Revista Brasileira de Fisiologia Vegetal, 10(2):97-100, 1998
COMMUNICATION
EFFECT OF NaCl-SALINITY ON THE EXPRESSION OF A
COTYLEDONARY α-AMYLASE FROM Vigna unguiculata1
Osmundo Brilhante de Oliveira-Neto2, Adriana Teixeira Damasceno3, Francisco de Assis
de Paiva Campos4, Enéas Gomes-Filho5, Joaquim Enéas-Filho6 and JoséTarquinio Prisco7
Laboratório de Fisiologia Vegetal, Departamento de Bioquímica e Biologia Molecular,
Universidade Federal do Ceará, Cx. Postal 1065, Fortaleza, CE, 60001-970, Brasil.
ABSTRACT - Pitiuba cowpea (Vigna unguiculata (L.) Walp.) seeds were sown in water (control) and
in 100 mM NaCl solutions, and cotyledonary α-amylase activities during seed germination and seedling
establishment in both the control and salt treatment were followed. A very low enzyme activity was
observed in the control at day 2 from planting, which increased rapidly after day 3, and reached its
maximum at day 5 after planting. The development of enzyme activity in the salt treatment was delayed
in relation to the control treatment. It was practically null up to the 3rd day, increased slightly at day 5 and
then started to increase rapidly to reach its maximal activity at the 7th day from planting. A purified
cotyledonary α-amylase was isolated from 5-day-old seedlings of the control treatment and monospecific antibodies against the purified enzyme were prepared. The antibodies were used to follow the
appearance of α-amylase during seed germination and seedling establishment under control and salt
stressed conditions. Immunoreactive proteins were detected initially at the 3rd day from planting, and
their concentration increased up to the end of the experimental period in the control treatment. In the
salt treatment they were detected only at day 5, and also increased up to the end of the experimental
period. These results correlate with the ones in which the development of α-amylase activity was followed
during seed germination and seedling establishment both under normal (control) and salt stressed
conditions. They suggest that this enzyme is de novo synthesized during seed germination and seedling
establishment and that salt stress delayed its synthesis.
Additional index terms: cotyledon starch mobilization; cowpeas; salt stress; seed germination and
seedling establishment.
EFEITO DA SALINIDADE-NaCl NA EXPRESSÃO
DE UMA α-AMYLASE COTILEDONÁRIA DE Vigna unguiculata
RESUMO - Sementes de feijão-de-corda (Vigna unguiculata (L.) Walp.) Pitiúba foram semeadas em
água (controle) e em soluções de NaCl a 100 mM e foram seguidas as atividades α-amilásicas
cotiledonárias ao longo da germinação e do estabelecimento da plântula. No dia 2 após o plantio,
observou-se uma atividade enzimática muito baixa no controle, a qual aumentou rapidamente depois
do dia 3 e atingiu seu máximo no dia 5. O desenvolvimento da atividade enzimática no tratamento
salino foi retardado em relação ao do controle. Foi praticamente nula até o 3º dia, aumentou ligeiramente no dia 5, e então começou a aumentar rapidamente, atingindo sua atividade máxima no 7º dia após
o plantio. Isolou-se, do tratamento controle, uma α-amilase, purificada a partir de cotilédones de plântulas
com 5 dias de idade e foram preparados anticorpos monoespecíficos contra a enzima purificada. Os
anticorpos foram usados para acompanhar o aparecimento da α-amilase, sob condições normais de
germinação e de estabelecimento da plântula e sob condições de estresse salino. No tratamento controle, as proteinas imunorreativas somente começaram a ser detectadas no 3º dia após o plantio, e
suas concentrações aumentaram até o final do período experimental. No tratamento salino elas só
foram detectadas no 5º dia, e também aumentaram até o final do período experimental. Estes resultados se correlacionam com os obtidos quando se estudou o desenvolvimento da atividade α-amilásica
ao longo da germinação e do estabelecimento da plântula, tanto sob condições normais (controle)
como de estresse salino. Eles sugerem que esta enzima é sintetisada de novo ao longo da germinação
e do estabelecimento da plântula e que o estresse salino retarda sua síntese.
Termos adicionais de indexação: estresse salino; feijão-de-corda; germinação e estabelecimento da
plântula; mobilização de amido nos cotilédones.
1
Recebido em 11/11/1997 e aceito em 23/06/1998.
Eng. Agron., M.S., Bolsista DCR/CNPq.
3 Estudante de Farmácia, Bolsista IC/CNPq.
4 Biólogo, M.S., Ph.D., Prof. Adjunto II, Pesq./CNPq
5 Eng. Quim., M.S., Dr., Prof. Adjunto IV, Pesq./CNPq.
6 Biólogo, M.S., Dr. 3ème Cycle, Prof. Adjunto IV, Pesq./CNPq.
7 Eng. Agron., M.S., Ph.D., Prof. Emérito, Pesq./CNPqCorresponding author.
2
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Oliveira-Neto et al.
98
INTRODUCTION
Cowpea (Vigna unguiculata) seeds contain 60%
starch in their cotyledons (Wien & Summerfield, 1984)
and this carbohydrate is the major reserve for germination
and seedling establishment in this species (Prisco et al.,
1981). During seed germination and seedling
establishment of both legumes and cereals carbohydrate
reser ves are degraded by amylases, starch
phosphorylases and glucosidases (Bewley & Black,
1994), and most of their hydrolysis products are then
transported to the embryo-axis to supply carbon and
energy for its growth (Juliano & Varner, 1969; Okamoto
& Akazawa, 1979; Koshiba & Minamikawa, 1981;
Jacobsen & Higgins, 1982; Sun & Henson, 1990, 1991).
When seeds are sown in saline environment the rate
and percentage of germination are decreased (Uhvits,
1946; Prisco & O’Leary, 1970). Even though NaCl salinity
inhibits seed germination, seedling growth is more
affected by salt stress than the former (Prisco, 1987).
This greater sensitivity during seedling growth is due
mainly to salinity effects on seed reserve mobilization
(Prisco & Vieira, 1976; Gomes Filho & Prisco, 1978;
Prisco et al., 1981; Gomes Filho et al., 1983). The delayed
mobilization could be due to delayed hydrolase activation
(solubilization), delayed de novo synthesis of these
enzymes or to inhibition of translocation of the hydrolysis
products from the reserve organs to the embryo-axis
(Prisco, 1987). Although de novo synthesis of
cotyledonary α-amylases from Vigna radiata (Morohashi
et al., 1989) and V. mungo (Koshiba & Minamikawa,
1983; Taneyama et al., 1995) has been demonstrated,
very little is known about the patterns of synthesis and
mobilization of the α-amylase isolated from V. unguiculata
(Bastos et al ., 1994). It is not known whether it is
synthesised de novo or whether it comes from a precursor which is rendered enzymatically active during the
germination process. In addition to this it is not known
how NaCl salinity interferes with this pattern. These
questions were addressed by determining the pattern of
α-amylase activity during seed germination and seedling
establishment under normal and salt stressed conditions,
as well as following the pattern of appearance of the
α-amylase protein in the same extracts that were
used for determining α-amylase activity.
MATERIAL AND METHODS
Plant material and seed germination conditions
Cowpea (Vigna unguiculata (L.) Walp.) seeds were
obtained from the Experimental Farm of the Universidade Federal do Ceará, Pentecoste, Ceará, Brazil. Seeds
were surface sterilised with 2.5% NaOCl for 5 minutes,
rinsed with sterile water, and sown in paper towels wetted
with distilled water or 100 mM NaCl solutions. The
germination conditions were the same as described by
Prisco & Vieira (1976).
Enzyme extraction and purification
The extraction and purification of α-amylase followed
the procedure of Bastos et al. (1994). Cotyledons from
5-day-old seedlings were frozen in liquid nitrogen, freeze
dried and ground into a powder. The crude enzyme
extract was obtained by suspending the freeze dried
powder in 0.1 M TRIS-HCl, pH 7.4, containing 5.0 mM
CaCl2 and 10 mM 2-mercaptoethanol, and stirring this
mixture for 2 h at room temperature. The ratio of freeze
dried powder to extraction buffer was 1:10 (m/v). The
slurry was filtered through cheese-cloth and centrifuged
at 10,000 g for 30 min at 4°C. The supernatant was
fractionated in ammonium sulphate. Most of the αamylase activity was present in the fraction that
precipitated with ammonium sulphate in the range 3060%. This fraction was dissolved in 50 mL sodium acetate
buffer, pH 5.4, containing 1.0 mM CaCl2 and 10.0 mM 2mercaptoethanol, and applied to an affinity column
made of Sepharose 6B-β-ciclodextrin (15 x 130 mm),
prepared according to Silvanovich and Hill (1976) and
equilibrated with the same buffer. The α-amylase bound
to the column was eluted by washing it with the same
buffer containing β-ciclodextrin (10 g L -1). The tubes
containing α-amylase activity were pooled, dialysed
against 10 mM TRIS-HCl buffer, pH 8.0, containing 1.0
mM CaCl2 and applied on a DEAE-Cellulose column (12
x 97 mm) equilibrated with the same buffer. The bound
α-amylase was eluted by washing the column with the
same buffer containing 0.6 M NaCl. The purity of the αamylase preparation was assessed by SDS-PAGE
(Laemmli, 1970) using a 5-20% linear gradient and 1.0
mm-thick gels.
Antibody preparation and immunoblotting analysis
Antibody against α-amylase was prepared according
to Harlow & Lane (1988) by injecting the purified protein
(α-amylase) subcutaneously in a 4 months old New
Zealand female rabbit. The material used for injection was
derived from the ion-exchange purification step. One
hundred µg of the purified enzyme were applied to a SDSPAGE slab gel. After staining with coomassie brilliant
blue R-250 the α-amylase band was cut out of the gel,
sonicated in a 1.3 mL Eppendorf tube containing 500 µL
of water. After sonication, 1.0 mL of Freund’s complete
adjuvant was added and, after emulsification, injected
into the rabbit. This operation was repeated three times,
at 15 day intervals. The IgG fraction was purified from
the serum by affinity chromatography in a Protein-A
Sepharose column (5 x 20 mm) according to Harlow &
Lane (1988). After PAGE-SDS electrophoresis of βmercaptoethanol treated samples, proteins were
transferred to a nitrocellulose membrane using a TE
Series Transphor Electrophoresis Unit (Hoefer Scientific
Instruments, San Francisco, CA, USA) as described by
Towbin et al. (1979), probed against polyclonal antibodies
raised against the protein and detected with alkaline
phosphatase-conjugated anti-goat immunoglobulins
antibodies.
α-Amylase activity and protein determination
α-Amylase activity was measured according to
Koshiba & Minamikawa (1981), and expressed as units
of activity (UA), being one UA defined as the amount of
enzyme required to produce 1 µmol of reducing sugar
R. Bras. Fisiol. Veg., 10(2):97-100, 1998.
Effect of NaCl-Salinity
99
per minute (Bastos et al., 1994). Protein determination
was according to Bradford (1976), using bovine serum
albumin as standard.
RESULTS AND DISCUSSION
The development of cotyledonary activity during seed
germination and seedling establishment in both control
and salt treatment is shown in Fig. 1. Cotyledons from
dry quiescent seeds (day 0) and cotyledons excised at
day 1 from both control and salt treatment did not show
any α-amylase activity. A very low enzyme activity
appeared in the control at day 2, increased rapidly after
day 3, and reached a maximum at day 5 from planting.
The development of cotyledonary α-amylase activity in
the salt treatment was delayed in relation to the control
treatment. It was practically null up to the 3rd day,
increased slightly at day 5 and then started to increase
rapidly to reach its maximal activity at the 7th day from
planting. The same general pattern of α-amylase activity
development during seed germination and seedling
establishment in both control and salt treatment were
observed previously (Prisco et al., 1981). While the
number of seeds with emerged radicles reached its
maximum at day 2 in the control and at day 4 in the salt
treatment (data not shown), the greatest increase in
cotyledonary α-amylase activity occurred after radicle
emergence in both treatments. This suggests that starch
mobilization in this species is a post germination event,
that is, it is necessary for seedling growth and
establishment (Bewley & Black, 1994).
The enzyme extracted and purified from cotyledons
excised from seedlings of the control treatment on the
5th day from planting (Fig. 2A) had the same molecular
mass as the cotyledonary α-amylase described
previously (Bastos et al., 1994). Early attempts to use
the purified enzyme to obtain antibodies against this
protein failed to produce mono-specific antibodies.
However, when the α-amylase band that was cut out
from the stained gel after running the purified enzyme
FIGURE 1- Cotyledonary α-amylase activity during seed
germination and seedling establishment. Seeds sown in water
(o) and in 100 mM NaCl solutions (o). The values represent the
mean enzyme activity of three different extracts run in triplicate;
see Material and Methods for definition of the units of enzyme
activity.
FIGURE 2 - Linear gradient (5-20%) SDS-PAGE of purified αamylase (A) and Western blot of α-amylase purified antibodies
tested against the enzyme crude extract (B). The numbers on
the left refer to molecular masses expressed in kDa; E = purified
α-amylase; M = molecular mass markers.
on SDS-PAGE was injected into the rabbit, the antibodies
produced were mono-specific. This specificity increased
by using the IgG fraction purified by affinity
chromatography instead of the total immunoglobulins
(Harlow & Lane, 1988). These antibodies had high titre
when tested against the purified cotyledonary α-amylase
from Vigna unguiculata (Fig. 2B).
These antibodies were then used to follow the
appearance of α-amylase during germination and
seedling establishment under control and salt stressed
conditions (Fig. 3). Immunoreactive proteins could be
FIGURE 3 - Western blot of purified α-amylase antibodies
against crude cotyledon extracts from control (A) and salt
stressed (B) seedlings at different stages during seed
germination and seedling establishment.
R. Bras. Fisiol. Veg., 10(2):97-100, 1998.
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Oliveira-Neto et al.
detected only after the 3rd day from planting, and their
concentration increased up to the end of the experimental period in the control treatment. In the salt treatment
the immunoreactive proteins were detected only at day
5, and also increased up to the end of the experimental
period. These results (Fig. 3) correlate well with the
development of α-amylase activity during along seed
germination and seedling establishment both under normal and salt stressed conditions (Fig. 1). Taken together
these data suggest that this Vigna unguiculata
cotyledonary α-amylase is de novo synthesized during
seed germination and seedling establishment as was
demonstrated for V. radiata (Koshiba & Minamikawa, 1983;
Morohashi et al., 1989) and V. mungo (Taneyama et al.,
1995). The data also demonstrate that salt stress delays
its synthesis.
ACKNOWLEDGEMENTS
To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Financiadora de Estudos
e Projetos (FINEP) for their financial support.
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