linking physiology, biochemistry and anatomy of c4...

LINKING PHYSIOLOGY,

BIOCHEMISTRY AND ANATOMY

OF C4 PHOTOSYNTHESIS

Susanne von Caemmerer

Australian National University

Carbon isotope discrimination as a tool:

• Rubisco fractionation

• C3-C4 cycle coordination

• CO2 diffusion

• Temperature dependence of C3 mesophyll

conductance

• Temperature dependence of C4 leakiness and

bundle sheath conductance

• Dry matter carbon isotope composition

C4 photosynthesis

Carbon isotope discrimination ()

• 12CO2 98.9% of atmospheric 13CO2 1.1%

• C3 photosynthesis discriminates against 13CO2 because

• 13CO2 diffuses more slowly

• Rubisco prefers 12CO2 (29‰)

• This discrimination is less during C4 photosynthesis

• Rubisco’s potential to fractionate is less and depends on bundle sheath resistance and C3/C4 cycle coordination

• first biochemical fractionation is hydration and PEPC (-5.7 ‰)

∆‰ =𝐶13

𝐶12𝑎𝑖𝑟

𝐶13

𝐶12𝑝𝑙𝑎𝑛𝑡 − 1

Carbon isotope discrimination () during

C3 photosynthesis

0.0 0.2 0.4 0.6 0.8 1.00

5

10

15

20

25

30

high light

low light

Carb

on isoto

pe d

iscrim

ination

(

o/ o

o)

Ratio of intercellular to ambient CO2

Ci/C

a

Tobacco

Evans et al. 1994, AJPP

Rubisco

increasing CO2 gradient with in the leaf

CO2

CO2

Stomatal

conductance

Mesophyll

conductance

Bowling et al. (2003) Agricultural and Forrest Meterology 118, 1-19

Measurements of carbon isotope discrimination ()

using Tunable diode laser (TDL) spectroscopy.

Measurements of carbon isotope discrimination ()

using Tunable diode laser (TDL) spectroscopy.

John Evans

Rubisco discrimination factor

• Diversity of Rubisco fractionation factor has been observed • (Rhodospirillum rubrum, cyanobacteria, higher plants)

• Is there variation amongst C3 species and differences between C3 and C4 Rubisco’s?

• Difficult in vitro measurements (McNevin et al. 2007)

• Can we make in vivo measurements exploiting transplastomically modified Rubiscos?

Transplastomic tobacco expressing

chimeric Rubisco’s

• Tob( Wt)

• Tob(bid)

• Lsu of F. bidentis (C4)

• Tob(flo)

• Lsu of F. floridana (C3-C4)

• Tob (Rr)

• R. rubrum

• Tob(L335V)

Whitney et al. (2011) PNAS 108, 14668

0

10

20

30

tob(Wt)

tob(bid)

tob(flo)

CO

2 a

ssim

ilatio

n r

ate

(m

ol m

-2 s

-1)

2%O2

0 200 400 600 800

10

20

30

tob(Wt)

tob(Rr)

tob(L335V)

Intercellular CO2 (bar)

Rubisco discrimination factors

0

10

20

30 tob(Wt)

tob(bid)

tob(flo)

b=29

(

o/ o

o)

0.0 0.2 0.4 0.6 0.8 1.00

10

20

tob(Rr)

tob(L335V)

b=23.8

b=13.9

Ratio of intercellular to ambient CO2

Ci/C

a

(

o/ o

o)

In vivo In vitro

tob(Wt) C3 29 28.5±0.7

tob(bid) C4 27.8±0.8

Tob(flo) C3-C4 28.6±0.6

Tob(Rr) 23.8±0.7

23.3±2.1

Tob(L335V) 13.9±0.7

12.3±1.6

Assuming mesophyll conductance, gm of Wt

Rubisco discrimination factors

• In vivo measurements confirmed in vitro

measurements for

• Rhodospirillum rubrum & L335V Rubisco

• In vivo Rubisco fractionation of chimeric Rubisco

with Lsu from F. bidentis and F. floridana are the

same as tobacco wild type Rubisco.

Carbon isotope discrimination as a tools

for measuring C3-C4 cycle coordination

CO2

CO2

Mesophyll cell Bundle sheath cell

C3

C4 Rubisco

PEPC

CH2O

Leakiness (): Bundle sheath leak rate/rate of CO2 supply from C4

cycle

(Farquhar 1983)

Carbon isotope discrimination as a tools

for measuring C3-C4 cycle coordination

CO2

CO2

Mesophyll cell Bundle sheath cell

C3

C4 Rubisco

PEPC

CH2O

Leakiness ()= Leak rate/CO2 supply rate

0.0 0.2 0.4 0.6 0.8 1.0

-5

0

5

10

15

20

25

30

C3

(

0/ 0

0)

Ci/C

a

C4 = 0.3

0.2

0

(Farquhar 1983) CAM: Griffiths et al. 2007 Plant Physiol

Transgenic Flaveria bidentis

anti-NADP malic enzyme (NME)

anti-Rubisco

CO2

CO2

Mesophyll cell Bundle sheath cell

C3

C4 Rubisco

PEPC

CH2O

Pengelly et al. (2012) Plant Physiol 160, 1070

-2

0

2

4

6

(

o/ o

o)

0 200 400 600 800

0.1

0.2

0.3

0.4

anti-NME

Intercellular CO2 (bar)

Le

akin

ess,

anti-Rubisco

Wild type

0 200 400 600 8000

10

20

30

40

anti-NME

Wild type

anti-SSu

Intercellular CO2 (bar)

CO

2 a

ssim

ilation r

ate

(m

olm

-2s

-1)

Pengelly et al. (2012) Plant Physiol 160, 1070

Comparison of anti-Rubisco and anti-NADP-ME

Flaveria transgenics

Calculating C4 cycle and leak rate

0

10

20

30

40

50

CO

2 a

ssim

ilation r

ate

(m

ol m

-2 s

-1)

10

20

30

40

C4 c

ycle

rate

(m

ol m

-2 s

-1)

0 200 400 600 8000

10

20

30

40

50 anti NME

Wt(NME)

anti Rubisco

Wt(anti Rubisco)

Bundle

sheath

leak r

ate

(m

ol m

-2 s

-1 )

Intercellular CO2 (bar)

Bundle sheath leak rate:

L=gbs(Cs-Cm)

Pengelly et al. (2012) Plant Physiol 160, 1070

Leakiness a measure of coordination of

C4 photosynthesis

• Leakiness is a useful measure of C3 / C4 cycle coordination

• Calculate C4 cycle and leak rate

• Calculation of bundle sheath CO2 requires knowledge of

bundle sheath conductance

• Leakiness is constant over a wide range of light and CO2

• Leakiness is similar between C4 species ( measured at high

light)

• (Henderson et al. 1992, Cousins et al. 2008)

Temperature dependence of bundle

sheath conductance

Temperature response of mesophyll

conductance (gm) in C3 species

• Mesophyll conductance

chloroplast surface area

per unit leaf (von Caemmerer and Evans 1991)

• CO2 permeability of cell

wall, plasmalemma,

cytosol and chloroplast

envelope

CO2

CO2

Stomatal conductance

Mesophyll conductance

Temperature response of mesophyll

conductance (gm)

10 15 20 25 30 35 40 450

10

20

30

Leaf temperature ( oC)

CO

2 a

ssim

ilation r

ate

(m

ol m

-2 s

-1)

380 bar CO2, 21 % O

2

1500 mol quanta m-2 s

-1,

Tobacco

Evans and von Caemmerer PC&E (2013)

10 20 30 400.0

0.2

0.4

0.6

0.8

1.0

Me

so

ph

yll

co

nd

ucta

nce

(m

ol m

-2 s

-1 b

ar-1

)

Leaf temperature (oC)

gm (@25 oC)=0.48±0.02 mol m-2 s-1 bar-1

15 20 25 30 35 400

10

20

30

40

50

Leaf temperature (oC)

Rice

Tobacco

Cotton

Soybean

Arabidopsis

Wheat

Ra

te o

f C

O2 a

ssim

ilatio

n

(m

ol m

-2 s

-1)

Diverse temperature responses of gm in

crop species

15 20 25 30 35 400.0

0.2

0.4

0.6

0.8

1.0

1.2 Rice

Tobacco

Cotton

Soybean

Arabidopsis

Wheat

Meso

ph

yll

co

nd

ucta

nce

,

gm (

mo

l m

-2 s

-1 b

ar-1

)

Leaf temperature (oC)

Modelling temperature dependence of

mesophyll conductance

10 20 30 400

1

2

3

10 20 30 400.0

0.5

1.0

gm

gmem

gliq

gm

em g

m (

mol m

-2 s

-1 b

ar-1

)

Temperature (oC)

gliq

tobacco

wheat

Eucalyptus

gm (

mol m

-2 s

-1 b

ar-1

)

Evans et al.: poster at St Louis

Role of aquaporin and carbonic anhydrase

Arabidopsis CA activity

(mol m-2 s-1 bar-1)

Wild type 1.60

PIP1,2 CO2

aquaporin

CA1 Chloroplast 0.39

CA2 cytosol 2.24

CA3 cytosol 2.24

10 20 30 400

5

10

15

20

CO

2 a

ssim

ilatio

n r

ate

(

mo

l m

-2 s

-1)

Leaf Temperature (oC)

wild type

PIP1,2

CA1

CA2

CA3

Role of aquaporin and carbonic anhydrase

10 20 30 400.0

0.1

0.2

0.3

Meso

ph

yll

co

nd

ucta

nce (

mol m

-2 s

-1b

ar-1

)

Leaf Temperature (oC)

wild type

PIP1,2

CA1

CA2

CA3

10 20 30 400

20

40

60

80

100

120

140

Ci-C

c(m

ba

r)

Leaf Temperature (oC)

wild type

PIP1,2

CA1

CA2

CA3

A/gm=Ci-Cc

Summary

• Diversity of temperature response of gm

• Balance between membrane and liquid phase

• Aquaporins, cytosolic and chloroplast CA

do not appear to affect gm in Arabidopsis

• Perhaps Arabidopsis not the best model

species for these experiments.

CO2

CO2

Temperature dependence of bundle

sheath conductance

Kiirats et al. Plant Physiol 2002;130:964-976

Amaranthus edulis PEPCmutant

Is there species diversity in the

temperature response of bundle sheath

conductance?

Amaranthus edulis

Chloris gyana

Zea mays

Species diversity in temperature dependence of

Leakiness ()

20 30 40 500

10

20

30

40

50

C. ciliaris

A. lappaca

P. dilitatum

S. bicolor

CO

2 a

ssim

ilation r

ate

(

mol m

-2 s

-1)

Leaf Temperature (oC)

20 30 40 500.0

0.1

0.2

0.3

0.4

0.5

C.ciliaris

A.lappaca

S. bicolor

P. dilitatum

Le

akin

ess,

Leaf Temperature (oC)

Dry matter carbon isotope

composition in diverse C4 species

Average annual rainfall (mm)

Less than 50

50-100

100-200

200-400

400-600

600-800

800-1000

1000-1200

Greater than 1200

Hattersley and Watson (1992) Diversification of photosynthesis. In

Chapman GP , Grass evolution and

domestication. Cambridge University Press

Dry matter differs between NADP-ME and NAD-ME

C4 grasses

Hattersley (1982)

plantair

Henderson et al. (1992); Ghannoum et al. (2002); Cousins et al. (2008)

Online measurements

0.0 0.2 0.4 0.6 0.8 1.00

2

4

6

Ci/C

a

on

line

( o

/ oo )

= 0.21

Species NADP NAD PCK Suberin

Monocots

S. bicolor Z. mays

U. panicoides

C. gayana

P. schinzii

E. corocana

Dicots A. rosea A. edulis F. trineriva F. bidentis G. globosa

Henderson et al (1992)

Cousins et al. (2008)

Comparison between online and dry matter

measurements

0 2 4 60

2

4

6

Dry

matte

r

(o/ o

o)

online (o/oo

)

Species NADP NAD PCK Suberin

Monocots

S. bicolor Z. mays

U. panicoides

C. gayana

P. schinzii

E. corocana

Dicots A. rosea A. edulis F. trineriva F. bidentis G. globosa

Henderson et al (1992)

Cousins et al. (2008)

NAD- ME species have a more negative dry

matter compared to NADP-ME

Why?

Summary

• Rubisco fractionation: similar between tobacco and chimeric

Flaveria Rubiscos.

• and leakiness provide information on C4 metabolic regulation.

• Species diversity in temperature response of C3 mesophyll

conductance, gm.

• CA and aquaporin don’t appear to influence gm in Arabidopsis

• Temperature dependence of C4 bundle-sheath?

• Species diversity in temperature response of leakiness

E:biosphereisotopesworkshop@gmal.com

Acknowledgement:

Spencer Whitney

Soumi Bala John Evans

Jasper Pengelly Australian Research Council