Biochemical networks related to
carbon accumulation in sugarcane
Marcos Buckeridge
Department of Botany,
Laboratory of Plant Physiological Ecology
IB USP
How are we approaching the
sugarcane metabolic network?
Types of experiments
1) Growing sugarcane under elevated CO2
2) Applying inhibitors of secondary metabolism to change
the cell wall
3) Studying the role of gibberellin in sugarcane seedling
growth and development
Technical approaches
1) Photosynthesis and Growth
2) Carbohydrate metabolism (sucrose and cell walls)
3) Gene expression
4) Cell Biology
5) Metabolic profiling towards a system approach
Growing sugarcane under
elevated CO2
N Assimilation and
micorrizal association
Starch, leaf area, biomass, root growth,
stress tolerance, defence and fertility
-
+
Plant
Water and
carbohydrate status
in leaves
Photosynthetic protein
-
+
+
e- transport
Transpiration
C assimilation
+
Conductance
Leaf
Respiration
-
PEPc & RUBISCO
ABI4
MITOCHONDRIA
NUCLEUS
Cell
STOMATA
Photosynthesis
Carbonic anhydrase
and RUBISCO
Buckeridge et al. 2008. Biologia & Mudanças Climáticas no Brasil. Rima Editora, São Carlos
Sugarcane leaves performe C4 photosynthesis
CO2
Mesophyll cells
CO2
Bundle Sheath
CO2
CO2
Débora Leite, Wanderley dos Santos, Amanda Souza & Marcos Buckeridge – Res. não publicados
De Souza et al. 2008
Plant Cell & Environment,
Volume 31, pg 1116
30% less
transpiration
60% higher
WUE
Productivity
Ambient
Ambient
Elevated
Elevated
2005
1 year in elevated CO2
50% more
Biomass
Microarray analysis of the CO2 experiments
3 months
Categories
Gene description
Ratio
(elevated/am
bient)
Development
light-induced protein
1,194
Photosynthesis
photosystem II protein K; psbK
1,315
Photosynthesis
Ferredoxin I; chloroplast precursor
1,26
Photosynthesis
photosystem I reaction centre subunit n,
chloroplast precursor
1,583
Cell wall metabolism
xyloglucan endotransglycosylase/hydrolase
2,582
Photosynthesis
Chlorophyll A-B binding protein
1,508
Stress response
ASR-like
1,735
Lipid, fatty-acid and
isoprenoid metabolism
AE9 stearoyl-ACP desaturase
3,59
Carbohydrate metabolism
beta-glucosidase isozyme 2 precursor
-2,189
Carbohydrate metabolism
putative glucose-6-phosphate
dehydrogenase
-1,232
Protein metabolism
translational initiation factor eIF-4A
-1,606
De Souza et al. (2008) Plant Cell & Environment, Volume 31, pg 1116
50
Elevated CO2
62,82%
CO2 Ambient
40
A umol m-2 s-1
30
20
10
0
0
500
1000
-10
1500
2000
2500
3000
PAR
250
Elevated CO2
CO2 Ambient
200
43,52%
CO2 assimilation and
electron transport rate (ETR)
of sugarcane under elevated
CO2.
A=CO2 assimilation
ETR
150
100
50
0
0
500
1000
1500
PAR
2000
Amanda Pereira de Souza, PhD thesis
2500
3000
5 months
Protein metabolism
large ribosomal protein 2
1,454
Carbohydr. metabolism/Photosynthesis
phosphoenolpyruvate carboxylase
1,245
Cell wall metabolism
Alpha-L-arabinofuranosidase
1,37
Protein metabolism
cathepsin B-like cysteine protease
1,349
Development
dormancy-associated protein
2,299
Transporters
Sugar transporter
1,252
Receptors
serine/threonine-protein kinase NAK
1,706
unknow
1,271
Protein metabolism
putative glutamate-tRNA ligase
1,504
Protein metabolism
cathepsin B-like cysteine protease
1,395
Protein metabolism
Aldo/keto reductase; Sigma-54 factor
1,229
putative nucleostemin (GTPase of unknown function)
1,397
Development
putative auxin-independent growth promoter
1,909
Transcription
pre-mRNA splicing factor
1,541
Nucleic acid metabolism
chromodomain-helicase-DNA-binding protein
0,17
Stress response
dehydrin
-1,42
unknow
-1,398
Secondary metabolism
caffeoyl-CoA 3-O-methyltransferase 1
-1,354
Carbohydrate metabolism
cell wall invertase
-1,615
Stress response
ferritin
-2,768
Protein metabolism
C2 domain-containing protein-like
-1,342
Pathogenicity
Thaumatin
-1,367
Transcription
auxin response factor 2
-1,352
Development
Lateral organ boudaries protein
-1,494
Cell cycle
kelch repeat-containing F-box family protein
-1,541
Cell cycle
cyclin H-1
-1,632
Leaves of sugarcane stained with
iodine
Débora Leite, Wanderley dos Santos, Amanda Souza & Marcos Buckeridge – Res. não publicados
Sugarcane culm
No starch is present in culms
Leite, Wanderley dos Santos, Amanda Souza & Marcos Buckeridge – Res. não publicados
Sugarcane Cell Wall
(Glucurono?)
and mixed linkabe beta-glucan
200
Oxg Xylogluan-ArabinoXylan #10
nC
1 - 3,783
180
Cana 0,5U 24H
HPAEC
Arabinoxylan
160
ECD_1
140
140
125
120
113
Oxg Xylogluan-ArabinoXylan #42
nC
Lichen. hem icel
ECD_1
11 - 16,175
2 - 5,908
100
100
80
88
8 - 20,092
75
60
40
63
3 - 9,200
5 - 13,017
7 - 16,608
6 - 15,142
20
9 - 21,092
11 - 22,025
12 - 29,200
50
13 - 18,442
10 - 21,492
4 - 11,542
38
0
25
-20
0,0
10,0
20,0
30,0
40,0
13
Augusto Crivellari & Marcos
Buckeridge – Res. não publicados
HPAEC
Beta-glucan
m in
55,0
3 - 3,958
14 - 20,308
4 - 5,367
1 - 2,667
6 - 8,192
8 - 912,517
2 - 3,433
5 - 5,992 7 - 10,058
- 14,592
-10
13,700
12 - 17,34215 - 21,908
0
m in
-20
0,0
10,0
20,0
30,0
40,0
55,0
Glucuronic acid
Sugarcane parenchyma
autofluorescence
dos Santos et al. 2009
Free ferulic acid
Free diferulic acid
Ferulic acid residue
esterified to an
arabinofuranose residue
Ferulic acid bridge
between two
polysaccharides
Cross-linked
Glucuronoarabinoxylans
dos Santos et al. 2008
Polysaccharides
Feruloyl
residues
Feruloyl
residues
dos Santos et al. 2009
Limit digest AX sugarcane
oligosaccharides
Elevated CO2
Ambient CO2
At 5 months, alpha
arabinofuranoside gene
was differencially
expressed (+37). This is
consistent with a
decrease in
arabinosylated
oligosaccharides
Amanda, Souza, Augusto Crivellari & Marcos Buckeridge – Res. não publicados
Applying inhibitors of secondary
metabolism to change the cell wall
Phenylpropanoids
Pathway
dos Santos et al. Functional Plant Science and Biotechnology, 2008
Light microscopy with polarization
Control
MDCA
PIP
10X
20X
A, B, C = 10×; D, E, F = 20×; A, D = control, B, E = MDCA 500 uM; C, F = PIP 100 uM
dos Santos & Buckeridge, res. não publicados
Xylanase activity on bagasse of treated culms
Xylanase activity
(mg free sugar min-1)
C u l m ( b a sa l p a r t )
2
1. 8
1. 6
1. 4
1. 2
1
0. 8
0. 6
0. 4
0. 2
0
c ont r ol e
[ 80]
[ 100]
[ 120]
T r a t me nt s ( M D C A uM )
dos Santos & Buckeridge, res. não publicados
Studying the role of gibberellin in
sugarcane seedling growth and
development
Andrea Brandão, PhD thesis
2d
Andrea Brandão, PhD thesis
4d
6d
7d
Biomass incorporation in sugarcane plants over
time of the presence of gibberellic acid ( M)
Total Plant Weight (mg)
5
a
4
b
bc
3
c
d
d
2
d
d
e
Control
Ga33 M
1
e
0
1
2
3
4
5
treatment time (days)
Andrea Brandão, PhD thesis
6
7
Sugarcane cell wall and elongation
control
Andrea Brandão, PhD thesis
3uM
30 uM
60 uM
0,5 cm
control
Vacuoles in meristematic cells of
sugarcane seedlings 6 days old
Andrea Brandão, PhD thesis
GA
Effect of GA3 on sucrose content of sugarcane seedlings
Andrea Brandão, PhD thesis
Expression of 100 genes of sugarcane by Real Time PCR
Hormones
Transcription
Factors
C metabolism
Cell Cycle
Cell Wall
Transcription Network
Hormones
Vacuole
Cell Cycle
Transcription
Network
Mitochnodria
Transcription
factors
Sucrose
SPP
INV
Sucrose-P
SPS
Glc & Fru
HK
UDP-GPP
UDPGlc
Cell
Wall
GROWTH
STORAGE
cytoplasm
Hexose-P
ADP-GPP
Triose-P
Photosynthesis
Starch
ADP-Glc
CO2
SUGARCANE
Chloroplast
cytoplasm
Sucrose
INV
Sucrose-P
SPS
Glc & Fru
HK
UDP-GPP
UDPGlc
Cell
Wall
GROWTH
SPP
ADP-GPP
Triose-P
Photosynthesis
Starch
ADP-Glc
CO2
STORAGE
Hexose-P
SUGARCANE
Chloroplast
Elevated CO2
What have we got up to now
We “attacked” three nodes within the metabolic network
1) C pathway - elevated CO2 changes photosynthesis and
carbohydrate metabolism and this can be seen through
gene expression and cell wall changes
2) Secondary metabolism - Inhibition of ferulic acid
production, induces changes in cell walls that are
apparently useful for applications in second generation
bioethanol
3) Hormone signalling - Gibberellin is one of the controllers
of carbohydrate metabolism, increasing cell expansion
(related gene expression) and vacuole formation
Now wee need the metaolomics to advance intro a systems approach so that we
can change carbon allocation and increase productivity for sugar, biofuel and
biorefinary products
Thank You
[email protected]
Students: Amanda P. de Souza, Andréa Brandão,
Augusto Crivellari, Débora Leite, Wanderley Dantas
dos Santos
Collaborators:
Gregório Ceccantini, Gilberto Kerbauy (IB-USP)
Gaucia Souza (IQ-USP)
http://msbuckeridge.wordpress.com
% of shoots in the seedlings of sugarcane
Control
3 M GA
7
61,25±0,03 c
83,33±0,17 b
% in relation
to control
+36
14
60,78±0,07 c
87,84±0,17 b
+44
21
50,17±0,17 c
82,22±0,44 b
+64
28
53,97±0,09 b
83,93±0,6 a
+55
Days