Enter title here for Powerpoint July 1 2013

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Enter title here for Powerpoint July 1 2013

Extra details here

The biomass, fibre and sucrose dilemma in realising the agronomic potential of sugarcane

Frikkie Botha 29 October 2013

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

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’.

Yield improvement in sugarcane

30

40

50

60

70

80

90

100

110

1920 1940 1960 1980 2000

TCH

YEAR

A

0

2

4

6

8

10

12

14

1920 1940 1960 1980 2000TS

H YEAR

B

5

7

9

1920 1945 1970 1995

TCH

/TSH

YEAR

C

Sugarcane

Dry matter (30%)

Water (70%)

Components of the sugarcane stalk (commercial varieties)

• 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

Saccharum officinarum

Saccharum spontaneum

Yield High Poor yielding Vigour Moderate Very good Tillering Poor Heavy Canes Thick Thin Roots Shallow Deep Sucrose High (sweet canes) Low Fibre Low High Abiotic Susceptible to frost, drought, salt Resistant to frost, drought, salt

Biotic Susceptible to most disease and insects

Resistant to most disease and insects

The two main progenitor species of “sugarcane”

• 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 genotype, o climate conditions, o location and rate of growth, o amount and type of fertilizers used on the crop o physical and chemical composition of the soil

o Once the secondary wall is formed no further expansion growth is possible

The Sugarcane Cell Wall (Fibre)

• 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).

Sink and Source relationship

CO2 + E (CH2O)n R1

R2

Biomass production R1 > R2 = Biomass accumulation

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

(CH2O)nx R3

R5

Biomass partitioning

+ E (CH2O)n

(CH2O)ny

R4

R6

(R3-R4):(R5-R6) = Biomass partitioning

CO2

CO2

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

Energy cane vs sugarcane

0

10

20

30

40

50

60

70

80

Sucrose Fiber Total

To

nn

es D

W/h

a

Sugarcane

Energycane

Fernando Reinach: Canavialis Brazil

Sink strength/priority drives carbon partitioning

Source (supply)

Sink 1 (culm)

Sink 2 (roots)

R1

R2

R1>R2

R2>R1

Supply

Demand

SUCROSE

SUCROSE

CO2

Supply

Demand

H2O Nutrients

P

X

Supply and demand

Plant group

Name Net assimilation rate µmol m-2 s-1

Reference

Sugarcane varieties

Chitton,Pindar, HQ409 29-61 Bull 1969

40 Australian varieties 16-54 Irvine 1967, 1975 8 Japanese varieties 25-44 Nose & Nakama 1990 N14 46* Allison et al. 1997 NiF4 34.3 Du et al.1999a Lahaina and H varieties 45-51* Meinzer & Zhu 1998 CP73-1547 31 Vu et al. 2006 Q138, Q183 30.5,35.5 Inman-Bamber et al. 2008 6 Brazilian varieties 41.3-60.7 Galon et al. 2009

Other Species Saccharum sinense 45.8 Meinzer & Zhu 1998

Saccharum robustum 49.2* Meinzer & Zhu 1998 Saccharum spontaneum 33.4-48.2 Nose et al. 1994 Sorghum bicolor 42.5 Ziska & Bunce 1997 Zea mays 52.4 Ziska & Bunce 1997 C4 plants 30-70 Larcher 2003 C3 Crop Plants 20-40 Larcher 2003

• In maize, maximum photosynthetic rates (~57 mol mí2 sí1) are observed at a leaf N of 80mmolmí2 , whereas sugarcane requires about 125 mmol mí2 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 mí2

• The key to high photosynthetic performance in sugarcane, therefore, is to maintain a high leaf N status or increase the PNUE.

Nitrogen use efficiency should be a key focus in sugarcane

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)

Percentage allocation of mobilised carbon from the internode to the developing shoot, roots and respiration. Values are the mean of three replicates ± SE.

Time (days) Shoot Roots Respiration Shoot Roots Respiration0 0 0 0 0 0 07 43.2 ± 1.4 32.0 ± 3.3 24.8 ± 2.4 43.2 ± 1.4 32.0 ± 3.3 24.8 ± 2.4 14 45.3 ± 1.5 12.3 ± 2.5 44.4 ± 2.8 41.6 ± 1.1 14.8 ± 2.8 43.7 ± 5.821 38.3 ± 1.3 13.3 ± 2.1 48.4 ± 5.3 47.8 ± 1.8 17.7 ± 2.1 34.5 ± 3.3

Dark Dark/Light

HCl

NaH214CO3

• 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.

R² = 0.9849

0

2000

4000

6000

8000

10000

12000

0 2 4 6 8

Upta

ke (B

q)

Time (min)

CO2 uptake

14 C- Pulse feeding

0102030405060708090100

0 30 60 90 120150

180

% L

abel

Time (min)

020406080

100120

90 180

% L

abel

Time (h)

Sucrose

Glu/FruA+OInsol

• Label is rapidly mobilised from the leaf • All the label is exported as sucrose

0

-2

Labelled leaf+1

+4

+7

+8

+11

00

-2

Labelled leaf+1

+4

+7

+8

+11

Sink strength = 1 > 3 > 4= 0 > 7 > 8 = -1 > 11 > -2 > -3

Sink strength

[Sucrose]

[Sucrose]

-3

3 Sugar Respiration

Sucrose

Fibre

R1

R2

R3 0 200 400 600

Carbon distribution (Bq)

6 weeks

6 hours

0 50 100

% Carbon distribution

0 50 100

0 50 100

0%

5%

10%

15%

20%

25%

30%

0 10 20 30 40

Labe

l los

t

Time (days)

30-3

Carbon partitioning

-3

3

Sugar Sucrose

Fibre

Respiration

R1

R2

R3

42%

8%

50%

30%

40%

30%

20%

75%

5%

Carbon partitioning

Hexokinase

Sucrose Synthesis

HP to TP

TP to HP

Respiration

Fibre Synthesis

Starch Synthesis

CO2 release

0 2 4 6 8 10

Internode 7 Internode 9 Internode 3

Cellular partitioning

• 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

trio

se-h

exos

e ph

osph

ate

cycl

ing

glucose

sucrose-6-P

VACUOLE

sucrose

fructose glucose

fructose

AI

F6P

F1,6P2

DHAP 3-PGA

TCA cycle and respiration (CO2 production)

glucose

G6P

UDPGlc SPS

sucrose

G1P

NI

SUSY

PFK PFP

sucr

ose

cycl

ing

CYTOSOL

Pi

PPi

UTP

PPi UGPase

fructose

sucrose

sucrose APOPLAST

CWI

MITOCHONDRIA

sucrose

0

0.5

1

1.5

2

2.5

3 5 7 9

SuSy

(Sy

nthe

sis :

Bre

akdo

wn)

Internode # Internode 14C-Glc/14C-Frc

Calculated enzyme ratio

SPS/SuSy

3 2.2 0.9 5 1.5 2.5 8 1.1 >20

15 1.0 SPS only

The SPS/SuSy story

The contributions by SPS and SuSy to synthesis

• 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?

Hexokinase activities

FLUX (nmol min-1 mg protein-1) Carbon cycling Suc to fruc Gluc to Suc Triose-P to Hex-P WT r r Internode 3+4 9.98 r 0.85 100.43 r 14.56 4.37 r 0.70 Internode 6+7 13.40 r 1.56 254.87 r 13.22 1.52 r 0.13 OPu506 Internode 3+4 90.12 r 9.56 901.33 r 88.99 1.68 r 0.42 Internode 6+7 42.3 r 5.67 456.11 r 50.87 0.92 r 1.28

0

2

4

6

Internode 3 Internode 6

WT OPU506

Triose-P cycling

Genotype He

xose

conc

entr

atio

n (P

mol

g-1

DW

)

Genotype

Sucr

ose

conc

entr

atio

n (P

mol

g-1

DW

)

0 50

100 150 200 250 300 350 400

WT TC 501 502 503 504 505 506 507 508 Q3 Q4 0 1000 2000 3000 4000 5000 6000 7000

*

0

100

200

300

400

500

600

WT TC 501 502 503 504 505 506 507 508 Q3 Q4 0 500 1000 1500 2000 2500 3000 3500 4000

* * *

*

* *

*

Impact of reduced PFP activity

Young Internode

Neutral Invertase SuSy Acid Inv CW Inv

nmol

/ min

/mg

prot

ein

0

20

40

60

80

NCo310 U1 U2

Maturing Internode

Neutral Invertase SuSy Acid Inv CW Inv

Young Int Maturing Int

Flux

into

suc

rose

nmol

/ min

/mg

prot

ein

0.0

0.5

1.0

1.5

2.0

2.5

3.0

NCo310 U1 U2

Young Int Maturing Int

Flux

into

glu

cose

nmol

/ min

/mg

prot

ein

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Reducing neutral invertase activity

• Recovery of CIN – GM clones problematic • Increase in sucrose content • 30% reduction in biomass accumulation • 50% reduction in bud germination

-50

200

400

600

800

1'000 nC

3

2

1

dete

ctor

resp

onse

]

DP3 Sucrose

Polymer

control

Clone 1

Clone 2 K2 = 1,1 -

K3 = 1,1,1 -

2

Kestotetraose

Kestopentaose

Sucrose

Kestose

Frucrose

_3 9 12_ _3 9 12_ _3 9 12_ _ 3 9 12_ _3 9 12_ Internode

Clon

e

1-2-

2-4

1-2-

3-5

1-2-

5-1

NCO

310

1-2-

6-12

Total Sugars

0

100

200

300

400

500

600

700

3 6 9 12 13 16Internode

nmol

/gra

m

NCo310

2153

2121

Conversion of vacuolar sucrose

H

Fibre

H

PP

ST

SP H2O Suc/

H2O

Vac

Suc Suc

H-P

T-P

Suc

Respiration

The Sugarcane story

• 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

kestose

H Fibre Sucrose

Sucrose storage

Respiration

H Sucrose

CO2

Tops

Roots

Leaf Stalk • 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

• 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)

The sugarcane story

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.

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