity is considered a primary factor limiting crop productivity (e.g.
Maron et al., 2008).
Due to the particular chemical nature of Al, its mechanisms of phytotoxicity and tolerance
are complex, and dependent on the interactions with other nutrients and soil properties (e.g.
Zheng et al., 2007, Maron et al., 2008). In general it is considered that Al3+ (e.g. AlCl3) is
the most phytotoxic form (e.g. Kinraide et al., 1992).
Rye (Secale cereale L.) is not a major crop, but it can resist in extreme conditions as poor
or acid soils. Among the cultivated Triticeae, rye is one of the most Al tolerant and
represents an important potential source of Al tolerance for improvement of wheat and
other cereals (Collins et al., 2008). Most studies on rye are focused on genes correlated
with resistance (e.g. Matos et al., 2005; Fontecha et al., 2007; Matos et al., 2007; Collins
et al., 2008; Shi et al., 2009). It has been proposed that the Al-resistance strategies can be
separated into a) exclusion of Al from the root apex and b) Al-tolerance (Collins et al.,
2008; Panda et al., 2009; for review see Horst et al., 2010). At the physiological level, root
growth inhibition is well described and referred as a marker used to classify crops
sensitivity to Al. Another sensitive indicator of Al injury is the induction of callose
synthesis (Tahara et al., 2005; Jones et al., 2006; Horst et al., 2010). Callose deposition
may lead to cellular damage by inhibiting intercellular transport through plasmodesmatal
connections (Kochian et al., 2005; Panda et al., 2009). This parameter was also indicated
as reliable for the classification of genotypes in terms of Al-resistance (Horst et al., 1997;
Massot et al., 1999; Meriga et al., 2003; Bhuja et al., 2004; Tahara et al., 2005).
Inside the root, most of the Al is located in the apoplast and the primary binding site of
Al3+ is probably the pectin matrix (Chang et al., 1999). This binding appears to be
positively correlated to callose formation and therefore to Al-sensitivity (Horst et al.,
1999). Some effects attributed to root inhibition are the Al disruption of cytoskeleton
dynamics, interaction with microtubules and actin filaments (e.g. Sivaguru et al., 2003;
Jardim, 2007). Also, induction of nutrient imbalances are reported such as Ca, B, K,or Mg
(e.g. Ma et al., 2002; Jiang et al., 2009; Panda et al., 2009; Silva et al., 2010). In particular,
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Aluminium toxicity in wheat and rye
an increase of membrane depolarization (Panda et al., 2009) and increase in cytosolic Ca2+
has been reported as response to Al exposure. The disruption of Ca2+ homeostasis may be
involved in the disorganization of the cytoskeleton and consequently inhibit division and
root elongation (Ma et al., 2002; Rengel and Zhang, 2003; Panda et al., 2009). Increases in
Ca2+ and several polyvalent metal cations (as Al) are requisites for callose synthesis (Horst
et al., 1995; 2010; Panda et al., 2009). As root growth involves both cell divisions and cell
expansion contributions, it is also important to identify the specific effects of Al in the
different root zones (e.g. meristematic and elongation cell zones). In maize, Ryan et al.
(1993) and Sivaguru and Horst (1998) showed that, respectively, the root apex, and the
transition zone were the most Al-sensitive zones. However, the available studies only focus
the apical region and omit the hairy root zone, an extremely important region for its role in
absorption. Therefore, it is still unknown if and how Al affects, for example, the
histological differentiation and reorganization in this region, and if tolerant and sensitive
genotypes differ in their responses to Al in this region.
Among the functional disorders, Al induced changes in reserves was also described in
wheat by Silva et al. (2010) and in sensitive Norway spruce seedlings (Nagy et al., 2004)
exposed to Al, despite why and how these reserves disappear is still unclear.
Al was shown to induce chromosome stickiness and breaks (e.g. Mohanty et al., 2004) and
to induce clastogenicity, detected by flow cytometry by Rayburn and Wetzel, (2002).
Furthermore, Al reduces cell division (e.g. Lazof and Holland, 1999). The accumulation of
Al in the nuclei supports a direct effect on cell division (e.g. Silva et al., 2000). Also,
Doncheva et al. (2005) demonstrated, for sensitive maize plants, that Al differently
affected the cell cycle dynamics depending on the root region, with inhibitions at S phase
in the proximal root meristem. The way Al affects the cell cycle dynamics and its putative
cytostatic effects and consequences to root growth inhibition remains therefore still a
matter of discussion.
From Montalegre regional rye population an Al tolerant line (AT6) and an Al sensitive line
(AS6) were obtained. The AT6 line tolerates up to 30ppm Al while the AS6 line doesn’t
show root regrowth at this concentration. These close lines are therefore unique models to
better understand genetic and phenotypic characteristics. We hypothesised that the
sensitive rye genotype has more severe blockages of the cell cycle and suffers more severe
disruptions of histological structure and function (e.g. ion accumulation) through the root
(from the root tip to the hairy root zone). These comprehensive analyses will provide a
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Aluminium toxicity in wheat and rye
global perspective of functional mechanisms in both rye lines regarding their tolerance to
Al.
Material and Methods
Plant material selection, growth and exposure to aluminium
The Montalegre regional rye population from Trás-os-Montes, Portugal (collection,
DGB/UTAD, Portugal) was used. Two lines were selected from this population, which
show different behavior to Al: AT6 (Al tolerant) and AS6 (Al sensitive). Seeds were rinsed
in distilled water and germinated (in the dark at 24 ºC) in petri dishes with moistened
Whatman paper until roots reached 0.5 cm long. Plantlets were then grown on a nutritive
solution (Polle et al., 1978), and after two days transferred to the same nutritive solution
containing 1.1 mM (30 ppm) AlCl3.6H2O. After 24h on this solution, half of the plants
were kept in the presence of Al for more 48 h (a total of 72h exposure). The other half was,
after 24h exposure, transferred to the same medium without Al for recovery during 48 h.
As control, another group of plants was maintained in nutritive solution without Al for 72
h. The pH was maintained at 4.0 throughout the assay.
Nutritional, Water and Organic Matter Determinations
For determination of water content and organic matter were used the following formulas:
water content = (fresh weight – dry weight / fresh weight) x 100; organic matter = (dry
weight – ashes weight / dry weight) x 100.
For root elemental quantification of Al, Ca, Si, P, K, Mg, Mn, Zn, Cu, B and Fe and for
leaf elemental quantification of Al, samples were treated as described by Silva et al.
(2010). Briefly, samples were collected at the following times: 24 h of exposure, 24 h and
48 h after recover. Fresh weight of roots was measured. After washing to remove root
adsorbed ions, samples were dried at 60 ºC until constant weight and then dried weight was
assessed. Elements were determined by inductively coupled plasma spectroscopy (ICPS,
Jobin Ivon, JY70 Plus, Longjumeau Cedex, France).
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Aluminium toxicity in wheat and rye
Histological, morphometric and reserve analyses
Histocytological analyses were performed according to Silva et al. (2010) on root samples
exposed for 24 h to Al, and 24 h and 48 h after Al removal (recovery). Sections from the
meristematic zone (MZ, 3 mm behind the root cap), from the elongation zone (EZ, 10-15
mm behind the root cap) (Sivaguru and Horst, 1998), and from the hairy root zone (HZ,
more than 30 mm behind root cap) were collected for analysis. Briefly, fresh samples were
fixed in 2.5% glutaraldehyde overnight at 4 ºC and then washed in piperazine-N,N’-bis-2ethanesulfonic acid (PIPES) buffer. Tissues were then transferred to 1.0 % (w/v) osmium
tetroxide in PIPES buffer, dehydrated using increasingly concentrated acetone solutions
(30 – 100 %, (v/v)) and embedded in an epoxi resin (Embed-812). Semi-thin sections (app.
1 µm) were stained with periodic acid-Schiff reaction (PAS) for carbohydrate detection.
Samples were analyzed with a Nikon Eclipse 80i light microscope (Nikon Co, Kanagawa,
Japan) and photographs were taken using a Leica DC 200 digital camera (Leica
Microsystems AG, Germany). Histological images were analyzed using the image
analyzing program UTHSCSA Image Tool, version 3.00 (University of Texas Health
Science Center, USA). For morphometric studies, the following parameters were
measured: root cross section area, pericycle and endodermis cell area and percentage of
endodermis cells with cell wall thickening. Measurements were based on at least three
different roots.
Callose detection
For callose detection were used roots after 24 h exposure, 24 h and 48 h after Al removal.
The same regions selected for histological analyses were chosen: MZ, EZ and HZ. Semithin sections were stained for 30 min with 0.1% aniline blue and then mounted in a drop of
50 % glycerine (Castro et al., 2008). Samples were observed with a Nikon Eclipse 80i
epifluorescence microscope equipped with a UV-2A filter cube (330–380 nm excitation;
Nikon Instruments, Inc., Kanagawa, Japan).
96
Aluminium toxicity in wheat and rye
Flow cytometric analysis
For FCM studies, nuclear suspensions of root apices were prepared according to Conde et
al. (2004) with minor modifications. Briefly, after chopping, the nuclei suspension was
filtered through an 80 µm nylon mesh and 50 µg/ml of propidium iodide (PI) (Fluka) and
50 µg/ml of RNAse (Sigma) were added to the samples. For DNA content estimation, 20
mg of Vicia faba L. (with 2C = 26.90 pg DNA) were added as internal standard, and cochopped with sample tissue. After a 5 min incubation period, samples were analysed in a
Coulter EPICS-XL flow cytometer (Coulter Electronics, Hialeah, Florida, USA), equipped
with an air-cooled argon-ion laser (15 mW operating at 488 nm). Before starting the
analysis, the instrument was checked according to Loureiro et al. (2007). Only samples
with at least 3,000 nuclei were considered for statistical analysis. The DNA ploidy level of
each sample was given as an index relative to the internal standard (V. faba), by analyzing
three replicates per condition.
The nuclear genome size of the samples was calculated according to the following
formula:
Sample 2C nuclear DNA content (pg) =
Wheat landrace G 0 /G 1 peak mean
× 26.90
V . faba G 0 /G 1 peak mean
For cell cycle analysis, flow cytometric files were transformed to single histogram files
with WinMDI ver. 2.9 software (freeware by Joe Trotter of the Scripps Institute, La Jolla,
CA.) and then analysed using Cylchred software (freeware developed by Terry Hoy of the
University of Cardiff).
Statistical Analysis
Values are given as mean ± standard deviation from at least six independent replicates as
calculated from data from independent experiences. Data normality distribution was tested
and whenever failed data transformation was performed for normalization. The comparison
between Al exposure periods and the control was made using a One Way ANOVA test to
data, followed by a Holmes-Sidak multiple comparison procedure (p < 0.05). For this
statistical analysis, the software package SigmaStat was used (SPSS 1995). Correlations
between Al accumulations vs water and Ca contents were tested by Pearson’s analysis.
97
Aluminium toxicity in wheat and rye
Results
Root growth
Under control conditions, roots of both lines were whitish and showed active growth. At
the end of the experiment (72 h) in the absence of Al, the AS6 line roots were 23.97 %
longer than the AT6 one. Under Al exposure, AS6 suffered a significant root growth
inhibition after 24 h compared to the control (p < 0.05), and this effect was never reverted
in this sensitive line. Contrarily, AT6 roots only suffered growth inhibition after 72 h of
continuous exposure to Al (p < 0.05) (Table 1).
Table 1
Variation in root water content (%), organic matter (%) and growth (cm) in tolerant (AT6) and sensitive (AS6) exposed
plants to 1.1 mM Al during 72 h and after 48 h in recovery. Values are given as mean ± standard deviation for each
condition.
AT 6
24h exp
Water content
Organic Matter
Growth
0 mM
1.1 mM
87.9 ± 7.40
93.6 ± 1.24
75.4 ± 7.40
83.6 ± 1.24
6.6 ± 0.59
6.1 ± 0.97
0 mM
1.1 mM
93.1 ± 2.87
93.7 ± 0.85
84.5 ± 2.87
84.2 ± 0.85
7.3 ± 1.16
7.1 ± 0.55
0 mM
1.1 mM
94.8 ± 2.98
91.5 ± 1.92
77.1 ± 2.98
85.1 ± 1.92
9.6 ± 0.82
6.4 ± 0.61 *
0 mM
1.1 mM
93.1 ± 2.87
92.8 ± 1.25
84.5 ± 2.87
83.4 ± 1.25
7.3 ± 1.16
7.7 ± 0.89
0 mM
1.1 mM
94.8 ± 2.98
94.1 ± 5.57
77.1 ± 2.98
79.9 ± 5.57
9.6 ± 0.82
9.2 ± 0.97
Water content
Organic Matter
Growth
48h exp
72h exp
24h rec
48h rec
AS6
24h exp
0 mM
1.1 mM
94.2 ± 2.68
94.4 ± 11.26
78.6 ± 2.68
68.0 ± 11.26 *
8.2 ± 0.51
6.3 ± 0.87 *
0 mM
1.1 mM
95.4 ± 1.07
93.6 ± 0.67 *
80.3 ± 1.07
87.0 ± 0.67
11.7 ± 0.89
7.6 ± 0.51 *
0 mM
1.1 mM
95.4 ± 3.52
93.4 ± 8.72 *
80.4 ± 3.52
91.9 ± 8.72
12.7 ± 0.88
7.3 ± 0.76 *
0 mM
1.1 mM
95.4 ± 1.07
94.3 ± 0.72
80.3 ± 1.07
75.9 ± 0.72
11.7 ± 0.89
8.2 ± 0.14 *
0 mM
1.1 mM
95.4 ± 3.52
94.2 ± 1.15 *
80.4 ± 3.52
81.3 ± 0.46
12.7 ± 0.88
9.5 ± 1.58 *
48h exp
72h exp
24h rec
48h rec
* Data are significantly different at p < 0.05 with respect to correspondingly control.
98
Aluminium toxicity in wheat and rye
Nutrient, organic matter and water contents
Together with root growth inhibition, water and organic matter contents decreased only in
AS6 roots (p < 0.05). The decrease was mostly evident for water content after 48 h, in both
exposure and recover conditions (Table 1).
Concerning nutrient elements, AS6 roots showed higher Al-induced nutrient imbalances
(Table 2). In this sensitive line, after 24 h it was detected increases in Fe, Si, Mn, Cu and,
mostly, Ca elemental contents (Table 2). However, this behavior wasn’t maintained
through the exposure period, and only Ca levels remained high during the exposure.
Comparatively, AT6 showed much less variation in root nutrient contents. After the first
24 h, variations were only detected in Mg and Cu contents. In particular, Ca levels were
just affected (decreased) during the recovery period (p < 0.05). In these roots, P levels
increased after 72 h exposure (p < 0.05). During the exposure and recovery periods in
general, AT6 presented levels of the analyzed elements closer to the ones of the control,
than the AS6 line (Table 2, 3).
Table 2
Nutrient (Ca, P, K, Mg, Al, Mn, Fe, Zn, Si and Cu) quantification in roots of AS6 plants exposed to 1.1 mM Al for 72 h
and after 48 h in recovery. Values are given as mean ± standard deviation for each condition and per dry weight (DW).
Ca (µg mg-1 DW)
P (µg mg-1 DW
0 mM
1.1 mM
72h exp
8.1 ± 0.37
12.4 ± 2.32 *
0 mM
1.1 mM
24h rec
0 mM
1.1 mM
48h rec
AS6
24h exp
0 mM
1.1 mM
K (µg mg-1 DW)
Mg (µg mg-1 DW)
5.9 ± 0.15
5.5 ± 0.48
42.1 ± 5.15
32.7 ± 5.55
4.9 ± 0.91
3.3 ± 0.50
0.12 ± 0.058
1.33 ± 0.065 *
6.4 ± 0.60
9.8 ± 2.02 *
5.7 ± 0.05
5.1 ± 3.21
47.0 ± 0.06
32.1 ± 20.84 *
4.6 ± 0.29
2.0 ± 1.16 *
0.17 ± 0.001
1.85 ± 1.025 *
7.4 ± 0.14
9.4 ± 0.24 *
6.3 ± 0.32
5.4 ± 0.12
49.7 ± 4.90
44.8 ± 8.54
4.7 ± 0.49
3.8 ± 0.04
0.31 ± 0.063
1.21 ± 0.448 *
6.4 ± 0.60
7.7 ± 0.53
5.7 ± 0.05
5.9 ± 0.54
47.0 ± 0.06
42.4 ± 2.58
4.6 ± 0.29
2.8 ± 0.17 *
0.17 ± 0.001
1.08 ± 0.063 *
Mn (µg mg-1 DW)
Fe (µg mg-1 DW)
Zn (µg mg-1 DW)
Si (µg mg-1 DW)
Al (µg mg-1 DW)
Cu (µg mg-1 DW)
24h exp
0 mM
1.1 mM
72h exp
0.03 ± 0.002
0.06 ± 0.010 *
1.31 ± 0.171
2.85 ± 0.247 *
0.26 ± 0.027
0.32 ± 0.024
0.51 ± 0.017
0.83 ± 0.149 *
0.16 ± 0.021
0.21 ± 0.022 *
0 mM
1.1 mM
24h rec
0.03 ± 0.002
0.02 ± 0.000 *
2.52 ± 0.534
0.81 ± 0.221 *
0.24 ± 0.025
0.24 ± 0.101
0.51 ± 0.006
0.57 ± 0.153
0.19 ± 0.020
0.19 ± 0.014
0 mM
1.1 mM
48h rec
0.03 ± 0.006
0.03 ± 0.005
1.65 ± 0.332
1.31 ± 0.412
0.32 ± 0.079
0.22 ± 0.014
0.56 ± 0.017
0.63 ± 0.015
0.17 ± 0.004
0.19 ± 0.003
0 mM
1.1 mM
0.03 ± 0.002
0.03 ± 0.008
2.52 ± 0.534
1.47 ± 0.693
0.24 ± 0.025
0.22 ± 0.027
0.51 ± 0.006
0.53 ± 0.043
0.19 ± 0.020
0.17 ± 0.023 *
* Data are significantly different at p < 0.05 with respect to correspondingly control.
99
Aluminium toxicity in wheat and rye
Both AS6 and AT6 control roots showed residual Al values. After 24 h, Al levels increased in both
root genotypes but at high levels in AS6. After 72 h of continuous exposure, Al accumulation in
AS6 roots showed high variability, ranging from 0.83 to almost 3.0 µ µgg-1 DW in some roots, with
average values of 1.68 µgg-1 DW. By this period, AT6 roots accumulated lower levels of Al (1.41.7 µgg-1 DW) and showed less variability in Al accumulation than AS6 (Table 2, 3). In both lines,
there was a decrease of Al content during recovery, being more accentuated in AT6.
Table 3
Nutrient (Ca, P, K, Mg, Al, Mn, Fe, Zn, Si and Cu) quantification in roots of AT6 plants exposed to 1.1 mM Al for 72 h
and after 48 h in recovery. Values are given as mean ± standard deviation for each condition and per dry weight (DW).
AT 6
24h exp
Ca (µg mg-1 DW)
P (µg mg-1 DW
K (µg mg-1 DW)
Mg (µg mg-1 DW)
Al (µg mg-1 DW)
0 mM
1.1 mM
72h exp
10.5 ± 3.08
7.8 ± 1.75
6.0 ± 0.24
5.2 ± 0.20
37.0 ± 1.82
36.7 ± 4.31
3.2 ± 0.37
2.7 ± 0.37 *
0.22 ± 0.041
0.99 ± 0.013 *
0 mM
1.1 mM
24h rec
9.0 ± 1.49
11.3 ± 1.29
3.3 ± 0.42
4.4 ± 0.53 *
20.3 ± 3.61
18.8 ± 1.12
1.5 ± 0.10
1.7 ± 0.28
0.26 ± 0.037
1.57 ± 0.135 *
8.0 ± 1.32
5.6 ± 1.16 *
4.9 ± 1.21
4.3 ± 0.54
35.8 ± 0.78
33.6 ± 2.45
2.5 ± 0.05
2.5 ± 0.23
0.25 ± 0.052
0.70 ± 0.103 *
9.0 ± 1.49
9.6 ± 2.62
3.3 ± 0.42
4.2 ± 1.11
20.3 ± 3.61
30.6 ± 0.29 *
1.5 ± 0.10
1.6 ± 0.02
0.26 ± 0.037
0.76 ± 0.120 *
0 mM
1.1 mM
48h rec
0 mM
1.1 mM
Mn (µg mg-1 DW)
Fe (µg mg-1 DW)
Zn (µg mg-1 DW)
Si (µg mg-1 DW)
0 mM
1.1 mM
72h exp
0.16 ± 0.131
0.04 ± 0.010
0 mM
1.1 mM
24h rec
Cu (µg mg-1 DW)
3.55 ± 2.418
1.79 ± 0.744
0.28 ± 0.045
0.20 ± 0.027
0.63 ± 0.191
0.55 ± 0.142
0.22 ± 0.060
0.13 ± 0.031 *
0.02 ± 0.000
0.02 ± 0.000
0.62 ± 0.239
0.86 ± 0.013
0.21 ± 0.010
0.21 ± 0.027
0.55 ± 0.107
0.77 ± 0.084
0.18 ± 0.046
0.18 ± 0.011
0 mM
1.1 mM
48h rec
0.02 ± 0.001
0.03 ± 0.014
0.75 ± 0.090
1.20 ± 0.671
0.17 ± 0.020
0.14 ± 0.004
0.64 ± 0.103
0.45 ± 0.034 *
0.18 ± 0.054
0.09 ± 0.014 *
0 mM
1.1 mM
0.02 ± 0.000
0.04 ± 0.023
0.62 ± 0.239
2.64 ± 1.909 *
0.21 ± 0.010
0.25 ± 0.118
0.55 ± 0.107
0.62 ± 0.139
0.18 ± 0.046
0.18 ± 0.030
24h exp
* Data are significantly different at p < 0.05 with respect to correspondingly control.
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Aluminium toxicity in wheat and rye
Table 4
Al quantification in leaves of AS6 and AT6 exposed plants to 1.1 mM Al for 72 h and after 48 h in recovery. Values are
given as mean ± standard deviation for each condition and per dry weight (DW).
Al (µg mg-1 DW)
AS 6
AT6
24h exp
0
1.1
72h exp
0
1.1
24h rec
0
1.1
48h rec
0
1.1
mM
mM
0.06 ± 0.004
0.08 ± 0.016
0.06 ± 0.003
0.14 ± 0.046 *
mM
mM
0.10 ± 0.005
0.13 ± 0.049
0.19 ±
0.11 ±
0.078
0.018
mM
mM
0.07 ± 0.003
0.12 ± 0.020 *
0.07 ±
0.06 ±
0.022
0.015
mM
mM
0.10 ± 0.005
0.17 ± 0.033 *
0.19 ± 0.078
0.09 ± 0.039 *
* Data are significantly different at p < 0.05 with respect to correspondingly control.
Concerning Al levels in leaves, AS6 showed trends of Al increase, particularly during the recovery
period (p < 0.05), while the Al levels in AT6 leaves only increased during the first 24 h of exposure
(p < 0.05), decreasing drastically during the recovery period (Table 4).
Histo-differentiation and reserves accumulation
Root histological analyses show that control AT6 roots presented, in general, thinner MZ and EZ
regions but thicker HZ regions than their AS6 counterparts. Under Al, the AT6 MZ area increased
133.9 %, while the one of HZ decreased app 21.2 %. These changes attenuated during recovery
(Table 5). AS6 roots showed similar changes after 24 h exposure, but with lower magnitude.
During recovery, differences were still detected in both lines, however with a tendency to reach the
control values (Table 5, 6).
Both lines suffered reduction in endoderm and pericycle cells area after 24 h exposure in HZ.
However, they behaved differently with recovery: AT6 presented a faster recover in some of the
parameters assessed. For example, the area of the endodermis cells increased to values closer the
control ones. Contrarily, in AS6 no changes were observed in the average endodermis cell area of
the HZ after 24 h recover. In AS6, the increases in endoderm and pericycle cell areas were
observed in EZ after 24 h exposure, contrarily to the AT6 EZ, that decreased (Table 6). On the
other hand, the increase of endodermis cell area in AS6 EZ after 24 h exposure was not
accompanied with an increase in endodermic cell wall thickness (Table 6, 7). After recover, both
lines were affected differently in HZ: AT6 roots presented a reduction of endodermic cells, with
cell wall thickened, while in AS6 cell wall thickness was stimulated after 24 h Al-exposure and
continued up to 24 h recover (Table 7).
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Aluminium toxicity in wheat and rye
Table 5
AT6 root area measurements in root meristematic (MZ), elongation (EZ) and differentiated (DZ) zone for Al exposed
plants during 24 h and 48 h recover. Values are given as mean ± standard deviation for each condition.
AT 6
24h exp
0 mM
MZ
EZ
HZ
1.1 mM
MZ
EZ
HZ
24h rec
0 mM
MZ
EZ
HZ
1.1 mM
MZ
EZ
HZ
48h rec
0 mM
MZ
EZ
HZ
1.1 mM
MZ
EZ
HZ
Root Area (mm2)
Endoderm cell Area (µm 2 ) Pericycle cell Area (µm 2 )
0.049 ± 0.0146
0.073 ± 0.0290
0.202 ± 0.0117
188 ± 84.1
473 ± 100.8
128 ± 38.2
253 ± 43.9
0.114 ± 0.0095 *
0.083 ± 0.0250
0.159 ± 0.0112 *
173 ± 59.6
297 ± 97.7 *
140 ± 42.5
186 ± 35.5 *
0.083 ± 0.0018
0.068 ± 0.0345
0.066 ± 0.0011
180 ± 62.1
126 ± 37.8
140 ± 57.9
85 ± 11.5
0.086 ± 0.0287
0.065 ± 0.0015
0.107 ± 0.0081 *
165 ± 38.5
272 ± 94.1 *
139 ± 32.6
207 ± 32.2 *
0.126 ± 0.0067
0.048 ± 0.0144
0.143 ± 0.0112
140 ± 57.3
246 ± 71.9
114 ± 24.8
182 ± 47.3
0.109 ± 0.0011 *
0.063 ± 0.0253 *
0.105 ± 0.0017 *
173 ± 45.0 *
354 ± 66.9 *
155 ± 42.8 *
192 ± 33.0
* Data are significantly different at p < 0.05 with respect to correspondingly control.
Table 6
AS6 root area measurements in root meristematic (MZ), elongation (EZ) and differentiated (DZ) zone for Al exposed
plants during 24 h and 48 h recover. Values are given as mean ± standard deviation for each condition.
AS6
24h exp
0 mM
MZ
EZ
HZ
1.1 mM
MZ
EZ
HZ
24h rec
0 mM
MZ
EZ
HZ
1.1 mM
MZ
EZ
HZ
48h rec
0 mM
MZ
EZ
HZ
1.1 mM
MZ
EZ
HZ
Root Area (mm 2 )
Endoderm cell Area (µm 2 ) Pericycle cell Area (µm 2 )
0.095 ± 0.0084
0.063 ± 0.0012
0.174 ± 0.0041
115 ± 18.1
365 ± 71.3
89 ± 13.9
316 ± 50.1
0.132 ± 0.0004 *
0.107 ± 0.0189 *
0.099 ± 0.0310 *
242 ± 58.8 *
206 ± 65.3 *
185 ± 34.0 *
143 ± 23.4 *
0.112 ± 0.0077
0.085 ± 0.0189
0.153 ± 0.0097
166 ± 45.1
277 ± 72.6
129 ± 32.5
179 ± 26.9
0.090 ± 0.0108 *
0.075 ± 0.0022
0.103 ± 0.0062 *
216 ± 56.8 *
189 ± 51.1 *
125 ± 28.2
140 ± 19.3 *
0.144 ± 0.0840
0.068 ± 0.0019
0.149 ± 0.0523
102 ± 22.7
309 ± 78.4
113 ± 14.8
195 ± 28.0
0.093 ± 0.0007
0.080 ± 0.0131
0.193 ± 0.0202
190 ± 55.9 *
313 ± 70.0
155 ± 28.1 *
207 ± 35.4
* Data are significantly different at p < 0.05 with respect to correspondingly control.
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Aluminium toxicity in wheat and rye
Control roots looked healthy and presented MZ with high meristematic activities. After 24 h
exposed to Al, cells of outer layers were degraded in both lines. Histochemical data showed that in
control roots both outer and inner cells layers strongly stained for lipids and that outer cell layers
and root cap accumulate carbohydrates (Fig 1A). After Al exposure, carbohydrates drastically
decreased in AS6, mostly in root cap (Fig 1B). After 24 h and 48 h Al removal (Fig 1C),
carbohydrates were again detected, although with much less extent than in the control. AT6 showed
a reduction in carbohydrates accumulation after Al exposure (Fig 1 D, E) but after 48h recover,
carbohydrates accumulation was restored (Fig 1F). Relatively to lipids, no evident alterations were
detected in both lines.
Table 7
Percentage of endodermic cells with cell wall thickened after 24 h Al exposure and 48 h in recovery. Values are given as
mean ± standard deviation for each condition.
Percentage of Endodermic cells with cell wall thickned
24h exp
AT 6
EZ
0 mM
1.1 mM
HZ
0 mM
1.1 mM
24h rec
48h rec
0.0 ± 0.00
0.0 ± 0.00
0.0 ± 0.00
0.0 ± 0.00
0.0 ± 0.00
40.5 ± 11.57 *
88.2 ± 13.69
100.0 ± 0.00
94.9 ± 6.60
80.2 ± 3.44 *
100.0 ± 0.00
49.6 ± 7.61 *
0.0 ± 0.00
0.0 ± 0.00
0.0 ± 0.00
0.0 ± 0.00
0.0 ± 0.00
10.9 ± 5.33 *
52.8 ± 0.81
87.7 ± 4.49 *
16.8 ± 9.72
85.9 ± 2.53 *
AS6
EZ
0 mM
1.1 mM
HZ
0 mM
1.1 mM
89.5 ± 10.53
100.0 ± 0.00
* Data are significantly different at p < 0.05 with respect to correspondingly control.
Fig. 1. Root apices microphotographs stained with PAS. A-C) AS6 roots: control root after 24 h culture (A), root exposed
for 24 h to Al (B) and root after 48 h in recover (C). D-F) AT6 root: control root after 24 h culture (D), root exposed for
24 h to Al (E) and root after 48 h in recover (F).
103
Aluminium toxicity in wheat and rye
Callose detection
Control roots presented a discrete accumulation of callose, generally observed as spots. Both lines
at MZ presented callose in protodermis and root cap cells. At EZ and HZ, callose deposition was
mostly restricted to epidermis and to few dots in outer cortical layer. After 24 h exposure, all root
zones of both lines presented an increase in callose deposition (Fig 2, 3).
Fig. 2. AS6 root microphotographs stained with aniline blue.
A-C) roots exposed for 24 h to Al: MZ (A), EZ (B) and HZ
(C); D) HZ after 48 h in recover. Bar: 50 µm.
104
Aluminium toxicity in wheat and rye
At MZ, callose distribution in sensitive and tolerant roots was more abundant than in the others
root zones and was located in root cap, protodermis and fundamental meristem cell layers
(specially in the outer layer) (Fig 2A, 3A). At EZ, callose was mostly detected in epidermis and in
the outer cortical layer in AT6 (Fig 3B), however, in AS6 was also detected in inner cortical layers
(Fig 2B). Relatively to HZ, for AS6 and AT6 roots, callose deposition was more abundant in the
cortical cell layers, being detected even in the inner layers (Fig 2C, 3C).
Fig. 3. AT6 root microphotographs stained with aniline blue.
A-C) roots exposed for 24 h to Al: MZ (A), EZ (B) and HZ
(C); D) HZ after 48 h in recover. Bar: 50 µm.
105
Aluminium toxicity in wheat and rye
With Al removal, callose deposition decreased in both lines. After 48 h recovery at MZ, callose
was detected mostly in root cap and in protodermis cells and at EZ it was deposited in epidermis
and in the outer cortical cell layer. At HZ, AS6 roots showed scarce callose deposition in epidermis
and in cortical cells layers (outer and inner layers) (Fig 2D). AT6 roots only presented rare callose
accumulation in epidermis cells, mostly associated with root hair (Fig 3D).
Flow cytometric analyses
The analyses by flow cytometry demonstrated that Al induced no significant changes in nuclei size
(FS) and complexicity (SS) in AS6 and AT6 (p > 0.05, data not shown). Control roots showed
consistent diploid nuclei, and the nDNA ranged between 16.39 ± 0.422 pg/2C and 16.79 ± 0.159
pg/2C for both lines.
Al exposure did not induce any major DNA ploidy changes, or variations in DNA content. To score
for possible clastogenic damage, the CV values of the G0/G1 peaks were analyzed. For AS6 roots, a
general increase of the CV values, ranging from 7 to 9 %, was detected after 72 h (p < 0.05) (Table
8).
The comparative analyses of Al effects on cell cycle dynamics was performed by quantifying the
proportion of cells in the different stages of the cell cycle (G0/G1: S: G2) in apical roots. Under
control conditions, both AS6 and AT6 apical roots showed on average higher rates of cells in
G0/G1.
Table 8
Nuclei’s full peak coefficient of variation (CV) for AT6 and AS6. Values are given as mean ± standard deviation for each
condition.
CV
AT6
AS6
24h exp
0 mM
7.9 ± 0.35
8.0 ± 0.735
1.1 mM 7.0 ± 0.75
9.4 ± 0.556
48h exp
0 mM
8.3 ± 1.42
8.8 ± 1.608
1.1 mM 7.7 ± 0.17
9.1 ± 0.589
72h exp
0 mM
6.2 ± 0.86
7.4 ± 0.094
1.1 mM 7.9 ± 0.29
9.0 ± 0.38 *
24h rec
0 mM
8.3 ± 1.42
8.8 ± 1.608
1.1 mM 8.0 ± 0.64
7.5 ± 1.1
48h rec
0 mM
6.2 ± 0.86
7.4 ± 0.094
1.1 mM 7.6 ± 0.70
8.1 ± 0.899
* Data are significantly different at p < 0.05 with respect to correspondingly control.
106
Aluminium toxicity in wheat and rye
Although no significant differences were detected in cell cycle progression, different trends were
seen in tolerant and sensitive lines. AT6 roots showed during all Al exposure period, a tendency for
blockage in G0/G1, mostly at the expenses of cells in S. In this genotype, the percentage of cells in
G0/G1 and in S phases reached, respectively, maximum and minimum values after 72h exposure.
Contrarily, in AS6 exposed roots, higher heterogeneity in terms of cell cycle progression was found
within the cells population, but overall during the first 48 h exposure, the percentage of cells in S
phase increased (Fig 4). During recovery, the AT6 apical roots showed G0/G1, S and G2 ratios more
similar to those presented by control ones, while in AS6, an increase in cells in S phase was
observed after 24 h (Fig 4).
AT6
100%
G2
90%
S
80%
G0/G1
70%
Nuclei
60%
50%
40%
30%
20%
10%
0%
0 mM 1.1 mM
0 mM 1.1 mM
0 mM 1.1 mM
0 mM 1.1 mM
0 mM 1.1 mM
24h exp
48h exp
72h exp
24h rec
48h rec
AS6
100%
G2
90%
S
80%
G0/G1
70%
Nuclei
60%
50%
40%
30%
20%
10%
0%
0 mM 1.1 mM
0 mM 1.1 mM
0 mM 1.1 mM
0 mM 1.1 mM
0 mM 1.1 mM
24h exp
48h exp
72h exp
24h rec
48h rec
Fig. 4. Cell cycle dynamics of AT6 and AS6 roots exposed to 1.1 mM Al during 24 h, 48 h and 72 h Al exposure (exp) or
24 h and 48 h with recovery (rec). The values given are the means of each population of cells in each of the cell cycle
stages (G0/G1, S and G2).
107
Aluminium toxicity in wheat and rye
Discussion and Conclusions
Rye is particularly tolerant to Al (Shi et al., 2009). The two lines of Montalegre regional
rye population are similar but differ in their tolerance to Al due the screening of tolerant vs
sensitive plants and selection for six generations, and provide an extremely valuable tool
for comparative studies and to better understand mechanisms involved in Al toxicity and
tolerance. Al reduced root growth sooner in the sensitive AS6, suggesting more severe
damages in this line, and the inability of this genotype to revert these effects even after
relatively short term (24 h) exposures.
It is demonstrated that, contrarily to AT6, Al decreased water content in AS6 roots (no
significant correlation), which was also not reverted by recovery treatments. In barley,
Támas et al. (2006) also correlated positively the uptake of Al with Al induced drought
stress (half of water content of control roots). Recently, Abdel-Basset et al. (2010) using
tobacco suspension-cultured cells showed that Al decreased cell osmolality, and the uptake
rate of sucrose and glucose soluble. In these suspension cells, sugar and, less, starch
contents decreased with Al exposure. These authors hypothesized that Al inhibits
elongation by reducing sugar uptake and, consequently the force for water uptake. These
findings are consistent with the decrease in water and starch contents in AS6. If there is no
sufficient uptake of sucrose and/or glucose, the cell may have to use its carbon source (if
available) as starch. This hypothesis is consistent with the decrease of starch and water
contents after exposure and even after Al removal. Contrarily, AT6 only demonstrated a
slight decrease of starch content after exposure, which was recovered after Al removal and
didn’t suffer any alteration in water content. So, this tolerant line seems to have different
mechanisms of Al regulation of the carbohydrate metabolism compared to the sensitive
line.
Together with the decreases of water and carbohydrate reserves, Al treated AS6 roots also
showed more severe imbalances of nutrients accumulation (e.g. the divalent cations as Ca,
Cu, Fe, Mn), besides higher Al accumulation. These changes, in particularly those of Ca
are described for other species under Al stress (Huang et al., 1992; Vanguelova et al.,
2007; Abdel-Basset et al., 2010). Increase of cytosolic Ca2+ is essential to callose
deposition (Sivaguru et al., 2005) and the disruption of Ca2+ homeostasis is also related
with cytoskeleton disorganization (Panda et al., 2009). Our data indicated alterations in Ca
108
Aluminium toxicity in wheat and rye
allocation and a possible increase of cytosolic Ca2+, since there was an increase in total Ca
contents, that was positively correlated with Al increase in AS6 (p < 0.05), and callose
deposition. Again, the tolerant line didn’t suffer alterations in total Ca levels after Al
exposure. However, accumulation of callose indicates alteration in cytosolic Ca2+. So, it
seems that Al exposure modifies cytosolic Ca2+ homeostasis but not Ca allocation in
tolerant AT6 rye plants. It was already demonstrated that Al exposure leads to changes in
cytosolic Ca2+ in rye plants (Ma et al., 2002). The fact that callose synthesis (dependent on
Ca-cytosolic levels) increased in both lines, and that only the AS6 showed significant
changes in Ca allocation, support that Al exposure affects more severely cytosolic Ca2+
contents, that may be accompanied by changes in Ca uptake/allocation. More comparative
studies are needed to provide better insight on the relations between Ca uptake vs cytosolic
Ca2+ alterations under Al exposure.
Al content reached higher levels in AS6 roots and leaves than in AT6. Also AS6 showed
trends of Al increase in leaves in all conditions, Al levels only increased in AT6 leaves
during the first 24 h of exposure, stabilizing thereafter. These data show that these lines
behaved differently in both Al uptake and translocation/allocation: AT6 has a more
efficient ability to prevent Al uptake and/or accumulation in the roots and to prevent its
translocation to upper parts. The exact mechanisms by which the tolerant AT6 line
prevents Al toxicity are still unknown, but among the factors contributing to Al external
and internal detoxification, organic acids (oxalate and/or citrate) are consistently reported
as they may form complexes with Al3+ in rye (Ma et al., 2001; Hayes and Ma, 2003; Sheng
et al., 2004; Tolrà et al., 2005). Less attention has been paid to the role of phenolics in the
detoxification process, but Tolrà et al. (2005) suggested, for R. acetosa, that phenolic
compounds rather than organic acids are involved in Al detoxification. In these rye lines, it
is under study if this AT6 produces more organic acids and/or phenolics compared to AS6,
and if putative changes may be correlated with Al contents. Also, AS6 roots showed higher
heterogeneity in their ability to accumulate Al (0.83-2.92 µg mg-1 DW), suggesting a less
structured general defense mechanisms in this line compared to AT6.
Al differently affected root histology in both lines, with evident degradation of outer cell
layers. Effects in root anatomy were more severe in AS6 roots that were not able to reverse
these effects when Al was removed. For both lines after 24 h exposure, an increase in root
area at MZ and a decrease at HZ was detected. In roots of C. japonica, C. obtuse and P.
109
Aluminium toxicity in wheat and rye
sylvetris root diameter was negatively related to Ca/Al molar ratio (Vanguelova et al.,
2007).
Al exposure alters Ca2+ homeostasis and in consequence, may inhibit cell division and root
elongation (Panda et al., 2009). These facts point to that in MZ, cell division is
compromised (probably due to disruption of Ca2+ homeostasis) whereas in HZ root
elongation appears to be stimulated. At EZ, no alteration in endodermis thickness was
detected in both lines after exposure. On the other hand, it seems that in AS6, endodermis
in HZ is more stimulated to differentiate than in AT6.
Overall, AS6 had higher Al contents, and at the HZ region (highly ion absorbing region),
Al exposure stimulated both callose and endodermis formation. Despite callose is often
related with higher Al-sensitivity, the Al interference in endodermis differentiation was
only recently reported (Silva et al 2010), and it should be clarified if this stimulation is due
to a higher entrance of Al in this particular region, compared to the highly tolerant line.
Moreover, and considering that Triticum aestivum (in general much more sensitive than
rye) showed an opposite profile concerning endodermis differentiation (Silva et al 2010),
this stimulation of endodermis should not be considered an indicator of sensitivity.
Alterations in root cell division and elongation due to Al exposure were already detected in
several species and is well documented (Nagy et al., 2004; Panda and Matsumoto, 2007;
Poschenrieder et al., 2008; Abdel-Basset et al., 2010; Horst et al., 2010). With respect to
cell cycle patterning less information is available. In Al-sensitive maize, Doncheva et al.
(2005) described a blockage of cells at the S phase after Al exposure. Also, in Vicia faba,
Yi et al. (2009) reported changes in the number of cells in each mitotic phase in Al treated
plants. Similar to Doncheva et al. (2005) data, we found a trend to blockage at the S phase
in the sensitive rye line, when exposed to Al. Contrarily, the cell cycle dynamics of Al
exposed AT6 roots followed a trend closer to control, but with a tendency to blockage at
the G0/G1 phase (mostly after 72 h exposure). Data strongly suggest that Al affects either
G0/G1-S transition checkpoint or the S-G2 transition checkpoint, depending on the
tolerance degree of the line. Checkpoints include monitoring the cell size and environment
prior proceeding from G1 to S, that all DNA has been synthesized before moving from S to
G2 (Gahan, 2007). A progression from G0/G1 to the S and G2 phases mean that cells
entered in replication/posreplication processes, and a blockage at one of these stages may
indicate a delay in cell cycle (mostly a blockage at the S phase) due to damages in the
110
Aluminium toxicity in wheat and rye
DNA replication/postreplication as demonstrated for other species (e.g.Andersen et al.,
2008).
In Al exposed rye roots, the CV of G0/G1 peaks increased significantly only in AS6 plants.
These data point to an Al-induced clastogenic effects, in the sensitive line. Yi et al. (2009)
also reported chromosome aberrations, and micronucleated cells in Al treated groups and
demonstrated that Al chloride is a clear clastogenic/genotoxic agent in Vicia root cells. One
must, however, take also into account that PI intercalation is also sensitive to chromatin
structure and template activity (Loureiro et al., 2007) and that Al can adversely affect them
(Matsumoto, 1991). Taken all together, our data support the occurrence of Al-induced
clastogenic damage in the rye sensitive line. On the other hand, Al exposure was not
sufficient, at the conditions tested, to induce variation in DNA content (hence DNA-ploidy
level) in both rye lines.
In conclusion, we demonstrated for this tolerant species, that Al exposure affected mostly
AS6 leading to decrease in root growth and regrowth, water contents and carbohydrates
accumulation. This suggests that the highly tolerant line AT6 has more effective
mechanisms to prevent insufficient uptake of sucrose and/or glucose and consequently
water uptake. Our data also suggest that cytosolic Ca2+ homeostasis is more sensitive to Al
exposure then Ca allocation. Both AT6 and AS6 lines differ in endodermis differentiation,
which also depends on root zone. However, a similar behaviour between the two lines was
observed in callose accumulation. Despite callose and endodermis seem to play
complementary roles to prevent Al entrance in the symplast, the relationship between Al
and endodermis thickening still remains unclear, and it seems that this tolerant species has
a different profile from other less tolerant species. These facts, combined with the different
effects of Al in cell cycle partitioning and clastogenicity between the two lines contribute
to better understanding the Al targets and tolerance mechanisms developed by tolerant rye
lines under Al stress.
Acknowledgements
FCT/MCT supported this work (POCI /AGR/ 58174/2004) and S. Silva (FCT⁄BD⁄ 32257⁄2006)
grants. Thanks are due to Armando Costa for technical support.
111
Aluminium toxicity in wheat and rye
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116
Aluminium toxicity in wheat and rye
CHAPTER III
LONG-TERM ALUMINIUM EXPOSURE
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118
Aluminium toxicity in wheat and rye
III.1: Antioxidant mechanisms in response to Al of tolerant and sensitive rye genotypes
This chapter was submitted as an original paper in a SCI journal:
Silva S, Pinto G, Pinto-Carnide O, Santos C. 2011. Antioxidant response in Al-treated rye
genotypes depend on organ, genotype and time exposure. Environmental and Experimental
Botany (submitted).
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120
Aluminium toxicity in wheat and rye
Abstract
Secale cereal L. plants are considered highly tolerant to Al, being capable to survive under
high Al concentrations. Two rye genotypes ‘D. Zlote’ (Al-tolerant) and ‘Riodeva’ (Alsensitive) were exposed to Al (1.11 and 1.85 mM) for 3 weeks. Results showed that, in
both roots and leaves, Al leaded to oxidative stress and other Al-induced injuries. For root
growth, ‘Riodeva’ was more affected than ‘D. Zlote’. Root parameters were in general
more affected than leaves. In leaves, CAT, SOD and G-POX played an important role in
ROS detoxify and results showed that the enhancement of APX, SOD and G-POX
activities contributed to the higher Al tolerance in ‘D. Zlote’ roots. Concerning AsA-GSH
cycle, response depended on organ, genotype (Al-tolerance) and exposure time: at the 2nd
week, leaves were more affected than roots, but at week 3, the AsA-GSH cycle in leaves
was closer to the one of the control, while in roots it declined; in ‘D. Zlote’ roots, an
enhancement of the AsA-GSH cycle was observed earlier, but declined at the 3rd week. We
suggest that, directly or indirectly, the root growth inhibition in ‘D. Zlote’ at the 3rd week
may be related to oxidation of AsA and GSH pools. Moreover, the ability of rye genotypes
to survive in Al environments may depend on the antioxidant system response. Finally
these results support the necessity to conduct long term experiments in order to better
understand functional responses to Al, and the genotypes best adapted to grow in acid
conditions.
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Aluminium toxicity in wheat and rye
122
Aluminium toxicity in wheat and rye
Introduction
In acidic environments, aluminium (Al) becomes available to plants and limits plant
growth and crop production. Among the cereals, rye (Secale cereale) is one of the most
tolerant species (Aniol and Gustafson, 1984). Research dealing with rye Al-tolerance is
limited (for review of Al toxicity see: Samac and Tesfaye, 2003; Panda and Matsumoto,
2007; Poschenrieder et al., 2008) and, up to authors’ knowledge, non studied rye behavior
in long-term Al-exposure. Thus, a deeper knowledge of the mechanisms employed by rye
to cope Al long-term exposure and toxicity will provide valuable tools in this field of
research.
The major morphological aspect of Al toxicity is the inhibition of root elongation. This
effect may be related with oxidative stress and changes in cell wall properties, as a
consequence of Al toxicity (Yamamoto et al., 2003; Zheng and Yang, 2005). Supporting
this hypothesis, Al exposure induced membrane damage by lipid peroxidation, which was
reported for several species, mostly in sensitive genotypes (e.g. Tabaldi et al., 2009;
Schuch et al., 2010; Yin et al., 2010). The reactive oxygen species (ROS) have the capacity
to oxidize cellular components such as lipids, proteins, enzymes, and nucleic acids, leading
to cell death. However, oxidative damages can be alleviated through enhanced antioxidant
capacity.
The AsA-GSH (ascorbate-glutathione) cycle has been regarded as a highly important
antioxidant pathway (Li et al., 2010). It is consensual that ascorbate peroxidase (APX),
monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and
gluthatione reductase (GR) are four key enzymes present in the AsA-GSH cycle. Together
with these antioxidant enzymes, ascorbate (AsA) and glutathione (GSH) are the most
abundant low molecular weight non-enzymatic antioxidants in plant cells participating in
ROS scavenging through the AsA-GSH cycle (Noctor and Foyer, 1998; Li et al., 2010).
Concerning Al toxicity, the AsA-GSH cycle has not been yet fully studied. In rice, Al
exposure (20 days) leaded to imbalances in the AsA-GSH cycle, which included alteration
in the enzymes activity (Sharma and Dubey, 2007) and in the non-enzymatic antioxidant
contents (Sharma and Dubey, 2007; Wang and Kao, 2007). Furthermore, Al-induced
alterations in AsA-GSH cycle were dependent on Al concentration and exposure time
(Sharma and Dubey, 2007).
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Aluminium toxicity in wheat and rye
AsA-GSH cycle can scavenge H2O2 produced in plants and can maintain an appropriate
oxidative and reductive environment through regulating ascorbate / dehydroascorbate
(AsA/DHA), reduced glutathione/oxidized glutathione (reduced GSH/GSSG) and
NAD(P)H/NAD(P) ratios. AsA is a major antioxidant, is present in most cellular
compartments (Chen and Gallie, 2006), serves as an electron donor and reacts with ROS.
It plays multiple roles in plant growth, as a regulator of cell division, expansion and as a
signal transduction molecule in plants (Green and Fry, 2005). GSH is important as an
antioxidant and redox buffer (Noctor and Foyer, 1998), is implicated in cell division (May
et al., 1998; Foyer et al., 2005) and, together with AsA, is also involved in gene expression
regulation (Wingate et al., 1988; Grene, 2002). It seems that its accumulation enhanced
some Al-tolerance: an Al-tolerant tobacco cell line presented higher AsA and GSH
contents than the Al-sensitive line (Devi et al., 2003) and in rice, treatments with AsA
alleviated Al-induced inhibition of root elongation (Guo et al., 2005a; Wang and Kao,
2007). However, the effect of Al exposure in AsA-GSH cycle is still almost unexplored.
Apart from AsA-GSH cycle, other antioxidant enzymes present an important role in the
antioxidant system, as catalase (CAT), guaiacol peroxidase (G-POX) and superoxide
dismutase (SOD). CAT and G-POX contribute to regulate the intracellular level of H2O2,
whereas SOD is responsible for the scavenging of O2- by the dismutation of O2.- to H2O2
(Blokhina et al., 2003). SOD is located in a large cell compartments and represents a first
line of defence against ROS. Al-induced variations in these antioxidant enzyme activities
were referred for many species (Ghanati et al., 2005; Guo et al., 2007; Liu et al., 2008ab;
Tabaldi et al., 2009; Yin et al., 2010).
We hypothesize here that Al exposure affects the oxidant status of rye plants. So, the aim
of this study was to compare the profiles of the antioxidant battery in response to Al
expose in two rye genotypes differing in their tolerance degree. For that, we analysed in
this comprehensive study the Al influence in ROS (H2O2), and in both AsA-GSH cycle and
other antioxidant enzymes (CAT, SOD, G-POX).This long term treatment will also give
insight in the concepts of “Al-tolerant” and “Al- sentitive” currently associated in literature
to root growth and callose deposition, in short term treatments.
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Aluminium toxicity in wheat and rye
Material and Methods
Plant material selection, growth and exposure to Al
Two rye genotypes, ‘Dankowski Zlote’ (Polish cultivar) and ‘Riodeva’ (Spanish inbred
line) were provided by University of Trás-os-Montes and Alto Douro (UTAD/Vila Real,
Portugal). ‘D. Zlote’ was classified as an Al-tolerant cultivar (e.g. Pinto-Carnide and
Guedes-Pinto, 1999; 2000) and ‘Riodeva’ as Al-sensitive (Gallego and Benito, 1997).
Seeds were disinfected, rinsed in distilled water and germinated (in the dark at 24 ºC) in
petri dishes. Germinated seeds were transferred for 4 days to a modified Hoagland
solution: KNO3 303 mg/L; Ca(NO3)2.4H2O 470 mg/L; MgSO4.7H2O 123 mg/L;
NH4H2PO4 14.4 mg/L, and the following micronutrients: Fe-tartrate 2.65 mg/L; H3Bo3
1.43 mg/L; MnCl2 0.905 mg/L; CuSO4.5H2O 0.04 mg/L; ZnSO4.7H2O 0.11 mg/L; H3Mo
0.008 mg/L (RW medium). For Al exposure, plants were transferred to the same nutritive
solution containing 1.11 mM or 1.85 mM of AlCl3.6H2O, corresponding to 0.26 mM and
0.42 mM of Al activity, respectively (Al activity was estimated by Geochem-EZ). Plants
were grown during three weeks on nutritive solution containing Al. A control group was
maintained in the nutritive solution without Al. Plants were grown in a culture room at 24
o
C with 16/8h photoperiod. The nutrient solution was continuously aerated and renewed
every 3 days. The pH was maintained at 4.0 throughout the assay (e.g. Silva et al., 2010).
Measurements were performed two and three weeks after the beginning of Al exposure.
Growth analysis
Leaves and roots of 5 plants were used for growth assessment. Plant (roots and leaves) and
root length was measured. For fresh biomass determination, plant (roots and leaves) and
root fresh weight was used.
Concentration of H2O2, MDA and total soluble proteins
Hydrogen peroxide was quantified as described by Zhou et al (2006). Samples were
homogenized with trichloroacetic acid (TCA) and centrifuged. The supernatant was
adjusted to pH 8.4 and the extract was divided into two aliquots. Eight µg of catalase
125
Aluminium toxicity in wheat and rye
(CAT) were added just to one of the aliquots and to both aliquots were added 0.5 ml of
colorimetric reagent. The reaction solution was incubated for 10 min at 30 ºC and the
absorbance was read at 505 nm. The amount of H2O2 was obtained against a H2O2 standard
curve (R2= 0.93).
Lipid peroxidation was determined by malondialdehyde (MDA) content according to
Santos et al (2001).
Soluble proteins were determined by the Bradford (1976) method using the Total Protein
Kit, Micro (Sigma-Aldrich, USA).
Enzymatic activity of AsA-GSH cycle enzymes
Enzyme extracts of ascorbate peroxidase (APX), were prepared by grinding frozen samples
with chilled potassium phosphate buffer (pH 7.5) containing ethylenediamine tetraacetic
acid (EDTA) and ascorbic acid (AsA). The homogenate was centrifuged and the activity
was assayed according to the method of Nakano and Asada (1981) by recording the
decrease in AsA content at 290 nm. The reaction mixture contained 50 mM potassium
phosphate buffer (pH 7), 0.5 mM AsA, 0.1 mM EDTA, 0.1 mM H2O2, and 0.1 ml enzyme
in a total volume of 1.5 ml and the reaction was started with the addition of H2O2. The
specific activity was calculated using the 2.8 mM-1 cm-1extinction coefficient.
For glutathione reductase (GR) activity, each frozen sample was homogenized with
extraction buffer and then centrifuged. The reaction mixture contained 0.2 M potassium
phosphate buffer (pH 7.5), 0.2 mM EDTA, 1.5 mM magnesium chloride, 0.25 mM oxized
glutathione (GSSG), 25 µM nicotinamide adenine dinucleotide phosphate (NADPH) and
50 µl enzyme extract (Loggini et al., 1999) and the reaction was initiated by the addition of
NADPH. The absorbance was recorded at 340 nm and the specific activity was calculated
using the 6.22 mM-1 cm-1extinction coefficient
For monodehydroascorbate reductase (MDHAR) frozen samples were homogenized with
extraction buffer and centrifuged. The reaction mixture contained 50 mM Hepes buffer
(pH 7.6), 2.4 mM AsA, 0.25 mM NADH, 50µl enzyme extract and 0.4 U ascorbate
oxidase (Murshed et al., 2008) and the reaction was started by adding of 0.4 U of AsA. The
activity was determined by measuring the decrease in the reaction rate at 340 nm. The
specific activity was calculated using the 6.22 mM-1 cm-1extinction coefficient.
126
Aluminium toxicity in wheat and rye
Contents of AsA-GSH cycle non-enzymatic antioxidants
Total AsA, reduced AsA and DHA (dehydroascorbate) concentrations were determined
following the procedure of Jin et al (2008). Briefly, frozen sample was extracted with TCA
and centrifuged. Total ascorbate (AsA + DHA) was determined in a reaction mixture
consisting of 0.1 ml of supernatant, 0.25 mL of 150 mM phosphate buffer (pH 7.4)
(containing 5 mM EDTA), and 0.1 ml of dithiothreitol (DTT) 10 mM. Ten min after, 0.05
ml of 0.5% (w/v) N-ethylmaleimide was added. The AsA was assayed in a similar manner
except that 100 µl of ultra pure water substituted DTT. Color was developed in both
reaction mixtures after the addition of 0.2 ml of 10% (w/v) TCA, 0.2 ml of 44% (v/v)
phosphoric acid, 0.2 ml of α,α’-dipyridyl in 70% (v/v) ethanol, and 0.1 ml of 3% (w/v)
FeCl3. After incubation at 40 ºC for 40 min the absorbance was read at 532 nm using AsA
as a standard. Standard curves for total AsA (R2= 0.94) and AsA (R2= 0.95) were
established. DHA was estimated from the difference between total AsA (AsA + DHA) and
AsA.
Total glutathione, GSSG and GSH were measured according to Ma et al. (2008) with some
modifications. Each frozen sample was homogenized with 5-sulphosalicylic acid and then
centrifuged. Total glutathione was measured in a reaction mixture (1 ml) consisting of 100
µl supernatant, 200 mM phosphate buffer solution (pH 7.2) containing 5 mM EDTA and 1
mM 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), 0.2 mM NADPH and 1 U GR. The
reaction mixture was then incubated at 27 ºC for 30 min and quantified at 412 nm. GSSG
was analyzed in a similar manner except that at 100 µl of supernatant was added 0.17 M 2vinylpyridine and 3.6 % trietanolamine. This mixture was then incubated at 27 ºC for 1 h.
Standard curves for GSH (R2= 0.99) and GSSG (R2= 0.95) were established. For each
sample, GSH was estimated from the difference between total glutathione and GSSG.
Activity of some enzymatic antioxidants
Enzyme extracts for catalase (CAT), guaiacol peroxidase (G-POX) and superoxide
dismutase (SOD) were prepared by grinding frozen samples with chilled potassium
phosphate buffer (pH 7.5) containing EDTA, and then centrifuged.
CAT activity was assayed by estimating the residual H2O2 using titanium reagent
(Teranishi et al., 1974). The reaction mixture consisted of 50 ml enzyme extract, 0.1 M
127
Aluminium toxicity in wheat and rye
phosphate buffer (pH 7.0), and 6 mM H2O2, which started the reaction. Reaction was
terminated adding 4 ml of titanium reagent. The absorbance was recorded at 415 nm. The
CAT activity was obtained against a CAT standard curve (R2= 0.91).
For G-POX determination the reaction mixture contained 10 mM phosphate buffer (pH
6.1), 12 mM H2O2, 96 mM guaiacol and 50 ml enzyme extract. Absorbance was recorded
at 470 nm (Castillo et al., 1984) and the specific activity was calculated using the 26.6
mM-1 cm-1extinction coefficient.
SOD activity was estimated by recording the enzyme induced decrease in absorbance of
formazone made by nitro-blue tetrazolium with superoxide radicals (Dhindsa et al., 1981).
The reaction mixture (1.5 ml) contained 13 mM methionine, 25 mM nitro-blue tetrazolium
chloride (NBT), 0.1 mM EDTA, 50 mM phosphate buffer (pH 7.8), 50 mM sodium
carbonate and 0.1 ml enzyme. Reaction was started by adding 2 mM riboflavin and placing
the tubes under two 15 W fluorescent lamps for 15 min. The absorbance was recorded at
560 nm, and one unit of enzyme activity was taken as that amount of enzyme, which
reduced the absorbance reading to 50 % in comparison with tubes lacking enzyme. The
SOD activity was obtained against a SOD standard curve (R2= 0.89).
Statistical analyses
For the measurement of all parameters, at least 4 replicates were used. Each replica
consisted in leafs and roots pools from 7-10 plants. Values are given as mean ± standard
error. The comparison between Al concentrations (0, 1.11, 1.85 mM) was made using One
Way ANOVA (SigmaStat for Windows Version 3.1, SPSS Inc., USA) test, and
comparisons between genotypes and between exposure periods were made using Two Way
ANOVA, followed by a Holms Sidak Comparison Test when data was statistically
different (p < 0.05).
128
Aluminium toxicity in wheat and rye
Results
Plant growth
Both genotypes presented a decrease trend in plant size (p ˂ 0.05) (Fig. 1A). When only
the root size was considered, significant differences (p ˂ 0.05) were detected after 2 weeks
in both genotypes, but were more accentuated in ‘Riodeva’ (Fig. 1B). At the 3rd week
exposure, the percentage of root growth inhibition (comparatively to control) becomes
similar in both genotypes. In treated roots of both genotypes, no significant differences
were detected between time exposures. Comparing the genotypes for root size, at the 2nd
week Riodeva and ‘D. Zlote’ controls were similar (p ≥ 0.05) but exposed roots were
significantly different.
Decreases in Al-treated plant and root biomass of both genotypes (p ˂ 0.05) were observed
at the 2nd week and aggravated after the 3rd week (Fig. 1C, D).
A
a
1,4
120
100
a
b
80
b
a
a
60
c
a
b
b
40
ab
ab
Plant biomass (g)
Plant length (cm)
a
20
a
a
1
0 mM
0,8
0,6
ab
0,4
ab
b
3 weeks
2 weeks
Riodeva
3 weeks
2 weeks
3 weeks
2 weeks
Riodeva
a
Root biomass (g)
60
50
a
40
10
a
a
b
a
b
b
b
a
0,5
0,4
a
a
0 mM
0,3
0,2
0,1
0
D
a
b
1.11 mM
a
b
c
b
c
3 weeks
D. Zlote
0,6
70
20
1.85 mM
b
D. Zlote
80
30
b
b
b
1.11 mM
b
a
0
2 weeks
Root length (cm)
1,2
0,2
0
B
C
1,6
140
b b
b b
b
1.85 mM
0
2 weeks
3 weeks
Riodeva
2 weeks
3 weeks
D. Zlote
2 weeks
3 weeks
Riodeva
2 weeks
3 weeks
D. Zlote
Fig. 1. Variation in length (A and B) and biomass (C and D) of the whole plant and roots of ‘Riodeva’ and ‘D. Zlote’
plants exposed to 1.11 mM and to 1.85 mM Al during 2 and 3 weeks. Values are given as mean ± standard error for each
condition. Data followed by different letters denote significant differences at p < 0.05 with respect to control.
129
Aluminium toxicity in wheat and rye
Concentration of H2O2 and total soluble proteins
Leaves and roots presented divergent behaviours (Fig. 2). In ‘Riodeva’ leaves, H2O2
content increased (after 2 weeks in plants exposed to 1.85 mM Al), in roots was observed a
H2O2 decrease for all tested conditions. In ‘D. Zlote’ leaves, no differences were detected
(p ≥ 0.05) but in roots it was evident a decrease in H2O2 contents at the 2nd and 3rd weeks.
0 mM
70
H 2O2 (µg g -1 FW )
1.85 mM
50
a
40
b
30
20
a
a a
a
a
a
a
a a a
10
H 2O2 (µg g -1 FW )
1.11 mM
60
0
2 weeks
3 weeks
2 weeks
Riodeva
45
40
35
30
25
20
15
10
5
0
a
a
a
a
ab
b
b
b ab
b
c
2 weeks
3 weeks
b
3 weeks
2 weeks
Riodeva
D. Zlote
3 weeks
D. Zlote
Roots
Leaves
Fig. 2. Variation in H2O2 content in leaves and roots of ‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11 mM and to 1.85
mM Al during 2 and 3 weeks. Values are given as mean± standard error for each condition. Data followed by different
letters denote significant differences at p < 0.05 with respect to control.
Malondialdehyde content increased only in ‘Riodeva’ plants. Both roots and leaves
presented enhancement at the 2nd week in plants exposed to 1.11 mM. At the 3rd week,
MDA increased in both Al concentrations for leaves and roots.
Table 1
Variation in malondialdehyde (MDA) in leaves and roots of ‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11 mM and to
1.85 mM Al during 2 and 3 weeks. Values are given as mean ± standard error for each condition.
Leaves
-1
MDA (mmol g FW)
2 weeks
Roots
3 weeks
2 weeks
3 weeks
Riodeva
0mM
1.11mM
1.85mM
11 ± 0.3
20 ± 2.4
13 ± 1.6
a
b
a
8 ± 0.2
18 ± 1.2
16 ± 1.7
a
b
b
0.7 ± 0.09 a
1.3 ± 0.20 b
0.9 ± 0.11 a
0.6 ± 0.04 a
1.3 ± 0.12 b
1.2 ± 0.14 b
12 ± 1.0
9 ± 1.6
11 ± 1.4
a
a
a
10 ± 2.4
10 ± 1.6
10 ± 0.8
a
a
a
1.0 ± 0.15 a
0.7 ± 0.22 a
0.8 ± 0.20 a
1.3 ± 0.12 a
0.8 ± 0.05 a
1.2 ± 0.26 a
D. Zlote
0mM
1.11mM
1.85mM
Data followed by different letters denote significant differences at p < 0.05 with respect to control.
130
Aluminium toxicity in wheat and rye
Concerning to soluble protein content, this parameter increased (p ˂ 0.05) only in leaves
and at the 2nd week for both genotypes. However, ‘Riodeva’ plants presented more
imbalances: in roots, presented a significant decreased at the 2nd (1.85 mM) and at the 3rd
weeks (1.11 mM) (Fig. 3).
Riodeva
D. Zlote
0mM
40
25
a a
1.85mM
a
b
a
a
15
a
a
10
b
ab
b
30
Protein (mg g-1 FW)
Protein (mg g-1 FW)
c
30
20
35
1.11mM
35
ab
25
20
a
a a
a
15
a a a
10
a a a
5
5
b
0
0
2 weeks
3 weeks
leaves
2 weeks
3 weeks
roots
2 weeks
3 weeks
leaves
2 weeks
3 weeks
roots
Fig. 3. Variation in soluble protein contents in leaves and roots of ‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11 mM
and to 1.85 mM Al during 2 and 3 weeks. Values are given as mean ± standard error for each condition. Data followed by
different letters denote significant differences at p < 0.05 with respect to control.
Enzymatic activity of AsA-GSH cycle enzymes
No variations in APX activity (Table 2) were detected in ‘Riodeva’ leaves and roots. In
‘D. Zlote’ plants, significant alterations in APX activity were detected only in roots and
after 3 weeks Al exposure: APX activity increased in both Al concentrations tested.
Glutathione reductase activity (Table 2) decreased in leaves of ‘Riodeva’ at the 3rd week
but no differences were obtained in roots. In ‘D. Zlote’, GR activity decreased (p ˂ 0.05) in
leaves at the 2nd week (1.11 mM) but, an enhancement was observed in roots at the 2nd and
3rd weeks (both Al concentrations).
For MDHAR activity, only leaves of ‘Riodeva’ at the 2nd week presented significant
variations (decreased) (Table 2). Relatively to roots, was detected an increase (p ˂ 0.05) in
MDHAR activity (when exposed to 1.85 mM Al) at the 2nd week for both genotypes.
131
Aluminium toxicity in wheat and rye
Table 2
Variation in ascorbate peroxidise (APX), glutathione reductase (GR) and monodehydroascorbat reductase (MDHAR)
activity in leaves and roots of ‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11 mM and to 1.85 mM Al during 2 and 3
weeks. Values are given as mean ± standard error for each condition.
leaves
APX (µmol g -1 FW s -1 )
2 weeks
roots
3 weeks
2 weeks
3 weeks
Riodeva
0 mM
1.11mM
1.85mM
0.21 ± 0.009 a
0.23 ± 0.014 a
0.21 ± 0.011 a
0.23 ± 0.013
0.28 ± 0.027
0.28 ± 0.01
a
a
a
0.61 ± 0.061
0.62 ± 0.067
0.72 ± 0.165
a
a
a
0.76 ± 0.088
0.47 ± 0.073
0.54 ± 0.075
a
a
a
D. Zlote
0mM
1.11mM
1.85mM
0.19 ± 0.015 a
0.21 ± 0.03 a
0.26 ± 0.017 a
0.21 ± 0.025
0.25 ± 0.014
0.35 ± 0.082
a
a
a
0.83 ± 0.19
0.75 ± 0.064
1.00 ± 0.133
a
a
a
0.56 ± 0.023
0.93 ± 0.097
0.85 ± 0.04
a
b
b
GR (nmol g -1 FW s -1 )
Riodeva
0 mM
1.11mM
1.85mM
2.9 ± 0.15
3.1 ± 0.22
2.3 ± 0.12
ab
b
a
14.4 ± 0.91
8.4 ± 0.53
9.1 ± 0.55
a
b
b
3.7 ± 0.26
4.0 ± 0.23
4.6 ± 0.19
a
a
a
8.2 ± 0.57
8.6 ± 0.97
11.3 ± 1.21
a
a
a
D. Zlote
0mM
1.11mM
1.85mM
13 ± 0.28
11 ± 0.37
13 ± 0.39
a
b
a
4.7 ± 0.66
5.4 ± 0.31
5.2 ± 0.43
a
a
a
6.5 ± 0.55
10.9 ± 0.17
9.7 ± 0.49
a
b
b
4.0 ± 0.09
6.0 ± 0.26
6.9 ± 0.44
a
b
b
Riodeva
0 mM
1.11mM
1.85mM
38 ± 5.7
20 ± 1.1
14 ± 6.1
a
b
b
8.0 ± 0.47
5.7 ± 0.66
13.1 ± 5.07
a
a
a
6.9 ± 0.60
6.3 ± 1.10
11.1 ± 0.96
a
a
b
12.4 ± 0.73
10.3 ± 0.84
14.8 ± 1.24
ab
a
b
D. Zlote
0mM
1.11mM
1.85mM
11 ± 0.3
13 ± 3.6
12 ± 0.5
a
a
a
4.5 ± 0.45
5.6 ± 0.66
4.6 ± 0.50
a
a
a
10.5 ± 0.14
12.6 ± 0.70
14.7 ± 1.51
a
ab
b
4.9 ± 0.54
4.9 ± 0.40
4.4 ± 0.31
a
a
a
MDHAR (nmol g -1 FW s -1 )
Data followed by different letters denote significant differences at p < 0.05 with respect to control.
Non-enzymatic antioxidant contents of AsA-GSH cycle
Glutathione content
Total glutathione (GSHt) levels varied in roots and leaves of ‘Riodeva’ and ‘D. Zlote’
plants (Fig. 4). In leaves, GSHt decreased (p ˂ 0.05) after 2 weeks in both genotypes. In
roots, GSHt content varied (p ˂ 0.05) in plants exposed to 1.85 mM Al: increased the 2nd
week in both genotypes but at the 3rd week just in ‘Riodeva’.
132
Aluminium toxicity in wheat and rye
Relatively to glutathione redox state (GSH/GSSG) (Fig. 4), ‘Riodeva’ plants showed a
more consistent behaviour: leaves had, in general, more oxidized forms while roots were
richer in the reduced ones.
In ‘D. Zlote’ roots, GSH/GSSG was more reduced at the 2nd week, whereas become
oxidized at the 3rd week. In leaves, alterations were more evident in plants exposed to 1.11
mM: after 2 weeks glutathione redox state was more oxidized and after 3 weeks was more
reduced.
0mM
50
1.11mM
µmol g-1 FW
40
1.85mM
a
30
20
a
10
b
a
a
b
a
b
a
b
a
a
b
ab
a
a
b
a
a
b
a
a
a
a
0
2 weeks
3 weeks
2 weeks
leaves
3 weeks
roots
Riodeva
1.9 0.3 0.2
2 weeks
0.9 0.6 0.5
1.0 0.6
3 weeks
leaves
GSHt
1.6
0.6 0.8 1.4
2 weeks
GSH/GSSG
3 weeks
roots
D. Zlote
1.9 0.6 1.7 0.7 1.8
0.4
0.8
1.8 3.0
1.8 0.8 0.4
Fig. 4. Variation in total glutathione (GSHt) contents and GSH/GSSG ratio in leaves and roots of ‘Riodeva’ and ‘D.
Zlote’ plants exposed to 1.11 mM and to 1.85 mM Al during 2 and 3 weeks. Values are given as mean ± standard error
for each condition. Data followed by different letters denote significant differences at p < 0.05 with respect to control.
Relatively to reduced glutathione content (Fig. 5A), the main trend was similar to the one
observed in total glutathione. In leaves, was detected a decrease (p ˂ 0.05) after 2 weeks
exposure in both genotypes, while after 3 weeks the GSH levels significant increased in
‘D. Zlote’ plants exposed to 1.11 mM Al. In ‘Riodeva’ roots, GSH content significantly
increased in both exposure times but only for 1.85 mM Al concentration. A similar
behaviour was detected in ‘D. Zlote’ roots at the 2nd week. However, at the 3rd week a
decrease in GSH content was observed in both Al concentrations.
Oxidized glutathione contents varied less than GSH contents (Fig. 5B). In ‘Riodeva’
plants, differences (p ˂ 0.05) were detected only in leaves: decreased after 2 weeks and
increased after 3 weeks. In ‘D. Zlote’ plants, GSSG levels increased in both leaves (1.85
mM) and roots (both Al concentrations) after 3 weeks.
133
Aluminium toxicity in wheat and rye
A
40
0mM
35
µmol g -1 FW
30
1.11mM
1.85mM
a
25
20
b
15
a
b
b
b
10
a
5
ab
a
a
a
a
a
b
a a
b
a
a
a
b
a
b b
0
2 weeks
3 weeks
2 weeks
leaves
3 weeks
roots
3 weeks
2 weeks
leaves
Riodeva
B
2 weeks
3 weeks
roots
GSH
D. Zlote
18
0mM
16
1.11mM
14
1.85mM
a
µmol g -1 FW
12
b
10
8
ab
b
a
a
b
6
a
a
a a
ab
a
a
b
b
a
4
a
a
a
a
a
2
b
b
0
2 weeks
3 weeks
2 weeks
leaves
3 weeks
2 weeks
roots
Riodeva
3 weeks
2 weeks
leaves
GSSG
3 weeks
roots
D. Zlote
Fig. 5. Variation in reduced glutathione (GSH) (A) and oxized glutathione (GSSG) (B) contents in leaves and roots of
‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11 mM and to 1.85 mM Al during 2 and 3 weeks. Values are given as mean
± standard error for each condition. Data followed by different letters denote significant differences at p < 0.05 with
respect to control.
Ascorbate content
AsA content increased in leaves of both genotypes when plants were treated with 1.85 mM
Al (Fig. 6). In ‘Riodeva’ leaves, the increase was detected at both exposure times, whereas
in ‘D. Zlote’ was observed only at the 3rd week. In roots, total ascorbate content varied (p ˂
0.05) only in ‘Riodeva’, which decreased at the 3rd week (Fig. 6).
AsA redox state (AsA/DHA) was dependent on plant organ and genotype (Fig. 6). In
leaves, become more oxidized in ‘Riodeva’ at the 2nd week, but there was an inversion at
the 3rd week, becoming more reduced. In ‘D. Zlote’ leaves, the AsA redox state was
similar in both exposure times (in 1.85 mM Al was more reduced). In roots, after 2 weeks
134
Aluminium toxicity in wheat and rye
exposure AsA/DHA ratio increased in both genotypes, whereas after 3 weeks was detected
a decline, which was more evident in ‘D. Zlote’.
0 mM
40
1.11 mM
35
b
1.85 mM
µmol g-1 FW
30
a
25
ab
a a
b
a
a
a
20
ab
15
b
a
a
a
10
b
a
5
a
a
a
a
a
a
a
c
0
2 weeks
3 weeks
2 weeks
leaves
3 weeks
roots
Riodeva
3.5 1.0 1.6
2 weeks
1.0 1.4 4.2
0.4 0.6 1.0
3 weeks
leaves
AsAt
0.1 0.05 0.5
2 weeks
AsA / DHA
3 weeks
roots
D. Zlote
5.5 5.4 18.0 5.2 5.1 8.2
0.08 0.7 0.4 0.2 0.04 0.2
Fig. 6. Variation in total ascorbate (AsAt) contents and in AsA redox state (AsA/DHA) in leaves (A) and roots (B) of
‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11 mM and to 1.85 mM Al during 2 and 3 weeks. Values are given as mean
± standard error for each condition. Data followed by different letters denote significant differences at p < 0.05 with
respect to correspondingly control.
Reduced ascorbate values increased (p ˂ 0.05) in leaves of both genotypes, mostly when
exposed to 1.85 mM Al. In roots, this parameter only varied in ‘D. Zlote’ plants: increased
after 2 weeks, but declined after 3 weeks Al treatment (Fig. 7A).
Contents of DHA showed higher imbalances in ‘Riodeva’ exposed plants than in ‘D.
Zlote’. At the 2nd week, ‘Riodeva’ leaves treated with 1.11 mM Al accumulated more
DHA. In ‘Riodeva’ roots, at the 3rd week, DHA contents decreased in both Al
concentrations.
135
Aluminium toxicity in wheat and rye
A
0 mM
35
1.11 mM
30
1.85 mM
b
b
µmol g-1 FW
25
b
ab
b
a
20
a
15
a
b
10
a
a
a
5
b
a
a a a
a
a
ab
a
a
b
b
0
2 weeks
3 weeks
2 weeks
leaves
3 weeks
2 weeks
roots
3 weeks
2 weeks
leaves
Riodeva
3 weeks
roots
AsA
D. Zlote
B
0 mM
16
1.11 mM
14
1.85 mM
µmol g-1 FW
12
8
b
a
b
a
a
6
4
a
a a
10
ab
a
a
a
a
a
a
a a
a
a
a
a a
2
b
c
0
2 weeks
3 weeks
2 weeks
leaves
3 weeks
2 weeks
roots
Riodeva
3 weeks
2 weeks
leaves
DHA
3 weeks
roots
D. Zlote
Fig. 7. Variation in reduced ascorbate (AsA) (A) and dehydroascorbate (DHA) (B) contents in leaves and roots of
‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11 mM and to 1.85 mM Al during 2 and 3 weeks. Values are given as mean
± standard error for each condition. Data followed by different letters denote significant differences at p < 0.05 with
respect to control.
Activity of some enzymatic antioxidants
Catalase activity varied in both ‘Riodeva’ and ‘D. Zlote’ genotypes (Table 3). In leaves,
differences were only detected after 3 weeks Al exposure: CAT values increased in both
genotypes leaves exposed to 1.85mM. In roots, ‘Riodeva’ CAT activity decreased at both
136
Aluminium toxicity in wheat and rye
exposure times, whereas in ‘D. Zlote’ decreased later (at the 3rd week) and only for the
lower concentration.
Superoxide dismutase activity (Table 3) varied only at the 3rd week. ‘Riodeva’ and ‘D.
Zlote’ leaves presented increases in SOD activity, whereas in roots, only ‘D. Zlote’ showed
variation in SOD activity (increased).
Relatively to guaiacol peroxidase activity (Table 3), ‘D. Zlote’ plants presented an
enhancement in its activity in both leaves and roots already at the 2nd week, which were
maintained after that. In ‘Riodeva’, G-POX activity variations were detected only at the 2nd
week: increasing in both organs in plants exposed to 1.85 mM Al.
Table 3
Variation in catalase (CAT) activity for 5 min, superoxide dismutase (SOD) activity and guaiacol peroxidase (G-POX)
activity in leaves and roots of ‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11 mM and to 1.85 mM Al during 2 or 3
weeks. Values are given as mean ± standard error for each condition.
leaves
-1
CAT (mg g FW)
2 weeks
roots
3 weeks
2 weeks
3 weeks
Riodeva
0 mM
1.11 mM
1.85 mM
5.8 ± 0.74 a
4.3 ± 0.09 a
4.5 ± 0.25 a
3.6 ± 0.26 a
4.4 ± 0.49 ab
5.9 ± 0.15 b
4.0 ± 0.27 a
3.9 ± 0.15 a
3.3 ± 0.10 b
4.8 ± 0.33 a
3.2 ± 0.14 b
2.9 ± 0.22 b
D. Zlote
0 mM
1.11 mM
1.85 mM
4.8 ± 0.13 a
5.6 ± 0.42 a
5.5 ± 0.23 a
3.8 ± 0.22 a
4.3 ± 0.36 ab
5.0 ± 0.12 b
4.6 ± 0.23 a
4.5 ± 0.78 a
4.8 ± 0.23 a
4.8 ± 0.33 a
3.4 ± 0.26 b
4.6 ± 0.32 a
-1
-1
SOD (mg g FW min )
Riodeva
0 mM
1.11 mM
1.85 mM
5.2 ± 0.49 a
8.6 ± 1.06 a
7.1 ± 1.08 a
6.3 ± 0.58 a
9.22 ± 0.4 b
7.85 ± 0.9 ab
12.5 ± 0.94 a
13.0 ± 1.34 a
10.2 ± 0.16 a
8.3 ± 1.54 a
11.6 ± 1.1 a
11.6 ± 1.5 a
D. Zlote
0 mM
1.11 mM
1.85 mM
3.8 ± 1.5 a
6.2 ± 0.74 a
7.2 ± 0.72 a
5.72 ±
7.18 ±
9.83 ±
4.09 ± 0.9 a
8.4 ± 0.69 a
8.6 ± 1.50 a
0.48 ± 0.1
6.75 ± 2.2
12.4 ± 2.5
-1
0.4 a
0.9 ab
1.1 b
a
ab
b
-1
G-POX ( µmol g FW s )
Riodeva
0 mM
1.11 mM
1.85 mM
0.9 ± 0.05 a
1.4 ± 0.11 ab
1.7 ± 0.26 b
1.3 ± 0.12 a
2.1 ± 0.27 a
1.9 ± 0.24 a
11.1 ± 0.59 a
13.7 ± 0.95 ab
15.6 ± 1.40 b
13.8 ± 0.71 a
12.9 ± 0.74 a
12.7 ± 1.36 a
D. Zlote
0 mM
1.11 mM
1.85 mM
1.7 ± 0.17 a
2.2 ± 0.25 ab
3.2 ± 0.45 b
0.9 ± 0.08 a
1.9 ± 0.46 ab
2.2 ± 0.31 b
9.8 ± 0.73 a
15.9 ± 1.11 b
18.9 ± 2.00 b
9.4 ± 0.83 a
15.5 ± 1.53 b
14.3 ± 1.07 b
Data followed by different letters denote significant differences at p < 0.05 with respect to control.
137
Aluminium toxicity in wheat and rye
Discussion and Conclusions
Plant redox status depends on the delicate equilibrium between ROS production and
scavenging at the proper site and time (Sharma and Dubey, 2007). In stressful conditions,
including metal toxicity, this equilibrium is highly dependent of the antioxidant machinery
activity. The present study was undertaken to contribute to a better knowledge of the
relationship between Al toxicity, oxidative stress and antioxidant system in rye. In general,
results strength that the oxidative status of the two rye genotypes was differently affected
by Al-treatment, and we discuss putative implications of these changes in the genotypes
degrees of tolerance.
The variation of H2O2 contents in Al exposed plants seems unclear: most literature refers
its increase, as in tobacco (Yin et al., 2010), duckweed (Radic et al., 2010) and barley
(Simonovicová et al., 2004), while in rice its variation was Al concentration dependent
(Sharma and Dubey, 2007). Although clear signals of oxidative stress were detected, H2O2
content decreased in almost all conditions in both rye genotypes. H2O2 levels decrease may
be a consequence of a) H2O2 consumption in oxidation processes, such as lipid
peroxidation; b) its detoxification via the increased activities of enzymes of the antioxidant
defence. Both mechanisms seem to coexist in ‘Riodeva’, whereas in ‘D. Zlote’, the last
strategy is more suitable, since various enzymatic and non-enzymatic antioxidants
increased and lipid peroxidation did not increase. One signal of Al toxicity is the increase
of lipid peroxidation, as reported for rice (Sharma and Dubey, 2007), wheat (Hossain et al.,
2005), potato (Tabaldi et al., 2009), triticale (Liu et al., 2008a), among others. In maize, Al
triggered lipid peroxidation in the sensitive line but not in the tolerant one (Giannakoula et
al., 2008). Also in maize, Boscolo et al. (2003) found that Al induced protein oxidation,
rather than lipid peroxidation, and suggested that the target of oxidative stress depends on
plant species. Similarly, only ‘Riodeva’ presented MDA increase and soluble protein
contents decrease supporting the assumption of Boscolo et al. (2003). Moreover, the
observed protein decrease with Al exposure may be a consequence of the enhancement of
ROS production and the consequent protein oxidation.
Enhancements of antioxidant enzymes in ‘D. Zlote’ roots were higher than those observed
in ‘Riodeva’. Works comparing Al-sensitive and Al-tolerant genotypes reported higher
enzymatic antioxidant activity in Al-tolerant ones (Du et al., 2010; Meriga et al., 2004).
SOD activity increase suggests an elevated content of superoxide radical. In Al exposed
138
Aluminium toxicity in wheat and rye
wheat roots, peroxide and superoxide formation was detected and SOD activity increased
in Al treated roots (Darkó et al., 2004). Higher SOD activity and consequent H2O2
production, leaded to the enhancement of enzymes that scavenge H2O2: in leaves both
CAT and G-POX (both genotypes), whereas in roots, APX and G-POX (‘D. Zlote’). By
not increasing in activity, CAT does not appear to be an efficient H2O2 scavenger in rye
roots, where Al accumulates more (Rengel and Reid, 1997; Liu et al., 2008b). This is in
agreement with what was found for rice by Sharma and Dubey (2007), which related CAT
inhibition with high Al concentration, and with the decrease of CAT activity in wheat
described by Hossain et al. (2005). On the other hand, it appears that G-POX is in the front
line to detoxify cells against H2O2 in both roots and leaves of ‘D. Zlote’. Several works in
Al, reported enhancements of G-POX activity in roots (Meriga et al., 2004; Simonovicová
et al., 2004; Hossain et al., 2005) and in both roots and shoots (Panda and Matsumoto,
2010). Another peroxidase that seemed to play an important role in ‘D. Zlote’ roots was
the APX. G-POX and APX are found throughout the cell and have higher affinity for H2O2
than CAT (Noctor and Foyer, 1998). Similarly to our findings, Sharma and Dubey (2007)
reported APX activity increase under Al and suggested that this enzyme plays an important
role in H2O2 detoxification in highly stressful conditions. In soybean roots, APX activities
increased as the Al levels and duration of treatment increased, significantly higher than
those in the sensitive genotype (Du et al., 2010). Taken all together, in rye roots, it seemed
that G-POX and APX were connected to the higher Al-tolerance observed in ‘D. Zlote’.
Leaves of both genotypes presented signals of GSH oxidation, after 2 weeks of Al
exposure. This tendency was reversed at the 3rd week in ‘D. Zlote’, but not in ‘Riodeva’.
GSH/GSSG decrease might be related to the GR activity decrease. Decline in GR activity
by Al treatment was also reported in pea (Panda and Matsumoto, 2010). In addition, in
’Riodeva’ leaves, GSH oxidation was also probably due to a decrease in DHAR activity,
since GSSG and DHA contents increased. Relatively to roots, at the 2nd week, both
genotypes presented a higher pool of reduced glutathione, but at the 3rd week ‘D. Zlote’
presents higher levels of the oxidized form. GSH enhancement under Al treatment was also
found in rice roots (Sharma and Dubey, 2007; Yang et al., 2007) and citrus leaves (Chen et
al., 2005). Contrarily to leaves, the decrease of GSH/GSSG ratio in ‘D. Zlote’ roots is not a
consequence of a decrease in GR activity (as it increased), but may be a consequence of
DHAR activity enhancement. GSH recycle by GR may not have been sufficient to
compensate GSH oxidation by DHAR. Some works corroborate this hypothesis since
139
Aluminium toxicity in wheat and rye
reported Al induced enhancement of DHAR activity (Tamás et al., 2006; Sharma and
Dubey, 2007). Increments in GR activity were also detected in barley (Tamás et al., 2006)
and rice (Sharma and Dubey, 2007).
The GSH/GSSG redox state showed clear differences between genotypes: in ‘Riodeva’, Al
long-term exposure affected mostly the leaves, while in ‘D. Zlote’ affected mostly the the
roots. The reduction of GSH pool apparently renders substantially more resistance to
stresses (Singh et al., 2006) as it induces the signal transduction and defence against ROS
(Fotopoulos et al., 2010). If GSH levels decrease, also decrease the ability to counteract the
negative effects of stress syndrome. So, a decline in GSH/GSSG ratio in ‘D. Zlote’ roots
shows its difficulty to continuously counteract the negative effects of long term Al
exposure and to adapt to stress conditions.
AsA is the most important antioxidant compound in the apoplast (Yin et al., 2010) and 3090% of total Al accumulation is predominantly in the apoplast of peripherical cells roots
(Liu et al., 2008b). Maintaining a high AsA content is important for detoxify Al-induced
ROS accumulation (Yin et al., 2010) and in rice roots feed with AsA, Al-tolerance
increased (Guo et al., 2005b). Rye leaves (both genotypes) presented increases of AsA
content (3rd week), however AsA/DHA ratio was more reduced in ‘D. Zlote’. AsA and
AsA/DHA ratio decreased simultaneous with high root growth inhibition. AsA acts as a
regulator of cell division and expansion, and as a signal transduction molecule. So,
decreases in it content may be directly and/or indirectly related to root growth inhibition
observed in ‘D. Zlote’.
As hypothesized by Shen et al (2004) the response of the glutathione system as an
ecophysiological reaction, can undergo different phases and may or may not reach
acclimation and further studies on longer exposure periods should be performed.
Al exposure leaded to oxidative stress and other Al-induced injuries in rye seedlings and
we concluded that: 1) rye roots were more affected by Al-toxicity than leaves; 2) in leaves,
CAT, SOD and G-POX played an important role in ROS detoxify; 3) the enhancement of
APX, SOD and G-POX activities in roots contributed to the higher Al tolerance in ‘D.
Zlote’ plants; 4) GR activity is negatively affected in leaves, but not in roots; 5) AsA-GSH
cycle response depended on organ and genotype; 6) the high root growth inhibition in ‘D.
Zlote’ at the 3rd week may be related to oxidation of AsA and GSH pools; 7) the ability to
survive in Al environments depends on the antioxidant system response capacity; 8) the
antioxidant response seemed to be a good tool to define Al-tolerance degree in rye
140
Aluminium toxicity in wheat and rye
genotypes. Furthermore, our results revealed that it is important to conduct long term
experiments in order to better understand the plants “tolerance” ability and which
genotypes are suitable to grow in acid conditions.
Acknowledgements
FCT/MCT supported this work (POCI /AGR/ 58174/2004) and S. Silva (FCT⁄BD⁄ 32257⁄2006)
grants. Thanks are due to Armando Costa and Barbara Correia for technical support.
141
Aluminium toxicity in wheat and rye
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III.2: Aluminium long-term
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This chapter was submitted as an original paper in a SCI journal:
Silva S, Pinto G, Dias C, Correia CM, Moutinho
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Aluminium toxicity in wheat and rye
148
Aluminium toxicity in wheat and rye
Abstract
Aluminium (Al) toxicity is considered one of the major problems for crop growth and
production on acid soils. The ability of crops to overcome Al toxicity varies among crop
species and cultivars. Among the Triticeae genus, rye (Secale cereale) is considered the
most Al-tolerant species. In the present work, two rye genotypes differing in Al tolerance
(‘Riodeva’: Al-sensitive and ‘Donkowsky Zlote’: Al-tolerant) were exposed to 1.11 and
1.85 mM Al during three weeks. Growth, water status and photosynthesis related
parameters (biomass, relative water content (RWC), gas exchange parameters, chlorophyll
a fluorescence, pigment contents and carbohydrates contents) were assessed. After three
weeks of Al-exposure, both genotypes presented similar decrease in leaf growth. Al
induced RWC decreased in both genotypes, but this effect was more remarkable in
‘Riodeva’. Al toxicity induced a decrease in net photosynthetic rate A only after three
weeks of exposure. In ‘D. Zlote’, A decrease was accompanied by stomatal closure, Chl a
content and qp reduction, but no alterations in RuBisCo or sFBPase activity were observed.
In ‘Riodeva’ plants exposed to 1.11 mM Al, A decrease was accompanied by Ci/Ca
increase (and increase of E and gs) whereas in plants exposed to 1.85 mM Al Ci/Ca was
not affected (decrease of E and gs). Nevertheless, for both conditions RuBisCo activity
decreased. A decrease did not limited glucose accumulation in neither of the rye genotypes.
This study revealed that Al induced earlier damages in the ‘Riodeva’ genotype, but both
genotypes showed long-term high susceptibility to Al. Furthermore, the photosynthetic
parameters proved to be a good tool to monitor Al-sensitivity and long-term exposure
showed to be crucial to evaluate Al-sensitivity.
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Aluminium toxicity in wheat and rye
150
Aluminium toxicity in wheat and rye
Introduction
Aluminium (Al) ions, mainly Al3+, are toxic to most plants, however its bio-availability is
mostly restricted to acid conditions (Poschenrieder et al., 2008). Soil acidity is increasing
due to atmospheric inputs of natural nitric and sulphuric acids mainly due by fertilization
practices and anthropogenic pollutants (Marschner et al., 1995). Therefore, tolerant plants
are needed to increase alternatives for crop production in acid soils. Among the cereal
species Al tolerance varies, being rye (Secale cereale) one of the most tolerant (Aniol and
Gustafson, 1984). Much has been done to understand mechanisms (e. g. organic acid
exudation, Al tolerant genes) that confer Al-tolerance in wheat (Ryan et al., 1992;
Matsumoto, 2005; Ryan et al., 2009) and in other species, such as barley, maize and
soybean (Samac and Tesfaye, 2003). Research dealing with rye Al-tolerance is scarce and
the few studies available focus only short term Al-exposure (Yang et al., 2005; Stass et al.,
2008). Thus, a deeper knowledge of the mechanisms employed by rye to cope with Al
long-term exposure and their consequent toxicity will provide valuable tools for future
plant improvement.
Root growth inhibition is the primary effect of Al toxicity and it is usually used as a
marker to estimate Al sensitivity. Other plant biomarkers are also used, as accumulation of
callose or Al specific tissue staining (Poschenrieder et al., 2008). Biomass reduction was
reported as a consequence of Al-exposure (Moustakas et al., 1997), revealing possibly
alterations in photosynthesis. There is a general lack of information about the direct or
indirect effects of Al-exposure on photosynthesis. Reductions in carbon dioxide (CO2)
assimilation rate due to Al toxicity are reported in some species, as maize (Lidon et al.,
1999), wheatgrass (Moustakas et al., 1996), tomato (Simon et al., 1994), sorgum (Peixoto
et al., 2002), citrus (Pereira et al., 2000; Chen et al., 2005; Jiang et al., 2008) and pinus
(Oleksyn et al., 1996). Moreover, the causes of the CO2 assimilation reduction (stomatal
and/or nonstomatal) are still a hotpoint in this research field. The few information available
indicate that Al-exposure induce damage of photosystem II (Zhang et al., 2007; ReyesDias et al., 2010), disturbance in chloroplasts ultrastructure (Moustakas et al., 1997),
decreases in Fv/Fm (maximum quantum efficiency) (Moustakas et al., 1996; Chen et al.,
2005), changes on light-harvesting pigments (Zkang et al., 2007; Azmat and Hasan, 2008)
and in the photosynthetic electron transport chain (Chen et al, 2005 a; Jiang et al., 2009 a).
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) is the first enzyme in the
151
Aluminium toxicity in wheat and rye
photosynthetic CO2 assimilation and it activation state determines the amount of RuBisCo
that can contribute to the overall rate of carboxilation (Crafts-Brandner and Salvucci,
2000). An impairment of the photosynthetic capacity due to reductions of the activity and
content of this enzyme was reported under several stress conditions (Mobin and Khan,
2007; Mateos-Naranjo et al., 2008; Dias et al., 2010). However, the effects of Al exposure
on RuBisCo content and activity are still unclear and the few reports available were
performed in citrus (Chen et al., 2005 b; Jiang et al., 2008) and in wild rice (Cao et al.,
2010). Jiang et al. (2009a; b) suggested that the influence of Al on RuBisCo activity
depends on phosphorus and borum concentration supplemented in the culture medium.
Changes in chlorophyll (Chl) contents due to Al-treatment is reported for several species,
as citrus (Chen et al., 2005 b; Jiang et al., 2008), tea plant (Yadav and Mohanpuria, 2009),
lentil (Azmat and Hasan, 2008), soybean (Zhang et al., 2007), among others. Furthermore,
Pereira et al. (2006) reported that Al decreased 5-aminolevulinic acid (ALA) dehydratase
activity in cucumber and in maize and Mihailovic et al. (2008) showed thet Al leaded to
inhibition of 5-ALA synthesis, both essential to Chl synthesis.
Stressful environment affect carbohydrates storage, translocation and metabolism. As final
products of photosynthesis, they are usually used as indicators of plant growth potential.
Carbohydrates increase had been correlated with Al tolerance in several species (Khan et
al., 2000; Tabuchi et al., 2004; Giannakoula et al., 2008).
It is widely known that species and genotypes within species greatly differ in their
tolerance to Al. Based in root regrowth, the rye genotype ‘Riodeva’ is considered Alsensitive, while Dankowski Zlote more tolerant: ‘Riodeva’ showed only 1.1 mm root
regrowth at 4 ppm (Gallego and Benito, 1997), whereas ‘D. Zlote’ presented root regrowth
of 1.3 mm at 40 ppm Al (Pinto-Carnide and Guedes-Pinto, 1999).
With the present work, we critically (re)evaluate the classification above referred, for
‘Riodeva’ and ‘D. Zlote’ when parameters other then root regrowth are used (e.g.
photosynthetic rate). Moreover, this work aims to identify the main Al photosynthetic
targets and in what extent the photosynthetic apparatus is affected by Al exposure. Thus,
‘Riodeva’ and ‘D. Zlote’ plants were compared for their photosynthetic responses to
different Al concentrations and exposure time. The evaluation of Al-induced effects
covered several parameters directly or indirectly related with carbon metabolism: relative
water content (RWC), gas exchange parameters, Chl a fluorescence, pigment contents and
152
Aluminium toxicity in wheat and rye
carbohydrate contents. Up to the authors’ knowledge, this is the first report on the effects
of long term Al exposure on plant performance of two rye genotypes.
Material and Methods
Plant material and growth culture
Two rye genotypes, Dankowski Zlote (Polish cultivar) and ‘Riodeva’ (Spanish inbred
line), which were classified as a more Al-tolerant (e.g. Pinto-Carnide and Guedes-Pinto,
1999; 2000) and an Al-sensitive (Gallego and Benito, 1997) genotypes, respectively, were
used. Seeds were disinfected, rinsed in distilled water and germinated (in the dark at 24 ºC)
in petri dishes. Germinated seeds were transferred for 4 days to a modified Hoagland
solution: KNO3 303 mg/L; Ca(NO3)2.4H2O 470 mg/L; MgSO4.7H2O 123 mg/L;
NH4H2PO4 14.4 mg/L, and the following micronutrients: Fe-tartrate 2.65 mg/L; H3Bo3
1.43 mg/L; MnCl2 0.905 mg/L; CuSO4.5H2O 0.04 mg/L; ZnSO4.7H2O 0.11 mg/L; H3Mo
0.008 mg/L (RW medium) For Al exposure, plants were transferred to the same nutritive
solution containing 1.11 mM or 1.85 mM of AlCl3.6H2O, corresponding to 0.26 mM and
0.42 mM of Al activity, respectively (Al activity was estimated by Geochem-EZ). Plants
were grown during three weeks on nutritive solution containing Al. A control group was
maintained in the nutritive solution without Al. Plants were grown in a culture room at 24
º
C with 16/8h photoperiod. The nutrient solution was continuously aerated and renewed
every 3 days. The pH was maintained at 4.0 throughout the assay (e.g. Silva et al 2010).
Measurements were performed two and three weeks after the beginning of Al exposure.
Growth and relative water content analysis
Leaves of 5 plants were used to measured growth and RWC. The leaf length and fresh
weight were measured. The leaf RWC was calculated using the following formula: RWC =
(fresh weight – dry weight / fresh weight) x 100.
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Aluminium toxicity in wheat and rye
Gas exchange and chlorophyll a fluorescence
In situ determinations of net photosynthetic rate (A), stomatal conductance (gs),
transpiration rate (E) and the intercellular (Ci) and atmospheric CO2 (Ca) concentration at
200 µmol photon m–2 s–1 were measured with a portable IRGA (LCpro+, ADC,
Hoddesdon, United Kingdom), operating in the open mode under growth chamber
conditions. The relative humidity of air entering the cuvette was set at 60% and the cuvette
temperature was 23 ºC. Flow rate of air through the cuvette was set at 300 µmol s–1. The
Ci/Ca was calculated according to von Caemmerer and Farquhar (1981). Measurements
were always performed in the 2nd and/or 3rd older leaf under ambient CO2 concentration.
Chlorophyll a fluorescence parameters were measured in situ with a pulse-amplitudemodulated fluorimeter (FMS 2, Hansatech Instruments, Norfolk, England) as described by
Öquist and Wass (1988). Maximum quantum efficiency of photosystem II (PSII) was
obtained (Fv/Fm = (Fm-F0)/Fm) according to the program of FMS 2, measuring the
fluorescence signal from a dark-adapted leaf, when all reaction centers are open, using a
low intensity pulsed measuring light source (F0) and during a pulse saturating light (0.7 s
pulse of 15000 µmol photons m–2 s–1 of white light), when all reactions centers are closed
(Fm). Leaves were dark-adapted for 30 min using dark-adapting leaf-clips (FMS).
Following Fv/Fm estimation, a pulse of actinic light was given (1500 µmol m–2 s–1) (20 s)
and light-adapted steady-state fluorescence yield (Fs) was averaged over 2.5 s, followed by
exposure to saturating light (15000 µmol m–2 s–1) for 0.7 s to establish F’m. The sample
was than shaded and a far-red pulse (5 s) was provided to determine F’0, which was used
for determination of photochemical quenching (qP = (F’m-Fs)/(F’m-F’0)), nonphotochemical quenching (qN = 1- F’v/Fv) and efficiency of electron transport as a measure
of the effective photochemical efficiency of PSII, ΦPSII =∆F/F’m = (F’m-Fs)/F’m (Bilger and
Schreiber, 1986; Genty al., 1989). As described by Krall and Edwards (1992), the electron
transport rate (ETR) was estimated as ETR = (∆F/F’m) x PAR x 0.5 x 0.84 (Bjorkman and
Demmig, 1987). Since measurements were done under constant PAR, ΦPSII and ETR are
equivalent.
For these parameters on average 6 plants were used.
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Aluminium toxicity in wheat and rye
Pigments analyses
Photosynthetic pigments (chlorophyll a (Chl a) and chlorophyll b (Chl b)) and carotenoids
were measured according to Sims and Gamon (2002). Chlorophylls and carotenoids were
extracted with acetone/50 mM Tris buffer at pH 7.8 (80:20) (v/v). After homogenization
and centrifugation, supernatant was used and absorbance read at 663 nm, 537 nm, 647 nm
and 470 nm (Thermo Fisher Scientific spectrophotometer, Genesys 10-uv S).
Determination of Calvin cycle enzymes activities: RuBisCo and sFBPase
Ribulose-1,6-bisphosphate carboxylase/oxygenase (RuBisCo, EC 4.1.1.39) activity was
determined spectrophotometrically (Thermo Fisher Scientific, Genesys 10-uv S) by
monitoring nicotinamide adenine dinucleotide (NADH) oxidation at 23 ºC and 340 nm
(Lilley and Walter, 1974). Leaf samples were homogenized in a chilled mortar with an icecold extraction buffer solution. The homogenate was centrifuged and the supernatant was
used. The reaction mixture (1 ml) contained 50 mM Hepes (pH 8.0), 10 mM KCl, 20 mM
MgCl2, 0.2 mM NADH, 2.5 mM ATP, 1 mM EDTA, 5 mM DTT, 5 mM Creatine
phosphate,
20
U/ml
Creatine
phosphokinase,
6
U/ml
glyceraldehyde
3-
phosphodehydrogenase and 6 U/ml 3-phosphoglycerate kinase. The reaction was started
with the addition of 0.6 mM of ribulose-1,5-bisphosphate (RuBP). To achieve maximum
activities, the extract was incubated in 20 mM MgCl2 and 10 mM NaHCO3 for 20 min on
ice prior to measurements.
Stromal
fructose-1,6-biphosphatase
(sFBPase)
activity
was
determined
spectrophotometrically by monitoring NADH phosphate oxidation at 23 ºC and 340 nm
(Dias et al., 2007). Leaf samples were homogenized in a chilled mortar with ice-cold
extraction buffer solution. The homogenate was centrifuged and the supernatant was used.
The reaction mixture (1 ml) contained 100 mM Tris-HCl (pH 8.8), 20 mM MgCl2, 1mM
EDTA, 0.5 mM NADP, 1.4 U/ml phosphoglucose isomerase (PGI) and 0.7 U/ml glucose6-phosphodesidrogenase and 10 mM DTT. The reaction was started with the addition of 4
mM of fructose -1,6-bisphophate.
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Aluminium toxicity in wheat and rye
Carbohydrates quantification
Glucose, fructose and sucrose were extracted from frozen leaf samples with 80% (v/v)
ethanol, at 80 ºC over 20 min as described by Correia et al. (2005). Soluble sugars were
quantified using a spectrometric enzyme-coupled assay according to Jones et al. (1977).
After extraction of soluble sugars, leaf samples were then used to starch analyses as
described by Correia et al. (2005).
Statistical analyses
For pigments, enzyme activities and carbohydrates quantifications 4 replicates were used.
Each replica consisted in leaf pools from 7-10 plants.
Values are given as mean ± standard error. The comparison between Al concentrations
and control was made using One Way ANOVA test and comparisons between
genotypes or between exposure times were made using Two Way ANOVA. When data
was statistically different, ANOVA test was followed by a Holms Sidak Comparison
Test (p<0.05).
Results
Growth and relative water content analysis
‘Riodeva’ plants exposed to 1.85 mM Al presented significant decrease of leaves size after
two weeks. In ‘D. Zlote’, leaves size decreased only after 3 weeks at the highest
concentration (Fig. 1A). After 2 weeks in both Al conditions, biomass decreased in
‘Riodeva’ and in ‘D. Zlote’ (Fig. 1B).
Leaf growth parameters (biomass and size) in control plants was significantly higher in ‘D.
Zlote’, but in exposed leaves, values were similar (p ≥ 0.05) for both genotypes. In general,
leaf growth values in exposed plants (‘Riodeva’ and ‘D. Zlote’) did not increased from the
2nd to 3rd week (p ≥ 0.05).
156
Aluminium toxicity in wheat and rye
Leaves size (cm)
A
a
50
40
30
a
1,2
a
a
b
a a
a a
b
b
a
1
Leaves biomass (g)
60
a
20
0,8
0,4
0 mM
1.11 mM
a
b
0,2
10
a
a
0,6
B
b b
bb
b b
3 weeks
2 weeks
3 weeks
1.85 mM
b
0
0
2 weeks
3 weeks
2 weeks
Riodeva
2 weeks
3 weeks
Riodeva
D. Zlote
D. Zlote
Fig. 1. Variation in leaves growth: size (A) and biomass (B) in ‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11 mM and
to 1.85 mM Al during 2 and 3 weeks. Values are given as mean ± standard error for each condition. Data followed by
different letters denote significant differences at p < 0.05.
‘Riodeva’ leaves presented a decline (p < 0.05) in RWC under both Al concentrations at
the 2nd week (Fig. 2). From the 2nd to 3rd week, RWC in ‘Riodeva’ leaves exposed to 1.11
mM continued to decrease (p < 0.05). In ‘D. Zlote’, a significant decrease in RWC values
was observed only in plants exposed to 1.11 mM Al after 2 weeks exposure (Fig. 2).
The two genotypes presented similar (p ≥ 0.05) RWC values.
0mM
1.11mM
100
a
b b
a b ab
a
b
a
a
a a
1.85mM
RWC (%)
80
60
40
20
0
2 weeks
3 weeks
2 weeks
Riodeva
3 weeks
D. Zlote
Fig. 2. Variation in relative water content in ‘Riodeva’ and ‘D. Zlote’ leaves of plants exposed to 1.11 mM and to 1.85
mM Al during 2 and 3 weeks. Values are given as mean ± standard error for each condition. Data followed by different
letters denote significant differences at p < 0.05.
Gas exchange and chlorophyll a fluorescence
Gas-exchange parameters (Table 1) presented significant alterations in ‘D. Zlote’ only after
3 weeks. Except for Ci/Ca rate, declines in all other parameters were observed (p < 0.05),
157
Aluminium toxicity in wheat and rye
being more accentuated in plants exposed to the highest Al concentration. ‘Riodeva’ plants
presented alterations in gas-exchange parameters at the 2nd week. At the 3rd week, plants
exposed to 1.11 mM presented enhancement of gs, Ci/Ca rate and E, whereas in plants
exposed to 1.85 mM E and gs decreased (Table 1). The gs, Ci/Ca and E values at the 3rd
week in 1.85 mM exposed plants were significantly lower to the values observed at the 2nd
week. In both genotypes, exposed plants presented A decrease at the 3rd week.
Differences between genotypes were detected at the 3rd week in E, gs and A.
Table 1
Gas-exchanges variations in ‘Riodeva’ and ‘D. Zlote’ leaves of plants exposed to 1.11 mM and to 1.85 mM Al during 2
and 3 weeks. Values are given as mean ± standard error for each condition (E: transpiration rate, gs: stomatal condutance,
A: net photosynthetic rate, Ci/Ca: intercellular to atmospheric CO2 concentration ratio)
E (mmolm -2 s-1 )
2 weeks
3weeks
gs (mmolm -2 s-1 )
2 weeks
3weeks
Riodeva
0 mM
1.11 mM
1.85 mM
2.5 ± 0.13
4.8 ± 1.48
7.1 ± 0.32
a
ab
b
3.4 ± 0.35
4.9 ± 0.44
2.1 ± 0.21
a
b
c
110 ± 6.55
236 ± 70.4
253 ± 11.8
a
ab
b
215 ± 14.5
301 ± 30.7
115 ± 13.0
a
b
c
D. Zlote
0 mM
1.11 mM
1.85 mM
6.9 ± 1.13
5.6 ± 0.19
5.2 ± 0.53
a
a
a
4.3 ± 0.26
2.3 ± 0.23
0.9 ± 0.18
a
b
c
317 ± 51.1
220 ± 9.14
200 ± 21.8
a
a
a
325 ± 29.9
158 ± 17.8
68 ± 12.4
a
ab
b
A (µmolm -2 s-1 )
2 weeks
3weeks
Ci/Ca
2 weeks
3weeks
Riodeva
0 mM
1.11 mM
1.85 mM
6.3 ± 1.04
5.1 ± 0.87
3.2 ± 0.17
a
a
a
18.5 ± 1.72
14.2 ± 1.38
8.4 ± 0.91
a
b
c
0.75 ± 0.036 a
0.84 ± 0.034 ab
0.90 ± 0.002 b
0.61 ± 0.041 b
0.76 ± 0.018 a
0.66 ± 0.041 ab
D. Zlote
0 mM
1.11 mM
1.85 mM
6.6 ± 1.41
3.9 ± 0.19
3.6 ± 0.50
a
a
a
16.7 ± 0.82
9.7 ± 1.37
7.9 ± 1.31
a
b
b
0.86 ± 0.010 a
0.88 ± 0.002 a
0.88 ± 0.005 a
0.75 ± 0.018 a
0.73 ± 0.029 a
0.62 ± 0.078 a
Different letters represent significant differences at p ˂ 0.05.
Concerning Chl a fluorescence parameters, Al exposure did not lead to significant
alterations in ΦPSII and, consequently in ETR values, in both genotypes (Table 2).
‘D. Zlote’ exposed plants, but not ‘Riodeva’, presented an enhancement of F0 values at the
2nd week. On the other hand, a decrease in this parameter was observed at the 3rd week in
‘D. Zlote’ and ‘Riodeva’ exposed leaves. The F0 values in exposed plants at the 3rd week
were significantly lower than those observed at the 2nd week.
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Aluminium toxicity in wheat and rye
Fv/Fm values decreased in ‘D. Zlote’ (at the 2nd week) and in ‘Riodeva’ plants (at the 3rd
week). In ‘Riodeva’, no significant differences were detected in qN and qP values, whereas
in ‘D. Zlote’, qN increased after 2 weeks and qP values decreased after 3 weeks, with no
differences between Al conditions (Table 2).
Table 2
Fluorescence parameters variations in ‘Riodeva’ and ‘D. Zlote’ leaves of plants exposed to 1.11 mM and to 1.85 mM Al
during 2 and 3 weeks. Values are given as mean ± standard error for each condition (F0: minimal fluorescence, Fv/Fm:
maximum quantum efficiency, ΦPSII: total photochemical efficiency, ETR: electron transport rate; qN: non-photochemical
quenching, qP: photochemical quenching).
F0
Fv/Fm
2 weeks
3weeks
2 weeks
3weeks
Riodeva
0 mM
1.11 mM
1.85 mM
D. Zlote
0 mM
1.11 mM
1.85 mM
255 ± 71.1
306 ± 43.5
263 ± 27.7
a
a
a
212 ± 70.9 a
709 ± 209.0 b
778 ± 93.1 b
55 ± 5.0
62 ± 6.7
53 ± 3.5
a
a
b
0.89 ± 0.017 a
0.89 ± 0.016 a
0.88 ± 0.017 a
0.88 ± 0.008 a
0.87 ± 0.010 ab
0.86 ± 0.017 b
148 ± 7.6
54 ± 32.6
49 ± 20.8
a
b
b
0.89 ± 0.029 a
0.85 ± 0.009 b
0.86 ± 0.005 b
0.86 ± 0.003 a
0.89 ± 0.026 a
0.88 ± 0.054 a
ΦPSIIR
ET R
2 weeks
3weeks
2 weeks
3weeks
0 mM
1.11 mM
1.85 mM
0.63 ± 0.028 a
0.59 ± 0.038 a
0.60 ± 0.029 a
0.73 ± 0.019 a
0.73 ± 0.009 a
0.69 ± 0.029 a
53 ± 2.3
50 ± 3.2
50 ± 2.5
a
a
a
61 ± 1.6
62 ± 0.8
58 ± 2.4
a
a
a
D. Zlote
0 mM
1.11 mM
1.85 mM
0.60 ± 0.038 a
0.53 ± 0.058 a
0.64 ± 0.015 a
0.75 ± 0.006 a
0.66 ± 0.053 a
0.75 ± 0.027 a
50 ± 3.2
45 ± 4.9
53 ± 1.3
a
a
a
63 ± 0.5
56 ± 4.5
63 ± 2.3
a
a
a
2 weeks
3weeks
2 weeks
0 mM
1.11 mM
1.85 mM
0.49 ± 0.098 a
0.34 ± 0.063 a
0.22 ± 0.047 a
0.20 ± 0.026 a
0.25 ± 0.053 a
0.28 ± 0.061 a
0.88 ± 0.022 a
0.84 ± 0.038 a
0.85 ± 0.035 a
0.88 ± 0.014 a
0.90 ± 0.012 a
0.86 ± 0.025 a
D. Zlote
0 mM
1.11 mM
1.85 mM
0.11 ± 0.037 a
0.69 ± 0.060 b
0.62 ± 0.021 b
0.31 ± 0.062 a
0.50 ± 0.121 a
0.52 ± 0.140 a
0.83 ± 0.037 a
0.81 ± 0.035 a
0.88 ± 0.010 a
0.95 ± 0.005 a
0.79 ± 0.052 b
0.85 ± 0.039 b
Riodeva
qN
qP
3weeks
Riodeva
Different letters represent significant differences at p ˂ 0.05.
159
Aluminium toxicity in wheat and rye
Pigments analyses
No significant alterations were observed in Chl a and Chl b contents (p ≥ 0.05) at the 2nd
week in exposed leaves of both genotypes (Table 3). However, after 3 weeks, a decrease in
both pigments was detected in ‘D. Zlote’ leaves exposed to 1.85 mM Al (Table 3).
Carotenoids contents increased only in ‘Riodeva’ plants when exposed to 1.85 mM Al at
the 2nd week (Table 3).
Control leaves of both genotypes presented similar pigment contents. However, ‘Riodeva’
leaves exposed to 1.85 mM, at the 2nd week, presented higher Chl a, Chl b and carotenoids
contents than ‘D. Zlote’ (p ˂ 0.05).
Table 3
Chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids contents in leaves of Riodeva and ‘D. Zlote’ plants exposed
to 1.11 mM and to 1.85 mM Al after 2 and 3 weeks. Values are mean ± standard error.
Chl a (µmol g-1 FW)
2 weeks
3 weeks
Chl b (µmol g-1 FW)
2 weeks
3 weeks
Riodeva
0 mM
1.11 mM
1.85 mM
1.1 ± 0.08
1.2 ± 0.13
1.4 ± 0.14
a
a
a
1.3 ± 0.09
1.5 ± 0.16
1.1 ± 0.12
a
a
a
0.42 ± 0.034 a
0.45 ± 0.048 a
0.54 ± 0.054 a
0.49 ± 0.037 a
0.46 ± 0.108 a
0.40 ± 0.045 a
D. Zlote
0 mM
1.11 mM
1.85 mM
1.1 ± 0.03
1.1 ± 0.06
0.9 ± 0.04
a
a
a
1.3 ± 0.09
1.0 ± 0.02
0.9 ± 0.05
a
ab
b
0.38 ± 0.006 a
0.39 ± 0.020 a
0.35 ± 0.019 a
0.47 ± 0.030 a
0.40 ± 0.027 ab
0.32 ± 0.033 b
Carotenoids (µmol g-1 FW)
2 weeks
3 weeks
70 ± 11.8 a
35 ± 12.9 a
128 ± 6.4 b
72 ± 1.3
74 ± 3.7
68 ± 3.5
a
a
a
87 ± 5.7 a
88 ± 17.7 a
94 ± 16.2 a
89 ± 9.4
76 ± 2.3
65 ± 4.6
a
a
a
Different letters represent significant differences at p ˂ 0.05.
Calvin cycle enzymes
Two enzymes of the Calvin cycle were measured, RuBisCo and stromal fructose - 1,6 bisphosphatase (sFBPase). Al exposure did not alter RuBisCo activity in ‘D. Zlote’ (p ≥
0.05) (Table 4). ‘Riodeva’ leaves showed a decrease (p ˂ 0.05) trend in exposed leaves at
the 3rd week (Table 4).
Relatively to sFBPase activity, a significant increase in the ‘Riodeva’ plants exposed to
1.85 mM Al after 3 weeks exposure was detected (Table 4).
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Aluminium toxicity in wheat and rye
Table 4
RuBisCo and fructose -1,6 - bisphosphatase (sFBPase) activity in leaves of ‘Riodeva’ and ‘D. Zlote’ plants exposed to
1.11 mM and to 1.85 mM Al after 2 and 3 weeks. Values are mean ± standard error.
RuBisCo (nmol g-1 FW s-1 )
2 weeks
3 weeks
Riodeva
0mM
1.11mM
1.85mM
D. Zlote
0mM
1.11mM
1.85mM
1.24 ± 0.171
1.62 ± 0.422
1.31 ± 0.629
a
a
a
3.77 ± 0.823 a
1.33 ± 0.180 b
0.82 ± 0.116 b
3.68 ± 1.732
0.58 ± 0.057
1.29 ± 0.511
a
a
a
1.97 ± 0.275 a
1.03 ± 0.629 a
1.19 ± 0.798 a
sFBPase (nmol g-1 FW s-1 )
2 weeks
3 weeks
21 ± 3.5 ab
19 ± 0.8 a
95 ± 22.5 b
148 ± 31.1 a
103 ± 14.3 a
164 ± 53.7 a
64 ± 26.6
151 ± 35.2
732 ± 60.1
a
a
b
197 ± 107.4 a
163 ± 48.2 a
129 ± 28.4 a
Different letters represent significant differences at p ˂ 0.05
Carbohydrates quantification
No significant differences in glucose, fructose and sucrose contents were observed in ‘D.
Zlote’ plants exposed to Al for two weeks (Fig 3). At the 3rd week, glucose and fructose
contents almost doubled in leaves of plants exposed to 1.85 mM Al. No differences were
observed in sucrose (Fig 3). Comparing the two exposure times, glucose contents
significantly increased in control and in plants exposed to 1.85 mM Al at the 3rd week.
Relatively to ‘Riodeva’, significant differences were detected already at the 2nd week:
glucose and fructose contents increased in exposed plants (Fig 3). From the 2nd to the 3rd
week, glucose contents continued to increase (p ˂ 0.05), with higher (p ˂ 0.05) incidence
in plants exposed to 1.85 mM (Fig 3).
Comparing the two genotypes, control plants did not differ in glucose, fructose and sucrose
contents, but ‘Riodeva’ Al-exposed plants presented higher (p ˂ 0.05) glucose and fructose
accumulation than ‘D. Zlote’ (in both exposure periods).
161
Aluminium toxicity in wheat and rye
D. Zlote
Riodeva
0mM
1.11mM
45,00
c
1.85mM
40,00
b
35,00
µmolg-1FW
b
b b
30,00
25,00
a
20,00
15,00
10,00
b
a
b
a
5,00
a
a
a
a aa
a
a
a
a a
a aa
a aa
ab
aaa
c
aaa
0,00
Glucose
Fructose
Sucrose
Glucose
2weeks
Fructose
Glucose
Sucrose
Fructose
Sucrose
2weeks
3 weeks
Glucose
Fructose
Sucrose
3 weeks
Fig. 3. Glucose, fructose and sucrose contents in leaves of ‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11 mM and to
1.85 mM Al after 2 and 3 weeks. Values are mean ± standard error. Different letters represent significant differences at p
˂ 0.05.
Starch contents decreased, when compared to control, in ‘Riodeva’ leaves exposed to 1.85
mM Al, at the 3rd week (Table 5). In ‘D. Zlote’, starch contents increased in both Al
concentrations, at the 2nd week exposure.
In general, both genotypes presented starch content decrease (p ˂ 0.05) from the 2nd to the
3rd week in Al-exposed plants. Furthermore, starch content was significantly higher in
‘Riodeva’ than in ‘D. Zlote’ plants.
Table 5
Starch contents in leaves of ‘Riodeva’ and ‘D. Zlote’ plants exposed to 1.11mM and 1.85mM Al after 2 and 3 weeks.
Values are mean ± standard error.
Starch (mmol g-1 FW)
2 weeks
Riodeva
0 mM
1.11 mM
1.85 mM
0.31 ± 0.036
0.27 ± 0.013
0.12 ± 0.008
a
a
b
0.11 ± 0.008 a
0.13 ± 0.008
0.18 ± 0.004 b
0.13 ± 0.006
0.18 ± 0.009 b
0.12 ± 0.011
Different letters represent significant differences at p ˂ 0.05.
a
a
a
D. Zlote
0 mM
1.11 mM
1.85 mM
0.25 ± 0.015
0.23 ± 0.006
0.26 ± 0.006
3 weeks
a
a
a
162
Aluminium toxicity in wheat and rye
Discussion and Conclusions
In literature, ‘D. Zlote’ is considered more Al-tolerant than ‘Riodeva’ (e.g. Gallego and
Benito, 1997; Pinto-Carnide and Guedes-Pinto, 1999), according to root regrowth ability.
In our report we present a more comprehensive study covering several physiological
parameters, focusing mainly on leaves. The results indicated that Al-treatment differently
affected the physiological/biochemical performance of rye genotypes.
Both Al concentrations negatively affected leaf growth in both genotypes. Al-induced
reductions of leaves size and biomass were reported for other species (e.g., Lidon et al.,
2000; Guo et al., 2007; Zhang et al., 2007; Konarska, 2010). Moreover, as detected in red
pepper (Konarska, 2010), Al also decreased width of leaves. In our work leaf number in
both rye genotypes was also reduced (data not shown).
Aluminium also decreased RWC in both rye genotypes, but in ‘Riodeva’ was persistent at
the 3rd week in some Al-treated plants. This water loss may be related to the increase of E
observed in ‘Riodeva’. Water deficit is one of the most commonly effects of metal stress,
including Al (Tamás et al., 2006; Tewari et al., 2008; Silva al., 2010). Some reports
suggested that Al binding to cell wall affected its water permeability and uptake of ions
(Horst, 1995) and these modifications can imitate drought stress (Tamás et al., 2006).
Thus, Al-induced drought stress may be a consequence of both high E and reduced water
uptake. So, the accentuated decrease in biomass should be regarded as a complex endpoint,
and besides water deficit, some authors proposed that nutritional imbalances may lead to
biomass decrease in Al exposed plants (Lidon al., 2000). Here we also support in Altreated rye plants that photosynthetic imbalances may be another cause for growth
decrease.
In general, during the first two weeks, Al does not induce severe effects on rye
photosynthesis, supporting the general classification of this species in literature as
“tolerant”, which is based on parameters such as root growth (Yang et al., 2005) and, root
regrowth (Kim et al., 2001; Hede et al., 2002). In ‘D. Zlote’ Al-exposed plants, by the end
of the experiment, the decreases of both A increment and stomatal closure (E and gs
decreased) were not accompanied by decreases of Ci/Ca, which remained constant,
suggesting that non-stomatal limitations to photosynthesis may also occur. One of the
putative limitations is a decrease in the Calvin cycle enzyme activities, which however
seems improbable since no changes in the maximal RuBisCo and sFBPase activity were
163
Aluminium toxicity in wheat and rye
observed. Moreover, for this genotype, the reduction of qP values and the constant values
of ΦPSII and ETR suggested that the decrease of A may be due to closure of PSII reaction
centers, rather than an impairment of the electron transport chain (Lu et al., 2009). In
general, the decrease in A in ‘D. Zlote’ did not limit sugars content. Similar results were
reported in other species exposed to Al (Guo et al., 2007; Abdalla, 2008; Giannakoula et
al., 2008).
In D Zlote, some parameters showed different profiles at week two and three. In particular
the increases in F0 and qN indicate that ‘D. Zlote’ plants had effective protective
mechanisms against photo-oxidative damage. The increase of non-photochemical
dissipation of excitation energy was also detected in wheatgrass and in wheat exposed to
Al (Moustakas and Ouzounidou, 1994; Moustakas et al., 1996). After 3 weeks of Al
exposure, the recuperation of qN to values near the control ones and a stabilization of F0
indicated a tendency to maintain the balance between the level of light energy absorption
and light energy utilization.
The decrease of Chl a is a common response of Al stress and may traduce a sensitivity to
Al-toxicity. A reduction in Chl a content (but not of Chl a/Chl b) observed in ‘D. Zlote’,
revealed elevated susceptibility to higher Al concentrations at 3 weeks exposure. In
agreement with our results, Chl content also decreased in Al exposed plants, as citrus
(Chen et al., 2005 a), soybean (Zhang et al., 2007), lentils (Azmat and Hasan, 2008), tea
plant (Yadav and Mohanpuria, 2009). Moreover, in blueberry, Reyes-Diaz et al. (2010)
concluded that long-term Al exposure led to significant changes in photosynthetic pigment
contents. These pigments reduction may be a consequence of nutritional imbalances (e.g.
Mg), ROS production and/or stimulation of chlorophyllase activity caused by Al exposure
(Drazkiewicz and Baszynski, 2010), and inhibition of Chl synthesis (Pereira et al., 2006).
The A reduction in ‘Riodeva’ plants exposed to 1.11 mM was accompanied by Ci/Ca
increase (increase of E and gs), contrary to the observed in ‘D. Zlote’. In contrast, at the
highest Al concentration, Ci/Ca values were constant, while A and RuBisCo activity
decreased. In ‘Riodeva’, RuBisCo activity alterations may be related to the reduced A.
Little is known about the effects of Al-toxicity in RuBisCo activity and content, and the
few reports available reported that RuBisCo activity was not affected by Al exposure
(Chen et al., 2005 b; Jiang et al., 2008), which is contradicted by this study. Similar to the
observed in ‘D. Zlote’ and ‘Riodeva’, Al-induced reduction of A was detected in soya
(Zhang et al., 2007), coffee (Pereira et al., 2000) and wheatgrass (Moustakas et al., 1996),
164
Aluminium toxicity in wheat and rye
suggesting that A could be regarded as a putatively interesting biomarker for Al toxicity in
plants in general.
Decreases in Fv/Fm are described for many abiotic stresses, including Al stress (Peixoto et
al., 2002; Lu et al., 2009; Drazkiewicz and Baszynski, 2010; Reyes-Diaz et al., 2010; Unal
et al., 2010). This parameter only decreased in ‘Riodeva’ but always with values above 0.8,
a standard value for healthy plants (Schreiber et al., 1995), so hampering any conclusion
on Al toxicity.
Sugars are the principal end products of photosynthesis. They are used to maintain leaf cell
turgor, act as nutrient and metabolite signaling molecules (Couée et al., 2006; Rolland et
al., 2006). A global analysis of our data show that, in ‘Riodeva’, the increase of glucose
contents is not correlated with high A, but seems to be a result of starch degradation, most
probably within a strategy of supplying plants soluble sugar needs, when photosynthesis
performance is affected.
Contrarily to the wide literature on Al effects in sugar contents in roots, less information is
available on the effects of Al on leaf sugars contents, (e.g. Guo et al., 2007; Abdalla, 2008;
Azmat and Hasan, 2008; Giannakoula et al., 2008). Under Al stress, sugars may be
particularly important to callose formation. Al-induced callose formation was reported for
many species and its accumulation occurs mostly in Al-sensitive genotypes (Bhuja et al.,
2004; Hirano et al., 2004; Tahara et al., 2005). The higher increase of glucose content in
‘Riodeva’ leaves, comparatively to ‘D. Zlote’, may be related to a higher callose
biosynthesis.
From this work it is evident that despite ‘Riodeva’ is described as Al-sensitive and ‘D.
Zlote’ an Al-tolerant genotype, both presented photosynthetic impairment and similar
decreases in leaf growth. Our results suggest that the main differences between genotypes
were: a) in general, Al induced earlier damages in ‘Riodeva’, but, both genotypes showed
high Al long-term susceptibility; b) ‘D. Zlote’ showed a high capacity to regulate its hydric
status; c) in ‘Riodeva’ both photochemical and biochemical processes were affected, while
in ‘D. Zlote’ plants only the photochemical processes was impaired.
In conclusion, the results indicate that ‘Riodeva’ is less Al-tolerant (as reported Gallego
and Benito, 1997) and, that ‘D. Zlote’ tolerance degree seems time dependent. Further
studies on the ability of this genotype to recover in longer exposures should be developed.
Taking in consideration the results as a whole, the photosynthetic parameters (e.g. A,
RuBisCo activity) proved to be a good tool to monitor Al-sensitivity and the
165
Aluminium toxicity in wheat and rye
implementation of long-term exposure experiments are crucial to understand the tolerance
mechanism involved in Al exposure. Finally we recommend further research in developing
a toolbox of best markers that, together with other parameters as root regrowth and callose
deposition may help to clarify the concepts of “Al-tolerant” and Al-sensitivity”.
Acknowledgements
FCT/MCT supported this work (POCI /AGR/ 58174/2004), S. Silva (FCT⁄BD⁄ 32257⁄2006) and
Celeste Dias (SFRH/BPD/41700/2007) grants. Thanks are due to Armando Costa for technical
support.
166
Aluminium toxicity in wheat and rye
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172
Aluminium toxicity in wheat and rye
CHAPTER IV
CONCLUDING REMARKS
173
Aluminium toxicity in wheat and rye
174
Aluminium toxicity in wheat and rye
Concluding Remarks
This PhD Thesis reports significant increments on the knowledge of Al-toxicity targets in
wheat and rye species. Emphasis was given to the comparative study of Al-tolerant and
sensitive genotypes in order to identify the mechanisms used to cope with Al-toxicity in
tolerant genotypes.
Short-term Al exposure
Al-induced effects after a short-term exposure depended on species, and on the genotype
within the species. The sensitive genotypes were more affected by Al and wheat is much
less tolerant to Al than rye. Al induced imbalances in different physiological aspects
(functional and metabolic).
Both species presented root growth inhibition, which was higher in the sensitive genotypes,
and only the tolerant ones recovered root growth after Al removal. The root nutritional
imbalances comprised more elements in wheat than in rye, but the behavior of some
nutrients was similar, mostly in sensitive genotypes: increase of Si and Ca and decrease of
K and Mg. The tolerant rye genotype maintained the nutritional levels closer to the control
ones, only P increased. The capacity to maintain nutritional balances seems to be an
important mechanism to cope with Al toxicity and to allow root growth recovers after Al
removal.
Endodermis differentiation pattern differed in treated and control plants, in both species.
Mostly in wheat, endoderm thickness seems to be, in addition to callose deposition, an
instrument to prevent Al entrance and it’s translocation to the shoots. Callose deposition
was detected in wheat and rye, but in rye, a similar distribution was observed in tolerant
and sensitive genotypes. After Al removal, in rye and in the tolerant wheat genotype,
callose accumulation decreased. These results showed that the inefficiency of callose
metabolism may jeopardise root growth under Al exposure.
In this work, it was reported that Al induced alterations in cell cycle patterning. Wheat and
rye tolerant genotypes presented an arrest in G0/G1 phase, whereas the sensitive genotypes
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Aluminium toxicity in wheat and rye
showed a tendency to arrest in the S phase. These data revealed that Al may induce arrest
of cells in specific cell cycle phases and that it depends on Al degree tolerance.
A similar behavior in both species was also observed in carbohydrate reserves. After Al
exposure, the sensitive genotypes presented starch content decrease. This suggests that the
highly tolerant genotypes had more effective mechanisms to prevent insufficient uptake of
sucrose and/or glucose.
Long-term Al exposure
In long-term treatments, we addressed some research areas less studied in rye, as AsAGSH cycle and carbon metabolism. These parameters proved to be a good tool to monitor
Al-tolerance, and long-term exposure showed to be crucial to evaluate “Alsensitivity/tolerance”.
Al exposure induced oxidative stress in rye (roots and leaves) and the antioxidant response
was organ, genotype and time dependent. Leaves and roots presented enhancement of SOD
and G-POX activity, but CAT and APX activity was organ dependent: CAT activity was
enhanced in leaves, whereas APX activity increased in roots. The enhancement of these
enzyme activities in roots was predominantly observed in ‘D. Zlote’, which may be related
to its higher Al-tolerance.
Concerning to AsA-GSH cycle, leaves showed earlier oxidation of AsA and GSH pools
than roots, and ‘D. Zlote’ was able to reverse AsA and GSH oxidation in leaves better than
‘Riodeva’. ‘D. Zlote’ roots presented higher AsA-GSH cycle response (increase in GR,
MDHAR, APX and perhaps DHAR activities, and increase in AsA contents) than
‘Riodeva’. However, at the end of the exposure, it presented higher oxidation of AsA and
GSH pools. AsA-GSH cycle response in ‘D. Zlote’ at the 2nd week, may be contributed to
the lower plant growth inhibition, while the oxidation of AsA and GSH pools at the 3rd
week may be related to the severe growth inhibition observed at that time exposure.
Furthermore, our results indicated that the ability of rye genotypes to survive in Al
environments may depend on the capacity of antioxidant system response.
Considering particularly leaf responses to Al-toxicity, several photosynthetic parameters
were analysed. Both genotypes presented photosynthetic impairment and similar decrease
in leaf growth. In ‘Riodeva’, both photochemical and biochemical processes were affected,
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Aluminium toxicity in wheat and rye
while in ‘D. Zlote’ plants only the photochemical processes were impaired: in ‘D. Zlote’, A
decrease was not accompanied by alterations in RuBisCo or sFBPase activity, as observed
in ‘Riodeva’. This study revealed that Al induced earlier damages in the ‘Riodeva’
genotype, but, both genotypes showed high Al long-term susceptibility.
Challenges for the future
Some physiological mechanisms used by plants to cope Al toxicity were already clarified
in the last decades. Moreover, it is clear that these Al-resistance mechanisms are regulated
by several genes. However, the knowledge is still rudimentary in most physiological and
molecular aspects of the Al-resistance mechanisms.
In this Thesis, we demonstrate that within the same species, different genotypes respond
differently in many parameters when plants are exposed to Al. We propose that some of
these responses contribute to higher Al-tolerance demonstrated by some genotypes. This
raises two interesting topics of further research:
a) Considering the high intra and interspecific variability found in Triticum and Secale
species, and considering the limited number of genotypes studied, it would be
interesting to extend the study to other genotypes/cultivars. This would provide
information confirming if the reported behaviors are genotype specific or can be
extrapolated to species or even plants in general.
b) It points to a possible differentiated gene regulation at different levels such as
oxidative stress, cell cycle checkpoints regulation and endodermis/callose. So, in
future research, deeper attention should be given to the genes controlling these
events/responses.
Furthermore, in the last chapter of this Thesis we demonstrate the importance of carrying
out studies for long term Al-exposure, an issue poorly studied in literature. Long-term
exposures are useful to clarify some important remaining questions in what concerns Al
toxicity:
a) Long-term Al exposure experiments are crucial as field toxicity of Al may be
imposed to the plant through its all life cycle. These long-term studies would
complement the majority of studies that have been conducted in young
seedlings for short exposure times.
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Aluminium toxicity in wheat and rye
b) Long-term Al exposure experiments would also provide a more complete and
realistic analysis of the different mechanisms of resistance in longer period of
the plants’ life cycle.
c) Moreover, Al partition and organs responses would be better understood
combining both information provided by short and long-term Al exposures.
d) Also the combination of short and long term studies would complement a better
choice of a toolbox of markers for Al toxicity.
e) Long-term Al exposure would also contribute to a better knowledge of some
physiological aspects that are already relatively well described in short-term
assays. In particular, the root organic acid exudation occurs a short period after
the beginning of the stress, but its occurrence after longer periods still remains
unclear. The gene regulation related with this organic acid exudation, have been
studied in some of the genotypes used here with promising results, and some
genes were already identified (eg., ALMT1, MATE). Elucidating genes that are
involved in organic acid exudation and their regulation would provide valuable
information on resistance mechanisms developed by the genotypes.
f) Finally, long-term experiments will open perspective for extrapolating the
experiments and data to “real scenarios”, i.e. to field experiments, namely in the
North region of Portugal, where the genotypes studied are well adapted.
As conclusion, the information given in this Thesis may be used as orientation in further
research covering several fields from e.g., physiology to molecular biology to study the Alresistance mechanisms in these two crops and eventually expand research to other
genotypes and species.
178
Errata
Silva, Sónia. 2011. Toxicidade do Alumínio em trigo e centeio. Tese de doutoramento em biologia.
Departamento de Biologia, Universidade de Aveiro, Aveiro, 2011.
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Abbreviations
Rye Al sensitive of Barbela line
Al-sensitive line of Montalegre population
Abbreviations
Rye Al tolerant of Barbela line
Al-tolerant line of Montalegre population
Abbreviations
Arabidopsis
Arabidopsis
Abbreviations
Mmultidrug and toxic compound
Multidrug and toxic compound
Abbreviations
CHM: Nonspecific protein
CHM: Cycloheximide - Nonspecific protein
18
principly
mainly
18
Al(OH)3
Al(OH)3
19
productionn
production
20
World
world
42
Triticum aestivum
Triticum aestivum
159 - Table 2
ΦPSIIR
ΦPSII