Event / Evento: II Workshop on Sugarcane Physiology for Agronomic Applications Speaker / Palestrante: Frederick C. Botha (Sugar Research Australia) Date / Data: Oct, 29-30th 2013 / 29 e 30 de outubro de 2013 Place / Local: CTBE/CNPEM Campus, Campinas, Brazil Event Website / Website do evento: www.bioetanol.org.br/sugarcanephysiology

1. Enter title here for Powerpoint
July 1 2013
The biomass, fibre and sucrose dilemma in realising
the agronomic potential of sugarcane
Extra details Botha
here
Frikkie
29 October 2013

2. Outline
• Background
–
–
–
–
•
•
•
•
The ‘Sugarcane Triangle’
Biomass composition of the culm
Genetic composition and selection pressure
Supply and demand in sugarcane
Is sugarcane the ideal biomass crop?
Carbon partitioning in the culm and seedling
What do we know about control?
Conclusions

3. Sugarcane Triangle
‘The Sugarcane Triangle, is the relationship between biomass,
fibre and sucrose. Many believe that Devil is at play here and
therefore call the area also as Devil's Triangle.
The facts however are quite far from what is generally said or
believed to be true. There are many publications, stories and
myths created through sheer imagination. True to say that in
some cases, conclusions got blurred’.

4. Yield improvement in sugarcane
110
A
B
14
100
12
90
10
TSH
70
8
60
6
50
4
40
2
30
1920
TCH/TSH
TCH
80
1940
1960
YEAR
1980
2000
0
1920
9
C
7
5
1920
1940
1945
1960
YEAR
1970
YEAR
1980
1995
2000

5. Components of the sugarcane stalk (commercial varieties)
Sugarcane
Dry matter
(30%)
Water
(70%)
• Fibre plus sucrose ~30%. When this goes much above 30% it is
non-plant matter or poor cane!!
• High fibre plus high sucrose- impossible
• Breeders and cropping systems always try to balance ratio of
fibre:sucrose
• Very complex physiological processes controlling this ratio
Fibre and sucrose make up 95% of the dry matter in the culm. The
remaining dry matter is probably crucial for survival and cannot be
used to enhance sugar content

6. The two main progenitor species of “sugarcane”
Saccharum officinarum
Yield
Vigour
Tillering
Canes
Roots
Sucrose
Fibre
Abiotic
Biotic
Saccharum spontaneum
High
Moderate
Poor
Thick
Shallow
High (sweet canes)
Low
Susceptible to frost, drought, salt
Susceptible to most disease and
insects
Poor yielding
Very good
Heavy
Thin
Deep
Low
High
Resistant to frost, drought, salt
Resistant to most disease and
insects

7. The Sugarcane Cell Wall (Fibre)
• The cell wall of sugarcane comprises cellulose (28%), hemicellulose
(58%), and pectin (8%)
• Type II walls which means that glucuronoarabinoxylans (GAX) is the
major cellulose/crosslinking glycan (CLG)
• The ratio between these different chemical components of fibre
depends upon multiple factors, including:
o
o
o
o
o
genotype,
climate conditions,
location and rate of growth,
amount and type of fertilizers used on the crop
physical and chemical composition of the soil
o Once the secondary wall is formed no further expansion growth
is possible

8. Sink and Source relationship
• Solute passage through plasmodesmata is passive.
Therefore, symplastic transport cannot, by itself,
establish a solute concentration gradient!
• Experimental manipulation of source/sink ratios
generally indicates that meristematic sinks are
source limited, whereas cell expansion and storage
sinks are sink limited(Smith and Stitt, 2007).

9. Biomass accumulation
CO2 + E
R1
Biomass production
R2
(CH2O)n
R1 > R2 = Biomass accumulation
R1 = photosynthesis
R2 = respiration
Plants respire approximately one-half of their fixed photosynthate in
providing energy and precursors for biochemical processes. Respiration us
therefore a significant drain on the carbon available for partitioning into
storage. Sugarcane ????
The energy and reducing equivalents produced during these steps serve as
vital co-mediators in a multitude of other chemical reactions necessary for
normal cell function.
Significant carbon losses occur during over-maturation and post-harvest
respiration of mature harvested cane (up to 10% of harvested sucrose)
Sucrolysis in the sugarcane culm is key for identify strategies and targets
for traditional breeding or genetic engineering to develop more desirable
attributes in sugarcane

10. Biomass partitioning
CO2
R4
(CH2O)nx
R3
(CH2O)n + E
R5
R6
CO2
(R3-R4):(R5-R6) = Biomass partitioning
(CH2O)ny
Biomass partitioning
Sucrolysis is sugarcane generally poorly studied. Probably would differ significantly from other
species (symport off loading and very high sucrose levels)
The sucrose storing capacity of sugarcane is characterised by pronounced substrate cycles,
sometimes called futile cycles because they involve both the continuous synthesis and
degradation of sucrose and the recycling of metabolic intermediates between the pools of hexose
phosphates and triose phosphates in the cytosol

11. Energy cane vs sugarcane
80
Tonnes DW/ha
70
60
Sugarcane
Energycane
50
40
30
20
10
0
Sucrose
Fiber
Total
Fernando Reinach: Canavialis Brazil

12. Sink strength/priority drives carbon partitioning
R1>R2
R1
Source
(supply)
R2
Sink 1
(culm)
Sink 2
(roots)
R2>R1

13. Supply and demand
CO2
Supply
Demand
SUCROSE
Demand
P
SUCROSE
H2O
Nutrients
X
Supply

14. Name
Plant group
Sugarcane
varieties
Net assimilation
rate µmol m-2 s-1
29-61
40 Australian varieties
8 Japanese varieties
N14
NiF4
Lahaina and H varieties
CP73-1547
Q138, Q183
6 Brazilian varieties
Other Species
Chitton,Pindar, HQ409
16-54
25-44
46*
34.3
45-51*
31
30.5,35.5
41.3-60.7
Saccharum sinense
Saccharum robustum
Saccharum spontaneum
Sorghum bicolor
Zea mays
C4 plants
C3 Crop Plants
Reference
Bull 1969
Irvine 1967, 1975
Nose & Nakama 1990
Allison et al. 1997
Du et al.1999a
Meinzer & Zhu 1998
Vu et al. 2006
Inman-Bamber et al. 2008
Galon et al. 2009
45.8
Meinzer & Zhu 1998
49.2*
33.4-48.2
42.5
52.4
30-70
20-40
Meinzer & Zhu 1998
Nose et al. 1994
Ziska & Bunce 1997
Ziska & Bunce 1997
Larcher 2003
Larcher 2003

15. Nitrogen use efficiency should be a key focus in sugarcane
• In maize, maximum photosynthetic rates (~57 mol m 2 s 1)
are observed at a leaf N of 80mmolm 2 , whereas sugarcane
requires about 125 mmol m 2 to exhibit the same peak A
value.
• The reason for the PNUE differences between sugarcane and
maize are unclear
• If sugarcane could be bred to have similar PNUE as maize,
then A could be increased about 25% at a leaf N of 80 mmol
m 2
• The key to high photosynthetic performance in sugarcane,
therefore, is to maintain a high leaf N status or increase the
PNUE.
Maintaining a high leaf N status is a major problem because it
promote growth over sugar accumulation and thus reduce crop
quality (‘Energy cane’ production)

16. Percentage allocation of mobilised carbon from the internode to the
developing shoot, roots and respiration. Values are the mean of three
replicates ± SE.
Dark
Time (days) Shoot
Roots
0
0
0
7
43.2 ± 1.4 32.0 ± 3.3
14
45.3 ± 1.5 12.3 ± 2.5
21
38.3 ± 1.3 13.3 ± 2.1
Dark/Light
Respiration Shoot
Roots Respiration
0
0
0
0
24.8 ± 2.4 43.2 ± 1.4 32.0 ± 3.3 24.8 ± 2.4
44.4 ± 2.8 41.6 ± 1.1 14.8 ± 2.8 43.7 ± 5.8
48.4 ± 5.3 47.8 ± 1.8 17.7 ± 2.1 34.5 ± 3.3

17. 0
R² = 0.9849
4000
Time (min)
2000
0
2
4
Time (min)
6
8
% Label
0
HCl
120
100
80
60
40
20
0
Sucrose
Glu/Fru
A+O
Insol
90
NaH214CO3
•
•
•
•
•
180
6000
150
8000
90
10000
120
12000
60
% Label
CO2 uptake
100
90
80
70
60
50
40
30
20
10
0
30
C- Pulse feeding
Uptake (Bq)
14
Time (h)
180
Labeling done on leaf 6
Uptake of CO2 was linear during the first 5 min of labelling (R2 0.98)
Fixation rate was 45 µmol C/m2/s.
Label is rapidly mobilised from the leaf
All the label is exported as sucrose

18. Sink strength
-2
0
+1
Labelled leaf
[Sucrose]
+4
+7
+8
+11
[Sucrose]
Sink strength = 1 > 3 > 4= 0 > 7 > 8 = -1 > 11 > -2 > -3

19. Carbon partitioning
0
50
100
0
50
100
0
50
100
6 weeks
Fibre
6 hours
R1
Respiration
R2
-3
Sugar
3
R3
0
200
400
600
Carbon distribution (Bq)
Sucrose
30%
Label lost
25%
3
0
-3
20%
% Carbon distribution
15%
10%
5%
0%
0
10
20
Time (days)
30
40

20. Carbon partitioning
42%
50%
8%
Respiration
R1
Sucrose
R2
R3
-3
30%
Sugar
3
30%
40%
5%
20%
Fibre
75%

21. Cellular partitioning
HP to TP
• The dominating metabolic flux is
sucrose synthesis, sucrose
breakdown, Hex-P and TP cycling
Metabolic modelling indicate that:
• CIN and Hexokinases have the
largest flux control coefficients
• Vacuolar loading would have a
large positive influence
• Reloading of the phloem would
be important
Sucrose Synthesis
TP to HP
Respiration
Hexokinase
0
2
4
6
8
10
Internode 7
Internode 9
Internode 3
CO2 release
Fibre Synthesis
Starch Synthesis

22. sucrose
sucrose
APOPLAST
CWI
CYTOSOL
PPi
PFP
G1P
G6P
F6P
PFK
Pi
F1,6P2
sucrose cycling
triose-hexose phosphate
cycling
fructos glucose
e
UTP
UGPase
PPi
SPS
UDPGlc
sucrose-6-P
sucrose
SUSY
NI
DHAP
3-PGA
fructose
VACUOLE
MITOCHONDRIA
glucose
fructose glucose
AI
sucrose
TCA cycle and respiration (CO2 production)
sucrose

23. SuSy (Synthesis : Breakdown)
The SPS/SuSy story
2.5
2
1.5
1
The contributions by SPS
and SuSy to synthesis
0.5
0
3
5
7
Internode #
9
Internode
14C-Glc/ 14C-Frc
Calculated
enzyme ratio
SPS/SuSy
3
5
8
15
2.2
1.5
1.1
1.0
0.9
2.5
>20
SPS only

24. Hexokinase activities
•
•
•
•
Rapid mobilisation of glucose and fructose
At least 5 hexokinase like activities with fructokinase
dominating
The role of FRK2 in sugarcane metabolism is not clear.
The only way that this enzyme could play a meaningful
part in fructose phosphorylation was if the fructose
concentration was less than 0.2 mM (even in young
internodes the concentration exceeds this limit by more
than 100 times.
Is this enzyme involved in sugar signalling?

25. Impact of reduced PFP activity
FLUX (nmol min-1 mg protein-1)
Suc to fruc
0.85
1.56
100.43
254.87
14.56
13.22
4.37
1.52
0.70
0.13
90.12
42.3
9.56
5.67
901.33
456.11
88.99
50.87
1.68
0.92
0.42
1.28
OPU506
4
2
0
Internode 6
600
Hexose concentration
( mol g-1 DW)
WT
Internode 3
Triose-P to Hex-P
9.98
13.40
Triose-P cycling
6
Gluc to Suc
500
*
*
400
*
300
503
*
Q3
200
100
0
WT
TC
501
504
505
506
Genotype
507
508
400
350
300
250
200
150
100
50
0
4000
3500
3000
2500
2000
1500
1000
500
0
Q4
*
*
502
*
7000
6000
5000
4000
3000
*
2000
1000
0
WT
TC
501
502
503
504
505
506
Genotype
507
508
Q3
Q4
Sucrose concentration
( mol g-1 DW)
WT
Internode 3+4
Internode 6+7
OPu506
Internode 3+4
Internode 6+7
Carbon cycling

26. Reducing neutral invertase activity
3.0
2.5
Flux into sucrose
nmol/ min/mg protein
Maturing Internode
Young Internode
80
NCo310
U1
U2
2.0
1.5
1.0
0.5
40
0.0
0.30
Young Int
Maturing Int
Young Int
Maturing Int
20
0.25
0
Neutral Invertase SuSy
•
•
•
•
Acid Inv
CW Inv
Neutral Invertase SuSy
Acid Inv
Recovery of CIN – GM clones problematic
Increase in sucrose content
30% reduction in biomass accumulation
50% reduction in bud germination
CW Inv
Flux into glucose
nmol/ min/mg protein
nmol/ min/mg protein
60
NCo310
U1
U2
0.20
0.15
0.10
0.05
0.00

27. Conversion of vacuolar
sucrose
Internode
3 9 12 3 9 12 3 9 12 3 9 12 3 9 12
Frucrose
Sucrose
Kestose
Kestotetraose
detector response]
1-2-6-12
700
Polymer
Clone 2
K2 = 1,1 -
2
200
K3 = 1,1,1 -
Clone 1
control
600
500
nmol/gram
3
400
-50
1-2-5-1
Total Sugars
600
1
1-2-3-5
Clone
Sucrose
DP3
NCO 310
800
1-2-2-4
Kestopentaose
1'000
nC
NCo310
2153
2121
400
2
300
200
100
0
3
6
9
12
Internode
13
16

28. The Sugarcane story
ST
Vac
kestose
PP
Suc
Suc/
H2O
Suc
Suc
H
H
H-P
T-P
Respiration
H2O
S
P
Fibre
• Maintaining a sucrose gradient
crucial for biomass production
• Sucrose concentration in the
culm between 0.5 and 0.9 M.
• Two major carbon cycles occur
even in mature internodes
• CIN plays an important role in
sucrose hydrolysis
• What is the signalling and
control pathways (FK)?
• Rapid labelling of Suc and
much slower for kestose; slow
loading or no loading?
• Fibre and respirqtion the
dominant demands in young
tissue

29. The sugarcane
CO
story
Tops
2
Sucrose
H
Sucrose storage
Leaf
• Under high input conditions
biomass accumulation is driven by
the solar radiation
• A constant radiation use efficiency
is not achieved throughout the crop
cycle (reduced growth phenomenon
(RGP)).
• Lower photosynthetic capacity
because of leaf nitrogen limitations
and poor PNUE
• Sucrose feedback control by the
sink tissues
• Increased respiration
• Active growth under especially under
limited water and nutrient supply reduce
availability of C for sucrose storage = high
fibre:sucrose
• Reduces available carbon for stalk and root
growth
H
Sucrose
Fibre
Stalk
Respiration
Roots
• Initial growth phase has a limited time
window and water stress or limited sunlight
will reduce internode growth.
• Mild stress conditions increases sucrose (high
sucrose :fibre).
• Vigorous growth (high nitrogen levels enough
water) will achieve the opposite (high
fibre:sucrose).
• Sucrose accumulation can suppress
photosynthesis (lower yield, vigour
ratoonability)

30. Conclusions
Sugarcane is one of the world’s most productive crops and its exceptional
ability to produce biomass makes it very attractive in a biomass-dependent
economy.
Surprisingly, the reported photosynthetic capacities of sugarcane are low
relative to other typical C4 species and frequently are equivalent to that of
C3 crops.
Several factors contribute to this phenomenon including lower
photosynthetic capacity because of leaf nitrogen limitations and feedback
control by the sink tissues that accumulate exceptionally high sugar levels.
The distribution of carbon between sucrose and fibre in the stalk is not
constant. In young actively growing tissue the majority of carbon is
allocated to fibre and energy production for growth. However, a
redirection of carbon to sucrose occurs during internode maturation.
Several potential control mechanisms have been studied abut no clear
picture is evident
An early switch to sucrose storage has a negative impact on biomass yield.
Key targets for further improvement of sugarcane should be improving
photosynthetic nitrogen use efficiency, or altering sink-source partitioning
of carbon and nitrogen.

32. http://www.wiley.com/WileyCDA/WileyTitle/productCd-0813821215.html
CONTENTS
1. Sugarcane: The Crop, the Plant, and Domestication
2. Anatomy and Morphology
3. Developmental Stages (Phenology)
4. Ripening and Postharvest Deterioration
5. Mineral Nutrition of Sugarcane
6. Photosynthesis in Sugarcane
7. Respiration as a Competitive Sink for Sucrose
Accumulation in Sugarcane Culm: Perspectives and Open
Questions
8. Nitrogen Physiology of Sugarcane
9. Water Relations and Cell Expansion of Storage Tissue
10. Water, Transpiration, and Gas Exchange
11. Transport Proteins in Plant Growth and Development
12. Phloem Transport of Resources
13. Cell Walls: Structure and Biogenesis
14. Hormones and Growth Regulators
15. Flowering
16. Stress Physiology: Abiotic Stresses
17. Mechanisms of Resistance to Pests and Pathogens in
Sugarcane and Related Crop Species
18. Source and Sink Physiology
19. Biomass and Bioenergy
20. Crop Models
21. Sugarcane Yields and Yield-Limiting Processes
22. Systems Biology and Metabolic Modeling
23. Sugarcane Genetics and Genomics
24. Sugarcane Biotechnology: Axenic Culture, Gene
Transfer, and Transgene Expression