Planta (1989) 178:258-266

Planta

9 Springer-Verlag 1989

Some relationships between contents of photosynthetic intermediates and the rate of photosynthetic carbon assimilation in leaves of Zea mays L. Richard C. L e e g o o d 1 and Susanne yon Caemmerer 2 1 Research Institute for Photosynthesis and Department of Plant Sciences, University of Sheffield, Sheffield SI0 2TN, UK, and 2 Department of Environmental Biology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, A.C.T. 2601, Australia

Abstract. The relationship between the gas-exchange characteristics of attached leaves of Z e a m a y s L. and the contents of photosynthetic intermediates was examined at different intercellular partial pressure of C02 and at different irradiances at a constant intercellular partial pressure of CO2. (i) The behaviour of the pools of the C4-cycle intermediates, phosphoenolpyruvate and pyruvate, provides evidence for light regulation of their consumption. However, light regulation of phosphoenolpyruvate carboxylase does not influence the assimilation rate at limiting intercellular partial pressures of CO2. (ii) A close correlation between the pools of phosphoenolpyruvate and glycerate-3phosphate exists under many different flux conditions, consistent with the notion that the pools of C4 and Ca cycles are connected via the interconversion of glycerate-3-phosphate and phosphoenolpyruvate. (iii) The ratio of triose-phosphate to glycerate-3-phosphate is used as an indicator of the availability of ATP and NADPH. Changes of this ratio with CO2 and with irradiance are compared with results obtained in C3 leaves and indicate that the mechanism of regulation of carbon assimilation by light in leaves of C4 plants may differ from that in C3 plants. (iv) The behaviour of the ribulose-l,5-bisphosphate pool with CO2 and irradiance is contrasted with the behaviour of these pools measured in leaves of C3 plants. Key words: C4 plant - Photosynthesis (metabolites) - Zea

(gas exchange)

Introduction

Studies of pools of photosynthetic metabolites and of photosynthetic fluxes in intact leaves of C4 Abbreviations: Pi=intercellular CO2 pressure; RuBP=ribulose-l,5-bisphosphate; PEP = phosphoenolpyruvate; triose-P = triose phosphates; PGA = glycerate-3-phosphate

plants have contributed substantially to our understanding of the regulation of photosynthetic carbon assimilation in these plants. However, the importance of these regulatory mechanisms can only be appreciated if they are combined with measurements of fluxes and metabolites in intact leaves. For example, the behaviour of the pool of phosphoenolpyruvate (PEP) in leaves during steadystate photosynthesis (Usuda 1987a, b; Leegood and von Caemmerer 1988) and during light transients (Furbank and Leegood 1984; Doncaster et al. 1989) shows that PEP-carboxylase activity is modulated by light in vivo and complements in-vitro evidence that the properties of PEP carboxylase change following illumination of leaves (Doncaster and Leegood 1987; Nimmo etal. 1987). Similarly, in C3 plants changes in the pool of ribulose-l,5-bisphosphate (RuBP) with changing irradiance have revealed that the supply of RuBP alone does not regulate the activity of RuBP carboxylase (Perchorowicz etal. 1981; Badger et al. 1984; Dietz and Heber 1984; von Caemmerer and Edmonson 1986). Studies of metabolite pools in C4 plants have emphasised the role played by diffusion ofmetabolites between the mesophyll and the bundle-sheath (Leegood 1985; Stitt and Heldt 1985b; Leegood and von Caemmerer 1988) and a close relationship between the pools of C3- and C4-cycle intermediates has become evident (Furbank and Leegood 1984; Leegood and yon Caemmerer 1988). This is also crucial to the co-ordination of the C3 and C~ cycles, particularly through modulation of the activity of PEP carboxylase, which is regulated both by the amount of its substrate, PEP (Leegood and von Caemmerer 1988) and by the concentration of the product of the C3 cycle, triose phosphate (triose-P) (Doncaster and Leegood 1987). The aim of this study was to measure metabolite pools in leaves of Z e a m a y s , an NADP-malic enzyme-type C4 plant, in relation to changes in

R.C. Leegood and S. von Caemmerer: Gas exchange and metabolies in Zea mays flUX b r o u g h t a b o u t by varying the irradiance and CO2 concentration. In particular, the relationship between the pools o f the C4 intermediate, PEP, and the C3 intermediate, glycerate-3-phosphate (PGA), was examined. Changes in the p o o l size o f R u B P with changing CO2 have been used to interpret concentrations o f CO2 in the bundle sheath, and the b e h a v i o u r o f the t r i o s e - P / P G A ratio (a p r o b e o f the relationship between the supply of, and d e m a n d for, A T P and N A D P H ) is comp a r e d in the leaves o f C3 and C~ plants. Materials and methods Plants. Zea mays L. cv. XL81 (DeKalb Seeds, Queensland, Australia) was grown in 5-1 pots (one plant per pot) in sterilised soil in a glasshouse during April of 1986 and 1987 in Canberra at a daytime temperature of about 30~ C and a nighttime minimum of 15~ C. Plants were fertilized twice a week with a Hewitt nitrate-type formulation containing 12 mM nitrate (Hewitt and Smith 1975, pp. 31-72). Mature leaves were taken from fourweek-old plants for gas-exchange measurements. Gas exchange. Gas-exchange measurements were made on attached leaves as already described (Badger et al. 1984; Leegood and von Caemmerer 1988). Assimilation rates were first measured in leaves at an irradiance of 1600 ~tmolquanta-m-2 s-1, 320 gbar COz in air and 28~ C. Those which displayed rates of photosynthesis appreciably different from the rest were discarded. In this way plant material was normalised to a uniform rate of carbon assimilation. When the irradiance was varied, the external CO2 concentration was varied to maintain internal COz (Pi) approximately constant. Metabolite measurements. Leaves were rapidly freeze-clamped in situ, extracted in HC104 and metabolites measured using the apparatus and procedures described previously (Leegood and yon Caemmerer 1988). Chlorophyll. Chlorophyll was measured by the procedure of Arnon (1949). The mean chlorophyll content of the Z. mays leaves was 392 rag. m-2 for experiments in which the intercellular partial pressure of CO2 was varied (April 1986) and 444 mg.m 2 for experiments in which the irradiance was varied (April 1987). Pool sizes of metabolites are expressed on a leafarea basis. Results and discussion D u r i n g steady-state CO2 assimilation at k n o w n intercellular CO2 pressure and irradiance, leaves were freeze-clamped and total metabolite pools were m e a s u r e d in relation to the assimilation rate. W h e n the irradiance was varied, ambient CO2 was adjusted so as to keep p~ a p p r o x i m a t e l y constant (Fig. 2). In Z. mays, only two metabolites o f the C4 cycle, P E P and pyruvate, r e s p o n d e d to changes in the assimilation rate which was shown by the very small pool sizes o f these c o m p o u n d s at low, as c o m p a r e d with high, CO2 (Figs. 1, 2). The large pool o f malate is k n o w n to be largely inactive in

259

photosynthesis ( H a t c h 1971, 1979) and consequently the b e h a v i o u r o f malate showed little obvious relationship to changes in CO2 flux in relation to changes in p~ or irradiance. The pools o f aspartate and alanine also showed no relationship to the assimilation rate, in contrast to previous observations (Usuda 1987a, b), even t h o u g h the pools o f these c o m p o u n d s were rather low by comparison with pools o f aspartate and alanine in leaves o f an a s p a r t a t e - f o r m e r such as Amaranthus edulis (Leegood and v o n C a e m m e r e r 1988). Oxaloacetate was present in a m o u n t s well below the limits o f accurate detection (less t h a n 0.1 gmol. m - 2 , results n o t shown) in contrast to leaves o f A. edulis (Leeg o o d and v o n C a e m m e r e r 1988). Changes in metabolites in relation to changes in the intercellular partial pressure o f C O 2. The pool o f P E P declined m a r k e d l y at sub-saturating Pi (less t h a n 100 gbar) as the assimilation rate (A) fell, in agreement with previous observations in Z. mays (Usuda 1987a) and A. edulis (Leegood and von C a e m m e r e r 1988). The a m o u n t s o f P G A , triose-P, p y r u v a t e and P E P all followed the assimilation rate a p p r o x i m a t e l y and thus declined markedly as Pi was decreased. The p o o l o f fructose-6p h o s p h a t e (Fru6P) also declined at low pi, b u t the content o f glucose-6-phosphate (Glc6P) remained constant, resulting in an increase in the ratio G l c 6 P / F r u 6 P in low CO2 (Leegood and von Caemmerer 1988). In contrast, the pools o f fructose-l,6bisphosphate (Frul,6bisP) and R u B P b o t h rose at intercellular CO2 pressures less than 100 ~tbar (Fig. 1). The pools o f PEP, P G A , triose-P, R u B P and F r u l , 6 b i s P all showed inflexion points at a Pi similar to the point o f inflexion in the A/p~ curve, while the a m o u n t o f p y r u v a t e decreased below a pi o f a b o u t 200 gbar. The metabolites which declined at low pi also showed a decline at high p~, even t h o u g h the assimilation rate did n o t decline at high p~. Changes in metabolites in relation to changes in irradiance. The pools o f P E P and P G A were almost constant at irradiances above 300 ~tmol q u a n t a . m - 2 . s -1, a feature which was also observed in Z. mays by U s u d a (1987b) and in A. edulis by L e e g o o d and von C a e m m e r e r (1988). The behaviour o f p y r u v a t e differed f r o m that o f PEP, because the a m o u n t o f p y r u v a t e fell appreciably at irradiances above and below 300 gmol q u a n t a . m -2. s - 1. Nevertheless, large pools o f all the metabolites were m a i n t a i n e d in very low light. The b e h a v i o u r o f the p y r u v a t e pool differs f r o m previous observations in Z. mays (Usuda 1987b) and A. edulis (Lee-

260

R.C. Leegood and S. von Caemmerer: Gas exchange and metabolies in Zea mays 300

80

(M~

60

9 o

/

9

9

200 200

i

E

150

40 100

o

E

250

10o

20

A

0

I

60

l

I

PGA

q

I

I

o

u

I

I

I

50 0

I

I

I

I

I

I

9 200

~

40

~

,

2000 9

9 100C

I00

20

PEP

trioseP

i

0

i

[

I

,

I

'E

I

0

i

I

i

l

rnalate

OJ I

.E

0

L

I

1

o

RuBP

E

I

I

aspartate

40 ~1500

30 1000

i

E

20q

lO

o

0

I

i

L

80 ~

1

I

0

Frul,6bisP 60

500

Cru6P

10

E

I

I

P~Q

I 9

I

~[

I

9

I

400 -O

9

OI

IO

I

alanine

300 ~0

~o

200 20

2o o 0

i 200

Pi (IJbar)

400

600

GIc6P

2tO0

L

o 9 o N I

Pi (pbar)

Fig. 1. Changes in the rate of CO2 assimilation ( A ) a n d contents of photosynthetic intermediates in leaves of Z. mays in relation to changes in the intercellular partial pressure of CO2 (Pi)- The irradiance was 1600 gmol quanta, m - 2. s - 1. Leaves were freezeclamped and extracted for metabolite analysis as described in Material and methods. The mean chlorophyll content of the leaves of Z. mays was 392 m g - m -2. Note that 10 g m o l . m 2 of a metabolite is equivalent to 26 n m o l . m g 1 chlorophyll. This content is approximately equal to an intracellular concentration of 1 m M if confined to the chloroplast or cytosol with a volume of 25 gl. m g - 1 chlorophyll. Fru6P = fructose-6-phosphate; Frul,6bisP = fructose-l,6-bisphosphate; Glc6P = glucose-6-phosphate

good and Caemmerer 1988) in which the pyruvate content remained constant at irradiances above 300 gmol quanta, m - 2. s - 1. The changes in the pyruvate pool that were observed in Z. mays (Fig. 2) actually resemble more closely the behaviour of alanine in Usuda's work. The amounts of triose-P, Fru6P and GIc6P all decreased with decreasing irradiance, although, in

I00

J 4OO

I

600

9

I~

,o

200

~00

L

600

Pi (pbar)

contrast to Fru6P, there was a considerable pool of Glc6P in low light. Consequently the ratio Glc6P/Fru6P rose at low irradiance (Leegood and yon Caemmerer 1988). The amount of PGA showed a less drastic decline than triose-P at low irradiance, reflecting the diminished availability of ATP and N A D P H for PGA reduction in low light (see below). Fructose-l,6-bisphosphate also showed a decrease with decreasing irradiance and lacked the stability seen in Z. mays by Usuda (1987b) or in bean leaves by Badger et al. (1984). The pool of RuBP showed some decrease with decreasing irradiance and was low at the lowest irradiance (Fig. 2) (compare Leegood and von Caemmerer 1988; Usuda 1987b). The regulation of carbon assimilation in maize. The relative stability of the PEP pool compared with the large changes in carbon fluxes observed with changing irradiance (Fig. 2) demonstrates that PEP carboxylase is regulated by light in vivo in

R.C. Leegood and S. von Caemmerer: Gas exchange and metabolies in Z e a m a y s 5C -r

9

100|I.-

26:l

9 9

400 9

t.0

N,. E

30

O

20

/ i

80~60

II

300

9 9

o0

200

E I0

t t

?

I I

320

100

PEP I

I

0

l

PGA I

Pj

/. 00L-P

9

2~,0

L

]

t

/

500F_ 200 300

160 80

I%"

9

9

o o

9

9

200

go

100

100

0

I

I

I

E 9

~o

pyruvofe I I

I

0

i

I

triose P

04'E

0

L

I

I

s

o

9

E

E

600

30

20 20 300

E

..t." o

10

alanine 0

[

I

I

[

i

l

I

aspartate

E

L

F r u l , 6 bisP 30

600

o 9

l

500

I

1000

lOe~

9 l

I

L

l

I

"~

1000

40 9

GIc6P

20 I

1500 2000

I (IJmol quanta.m-?s -I}

I

60

20 q

9

I

100 80

300 9

Fru6P

RuBP

9

0

0

I

I

L

L

t

500

1000

1500

2000

S00

I (IJmol quanta.m-Z.s-u)

Fig. 2. Changes in the rate of CO2 assimilation (A), the intercellular partial pressure of COz (Pl) and contents of photosynthetic intermediates in leaves of Z. m a y s in relation to changes in the irradiance (/). Leaves were freeze-clamped and extracted for metabolite analysis as described in Material and methods. The mean chlorophyll content of leaves of Z. m a y s was 444 mg. m - 2 Note that 10 g m o l . m - z of a metabolite is equivalent to 23 n m o l . m g - ~ chlorophyll. This content is approximately equal to an intracellular concentration of 1 m M if confined to the chloroplast or cytosol with a volume of 25 g l . m g - 1 chlorophyll. Fru6P = fructose-6-phosphate ; F r u l ,6bisP = fructose- 1,6-bisphosphate; G l c 6 P = g l u c o s e - 6 - p h o s p h a t e

Z. mays as in A. edulis (Leegood and yon Caemmerer 1988). The PEP content declined as the assimilation rate declined at low CO2, showing that PEP carboxylase may be limited not only by the intercellular partial pressure of CO2 but also by the concentration of PEP. For example, a content of PEP of 20 gmol.m 2 corresponds to an intra-

t

1500 2000

I (pmol quanta.m-~s -u

cellular concentration of about I m M (assuming that 50% of the PEP is in the cytosol (Usuda 1988)), which compares with an So.s (PEP) of 110 m M for PEP carboxylase measured in a simulated cytosolic environment (Doncaster and Leegood 1987). In C3 plants, the initial slope of the A/pi curve may, or may not, be affected by changes in the irradiance, depending upon whether the activation state of RuBP carboxylase alters with changes in irradiance (Terry and Farquhar 1984). Figure 3 shows that in Z. mays a large decrease in irradiance fi'om full to half sunlight had no effect upon the initial slope, although it reduced the point of inflexion to a lower partial pressure of CO2. It is clear that light modulation of PEP-carboxylase activity does not override the CO2 limitation so as to change the initial slope of the A/pi relationship in Z. mays. The dependence of the assimilation rate upon CO2 at low Pi may therefore be

262

R.C. Leegood and S. yon Caemmerer: Gas exchange and metabolies in Zea mays

60

.~,~If

-

100 - -

I == 20001Jmol quanta 9 m- ,2s- i

D

D

50

DD

O

~. f E -2-" O E ,::[

D9

n

1,0

E000#mel qu0nt0-m-2s4

E

30

E

20

n I.J [3-

n~

O o~

a go o

o

5(-o

C13 D 9 []

9

0o o

9

9

9 9

o

,oo

g

10 o

o

l

I 100

I

I 200

I

I

300

I ~00

Pi (pbar)

Fig. 3, The relationship between the steady-state COz assimilation rate (A) and the intercellular partial pressure of CO: (Pl) in Z. mays leaves at two irradiances, e, 2000 gmol quanta, m - 2. s - l ; A, 1000 gmol q u a n t a . m - Z . s -a

assigned to a dependence of PEP-carboxylase activity on CO2 and we can conclude that, even at half sunlight, it remains limiting. The stability of the pyruvate pool in relation to irradiance (Fig. 2) has previously been observed in leaves of A. edulis (Leegood and von Caemmerer 1988). In Z. mays the pool of pyruvate was between two and four times larger than in leaves of A. edulis, which probably reflects the requirement for intercellular transport of pyruvate in Z. mays. The decrease in pyruvate as the irradiance was increased (except at very low irradiance) also demonstrates regulation of pyruvate consumption by light-modulation of pyruvate, Pi dikinase (Burnell and Hatch 1985), by light-regulation of the active uptake of pyruvate into the stroma of mesophyll chloroplasts (Flfigge et al. 1985) or by the provision of ATP. The reason for the decline in these and some other metabolites (except RuBP and Frul,6bisP) at very high Pi is not immediately apparent, although it could be a consequence of the onset of CO2 inactivation by stromal acidification (Dietz and Heber 1984). In Z. mays a PGA/triose-P shuttle operates between the bundle sheath and the mesophyll cells as some of the PGA formed in the bundle sheath has to be reduced in the mesophyll cells (Hatch and Osmond 1976). This shuttle also constitutes a mechanism by which the fluxes through the Calvin cycle can be co-ordinated with the fluxes of carbon through the C4 cycle and into sucrose. Pools of C4 intermediates are connected to pools of C3 intermediates because of the interconversion of PGA and PEP (Huber and Edwards 1975; Fur-

0 0

I

I

200

400

PGA

(IJ mol. m -2)

Fig. 4. The relationship between the steady-state contents of P G A and PEP in leaves of Zea mays and Amaranthus edulis with varying intercellular partial pressures of CO2 and irradiance. Maize: pi varied (o), irradiance varied (m); A. edulis: Pi varied (I), irradiance varied (I). Note that the same relationship holds within, as well as between, treatments. Data for A. edulis from Leegood and yon Caemmerer (1988), for Z. rnays from Figs. 1 and 2

bank and Leegood 1984). Figure4 shows that there was a strong positive correlation between the pools of PEP and PGA in leaves of Z. mays and A. edulis under many different flux conditions. The average ratio for PGA/PEP of 4 accords well with the observed equilibrium constant (between 1 and 4) for the phosphoglycerate mutase/enolase couple (Newsholme and Start 1973, p 263) and indicates that this reaction is close to equilibrium in vivo. This close relationship means that the decline of the pools of C4 intermediates such as PEP with decreasing CO2 may simply reflect the decreased availability of PGA in the mesophyll. This will be due to the decreased rate of PGA production and hence decreased export of PGA from the bundle sheath when the photosynthetic rate is low, as well as increased conversion of PGA to triose-P in the mesophyll when light is saturating photosynthesis and the supply of ATP and NADPH is abundant (see below, Fig. 5 b). A strong relationship between the contents of PGA and pyruvate has also been noted in Z. mays (Usuda 1987b), but this probably also reflects ATP availability (Doncaster et al. 1988). Triose phosphate is a strong activator of PEP carboxylase (Doncaster and Leegood 1987) and, together with PGA-led changes in PEP concentration, will influence PEP-carboxylase activity in vivo, achieving a co-ordination of the rate of CO2 pumping by the C4 cycle with the rate of sucrose

R.C. Leegood and S. von Caemmerer: Gas exchange and metabolies in Zea mays

263

2.5n 2.0 -5

E 1.5

9

O

E \

--

O

10

. ..,x

0

.../

9

E

E

05

friose P/PGA

0

I

i

I

i

200

a

400

Pi ~.00--

s+e

1.

triose P/PGA

9" I

I

0

600

[pbar)

i

500

I

1000

I

1500 2 0 0 0 2500

Fig. 5a--d. Changes in the ratio of triose-P to PGA in leaves of Z. mays in relation to changes in the intercellular partial pressure of CO2 (a) and the irradiance (b) and in leaves of bean, Phaseolus vulgaris, in relation to changes in the intercellular partial pressure of COz (e) and the irradiance (d). Ratios for Z. mays were calculated from Figs. 1 and 2; ratios for P. vulgaris leaves in 2% Oz (o) and 20% 02 (o) were calculated from the data of Badger et al. (1984)

] (#rnol quanta, m-.2s-1)

b

triose P/PGA o

triose P / P G A 0.8

~ 0 1 .if 9 E

\

O

O

0.6i

o~

0 s,~,%9

E 0 93;-9 9 0

C

o 0

o 9

0

04 o9

9 ~ 00o 9 9 I o

Oo I

02 9o

600

300

Pi

(pbar)

9 o I

"]

900

1200

o

'0

9 9

9

! o

d

synthesis, as suggested by Leegood and yon Caemmeter (1988). Since sucrose synthesis occurs in the mesophyll cytoplasm, the fate of triose-P within the mesophyll must also be under strict control, as two-thirds of the total triose-P which is formed in the mesophyll must be returned to the bundle sheath in order to regenerate RuBP. The rate of conversion of triose-P to sucrose may be largely controlled by the availability of triose-P above a certain threshold concentration (Stitt and Heldt 1985a), and it is important to note that triose-P and Fru6P (a substrate for sucrose-phosphate synthetase and also an activator of PEP carboxylase) both follow the assimilation rate in relation to irradiance and the intercellular partial pressure of CO2 (Figs. 1, 2) and will thereby limit the rate of sucrose synthesis by simple availability of substrate (see also Usuda et al. 1987). The rise in the amount of Frul,6bisP at low Pi (Fig. 1) also indicates control over the entry of triose-P into the Calvin cycle (see Leegood and von Caemmerer 1988 for discussion). The difference in the behaviour of triose-P and Frul,6bisP probably also reflects their differing intercellular distribution, with triose-P predominating in the mesophyll cells (Leegood 1985; Stitt and Heldt 1985a, b). The reduction of PGA to triose-P. Electron transport provides ATP and N A D P H for the reduction of PGA to triose-P. The ratio between the amounts

[

I

I

500

1000

1500

2000

I (pmol quanta.m-?s -I) of triose-P and PGA can be taken as an indication of the strength of this driving force provided by electron transport, which Heber et al. (1986, 1987) have termed the assimilatory force (FA). If the reactions catalysed by the enzymes PGA kinase and glyceraldehyde-phosphate dehydrogenase approach thermodynamic equilibrium, as they do in isolated chloroplasts (Dietz and Heber 1984), then : [PGA][ATP][H+][NADPH]

k-

o s 1(~-6

and FA, the assimilatory force = [ATP][NADPH] _ [triose-P] 9K [ADP] [Pi] [NADP] - [PaA] [U +l In leaves of C4 plants, near-equilibrium is also likely to be achieved in this reaction since both mesophyll and bundle-sheath compartments contain high activities of PGA kinase and glyceraldehydephosphate dehydrogenase, and isolated cells and chloroplasts from both the mesophyll and the bundle sheath reduce PGA at high rates (e.g. Slack et al. 1969; Ku and Edwards 1975; Day et al. 1981 ; Jenkins and Boag 1985). Ratios of triose-P/PGA for Z. mays and leaves of Phaseolus vulgaris are shown in Fig. 5(a-d) as a function of the intercellular partial pressure of COz and the irradiance. The data for P. vulgaris are calculated from measurements on intact leaves by Badger etal. (1984). In C3 plants, the ratio

264

R.C. Leegood and S. von Caemmerer: Gas exchange and metabolies in Zea mays 7[

o

o

_o

Z. m a y s

6[ A, 5[

9

~"

~c

o

3C

,~

2(

E E

o

9

edufis

9

9 9

R.

$OtiVUS

1(

C 20( i

E

1~(

o

E

::3-

12( -

~e~

9

n m

8c

rr

L,C 9 0

I

~oo

~

2~oo

Joo

L.O0

Pi

IA

I

I

I

I

SO0

600

700

eO0

900

~000

(pbor)

Fig. 6. A comparison of CO2 assimilation rates (A) and amounts of RuBP in leaves of two C4 species, Zea mays (o) and Amaranthus edulis (A), with leaves of radish, Raphanus sativus (e), in relation to the intercellular partial pressure of CO2 (Pi). Data are taken from Fig. 1 (for Z. mays), Leegood and von Caemmerer (1988) (for A. edulis) and from von Caemmerer and Edmonson (1986) (for radish)

triose-P/PGA rises at low C O 2 (Fig. 5a) and thus meets the simple expectation that when the CO2 limits the assimilation rate the quotient (ATP/ ADP. Pi)(NADPH/NADP +) will rise (Heber et al. 1986, 1987). In spinach leaves the quotient (ATP/ ADP. Pi)(NADPH/NADP +) is much lower in the chloroplast at low, as compared with high, irradiance (Prinsley et al. 1986). However, chloroplastic triose-P/PGA ratios in spinach leaves can increase as the irradiance is decreased either at high CO2 (Dietz and Heber 1986) or at ambient CO2 (Heber et al. 1986, 1987). Figure 5d shows that in intact leaves of P. vulgaris a similar phenomenon is observed. The triose-P/PGA ratio falls at intermediate irradiance, but rises as the irradiance is further decreased. Thus in leaves of C3 plants decreases in photosynthetic flux as the irradiance is decreased must either be accompanied by an increase in the production of ATP and NADPH (which is unlikely) or else the consumption of ATP and NADPH must be diminished by an increase in the resistance to flux (Heber et al. 1986, 1987). Regulation of consumption can be explained if the enzymes of the Calvin cycle become less active in low light (Heber et al. 1986, 1987). Evidence of

this can be seen in the rise in the pool of Frul, 6bisP in C3 plants as the irradiance is decreased (Dietz and Heber 1984) showing that Calvin-cycle fluxes are regulated by modulation of enzymes as the irradiance is decreased. In contrast, the Frul, 6bisP pool falls in Z. mays when the flux decreases in low light (Fig. 2). In Z. mays the triose-P/PGA ratios were very much higher than those observed in the leaves of C3 plants (Fig. 5a, b). These reflect the high ATP/ ADP ratios generated in the mesophyll cells of Z. mays leaves in the light (Leegood 1985; Stitt and Heldt 1985b). The triose-P/PGA ratios rose as Pi decreased, as in the leaves of C3 plants (Fig. 5 a), and fell as the irradiance was decreased (Fig. 5 b). Similar observations have been made in leaves of A. edulis (Leegood and von Caemmerer 1988). If the above explanation for the behaviour of trioseP/PGA ratios in C3 plants is accepted then these observations indicate that regulation of photosynthetic flux in the steady-state in relation to light in C4 plants may be less reliant upon direct modulation of enzyme activity by light (i.e. regulation by consumption) than it is in C3 plants. Characteristics of gas-exchange in relation to the RuBP pool. Figure 6 illustrates a comparison between CO2 assimilation rates and amounts of RuBP in leaves of two C4 plants, Z. mays and A. edulis, and in leaves of a C3 plant, radish (Raphanus sativus)o in relation to the intercellular partial pressure of COz (Pi). In leaves of C3 plants, the response of the CO2 assimilation rate to the intercellular concentration of CO2 (P0 can be interpreted as being limited by the activity of RuBP carboxylase-oxygenase at low Pi and at higher p~ by the capacity of the system to regenerate RuBP (Farquhar et al. 1980). In C4 plants the response of the CO2 assimilation rate to Pi is characterised by a steep initial response to CO2 at low p~, followed by saturation of the assimilation rate at a lower Pi of about 100 labar, compared with C3 plants which tend to saturate above 200 ~tbar. At low pi, CO2 assimilation in C4 plants is likely to be determined by the amount and the CO2-dependence of PEP carboxylase. Saturation of the rate of CO2 assimilation at high p~ could be caused by a limitation by the activity of RuBP carboxylase, by the capacity of the system to regenerate RuBP in the bundle-sheath cells, or by the capacity to regenerate PEP in the mesophyll cells. The relationship illustrated in Fig. 6 serves to demonstrate that, in accordance with the kinetics of gas exchange, the large rise in the RuBP pool occurs at very much lower intercellular partial pressures

R.C. Leegood and S. von Caemmerer : Gas exchange and metabolies in Zea mays

of C O 2 in Z. mays and A. edulis than it does in C3 plants (of which radish provides a typical example), and it thus emphasises very strongly the CO2concentrating function of C~ photosynthesis. In a C4 leaf, far less RuBP carboxylase is required to achieve CO2 assimilation rates similar to those in a C3 leaf (Wong et al. 1985 ; Sage et al. 1987). The amounts of RuBP in Z. mays leaves were also lower than in the leaves of C3 plants under comparable conditions (Badger et al. 1984; yon Caemmerer and Edmonson 1986). At the point of inflexion in the A/pi curve, the content was about 20 gmol. m - 2 (equivalent to 46 n m o l . m g - 1 chlorophyll) and declined to approx. 5 ~tmol.m-2 in high CO2. This may be compared with a calculated concentration of RuBP-carboxylase binding sites of 11 g m o l . m -2 (Leegood and von Caemmerer 1988), which contrasts with a binding-site concentration of about 30 Ixmol.m -2 in radish (von Caemmerer and Edmonson 1986). Therefore, under most conditions (including ambient CO2) the amount of RuBP in Z. mays leaves would appear to be above the binding-site concentration, although in high CO2, RuBP pools may fall below binding-site concentrations. Usuda (1987a) found amounts of RuBP in Z. mays to be above bindingsite concentrations, but did not measure pool sizes at high CO2 concentrations. The comparison of RuBP-pool sizes in C3 and C4 leaves shows that RuBP-pool sizes at high CO2 concentrations and RuBP-carboxylase binding sites are correlated. The RuBP pool rose over the entire CO2-response curve with declining Pi both in Z. mays (Fig. 1 ; Usuda 1987a) and in A. edulis (Leegood and von Caemmerer 1988, Fig. 6) but the rise was much more pronounced at CO2 pressures less than 100 gbar. The fact that RuBP does not rise as dramatically with decreasing Pi in Z. mays and A. edulis as it does in leaves of R. sativus may in part be due to the fact that even at ambient CO2 concentrations of 5-10 lxbar, bundle-sheath CO2 concentrations may be above 200 gbar, but the rise in RuBP at low pi must reflect subsaturating levels of CO2 in the bundle sheath (Collatz 1978). Furbank and Badger (1982) noted a rise in 180 2 uptake at low external CO2 pressures in several C4 plants, which is most likely to be the result of an increase or oxygenation of RuBP. This occurs despite a lack of O2 sensitivity of photosynthesis even at low intercellular partial pressures of CO2 (Edwards et al. 1985). Under these conditions, PEP carboxylase is likely to limit the rate of CO2 supply to the bundle sheath and changes in 02 concentration result primarily in changes of bundle-sheath CO2 and O2 concentration but not

265

in a change in rate. This can be seen in Table 2 of Berry and Farquhar (1977). The RuBP pool did not show as great a response to irradiance as did the rate of CO2 assimilation. A similar result in C3 leaves can be explained by changes in RuBP-carboxylase activation state (Perchorowicz et al. 1981 ; Seemann et al. 1985). Changes in the activation state of RuBP carboxylase with irradiance are less likely to occur in C4 plants in so far as no changes in activity between light and dark have been observed (Vu et al. 1984; Usuda 1985; Servaites et al. 1986). The relatively high RuBP pools at low irradiance may merely reflect lower bundle-sheath COz concentrations at low irradiance (Furbank and Hatch 1987). Visits by R.C.L. to Canberra were sponsored by the British Council under the Academic Links and Interchange Scheme and by the Nuffield Foundation and facilitated by Barry Osmond. This work was also supported by the Science and Engineering Research Council, U.K. (Grant No. GR/D/02577). We would like to thank Murray Badger (Dept. of Environmental Biology, A.N.U., Canberra, Australia.) and Tom Sharkey (Desert Research Institute, Reno, Nev., USA) for providing the data for bean leaves in Fig. 5.

References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgar&. Plant Physiol 24, 1-15 Badger, M.R., Sharkey, T.D., von Caemmerer, S. (1984) The relationship between steady-state gas exchange of bean leaves and the levels of carbon-reduction-cycle intermediates. Planta 160, 305-313 Berry, J.A., Farquhar, G.D. (1977) The CO2-concentrating function of C4 photosynthesis. A biochemical model. In: Proc. IV Int. Congr. on Photosynthesis, pp. 119-131, Hall, D.O., Coombs, J., Goodwin, T.W., eds. Biochemical Society, London Burnell, J.N., Hatch, M.D. (1985) Light-dark regulation of leaf pyruvate, Pi dikinase. Trends. Biochem. Sci. 10, 288 291 Collatz, G.J. (1978) The interaction between photosynthesis and ribulose-P2 concentration effects of light, CO2 and 02. Carnegie Inst. Washington Yearb. 77, 248-251 Day, D.A., Jenkins, C.L.D., Hatch, M.D. (1981) Isolation and properties of functional mesophyll protoplasts and chloroplasts from Zea mays. Aust. J. Plant Physiol. 8, 21-29 Dietz, K.-J., Heber, U. (1984) Rate-limiting factors in leaf photosynthesis. I. Carbon fluxes in the Calvin cycle. Biochim. Biophys. Acta 767, 432-443 Dieth, K.-J., Heber, U. (1986) Light and CO2-1imitation of photosynthesis and states of the reactions regenerating ribulose 1,5-bisphosphate or reducing 3-phosphoglycerate. Biochim. Biophys. Acta 848, 392-401 Doncaster, H.D., Adcock, M.D., Leegood, R.C. (1989) Regulation of photosynthesis in leaves of C4 plants following a transition from high to low light. Biochim. Biophys. Acta 973, (in press) Doncaster, H.D., Leegood, R.C. (1987) Regulation of phosphoenolpyruvate carboxylase activity in maize leaves. Plant Physiol. 84, 82-87 Edwards, G.E., Ku, M.S.B., Monson, R.K. (1985) C4 photosynthesis and its regulation. In Photosynthetic mechanisms

266

R.C. Leegood and S. von Caemmerer: Gas exchange and metabolies in Zea mays

and the environment, pp. 287 327, Barber, J., Baker, N.R., eds. Elsevier, Amsterdam Farquhar, G.D., von Caemmerer, S., Berry, J.A. (1980) A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta 149, 78-90 Fliigge, U.-I., Stitt, M., Heldt, H.W. (1985) Light-driven uptake of pyruvate into mesophyll chloroplasts from maize. FEBS Lett. 183, 335-339 Furbank, R.T., Badger, M.R. (1982) Photosynthetic oxygen exchange in attached leaves of C4 monocotyledons. Aust. J. Plant Physiol. 9, 553-558 Furbank, R.T., Hatch, M.D. (1987) Mechanism of C4 photosynthesis : the size and composition of the inorganic carbon pool in bundle sheath cells. Plant Physiol. 85, 958-964 Furbank, R.T., Leegood, R.C. (1984) Carbon metabolism and gas exchange in leaves of Zea mays L. Interaction between the C3 and C4 pathways during photosynthetic induction. Planta 162, 457-462 Hatch, M.D. (1971) The C4-pathway of photosynthesis. Evidence for an intermediate pool of carbon dioxide and the identity of the donor C4-dicarboxylic acid. Biochem. J. 125, 425-432 Hatch, M.D. (1979) Mechanism of C4 photosynthesis of Chloris gayana: Pool sizes and kinetics of 14COz incorporation into 4-carbon and 3-carbon intermediates. Arch. Biochem. Biophys. 194, 117-127 Hatch, M.D., Osmond, C.B. (1976) Compartmentation and transport in C4 photosynthsis. In: Encyclopedia of plant physiology, N.S. vol. 3: Transport in Plants IlI, pp. 114184, Stocking, C.R., Heber, U. eds. Springer, Berlin Heber, U., Neimanis, S., Dietz, K.-J., Viil, J. (1986) Assimilatory power as a driving force in photosynthesis. Biochim. Biophys. Acta 852, 144-155 Heber, U., Neimanis, S., Dietz, K.-J., Viii, J. (1987) Assimilatory force in relation to photosynthetic fluxes. Advances Photosynth. Res. 3, 293 299 Hewitt, E.J., Smith, T.A. (1975) Plant mineral nutrition. English University Press, London Huber, S.C., Edwards, G.E. (1975) Regulation of oxaloacetate, aspartate and malate formation in mesophyll protoplast extracts of three types of C4 plants. Plant Physiol. 56, 32z1~331 Jenkins, C.L.D., Boag, S. (1985) Isolation of bundle sheath cell chloroplasts from the NADP-ME type C4 plant Zea mays. Capacities for CO2 assimilation and malate decarboxylation. Plant Physiol. 79, 84-89 Ku, M.S.-B., Edwards, G.E. (1975) Photosynthesis in mesophyll protoplasts and bundle sheath cells of various types of C4 plants. IV. Enzymes of respiratory metabolism and energy utilizing enzymes of photosynthetic pathways. Z. Pflanzenphysiol. 77, 16-32 Leegood, R.C. (1985) The intercellular compartmentation of metabolites in leaves of Zea mays. Planta 164, 163-171 Leegood, R.C., von Caemmerer, S. (1988) The relationship between contents of photosynthetic metabolites and the rate of photosynthetic carbon assimilation in leaves of Amaranthus edulis L. Planta 174, 253-262 Newsholme, E.A., Start, C. (1973) Regulation in metabolism. John Wiley, London Nimmo, G.A., McNaughton, G.A.L., Fewson, C.A., Wilkins, M.B., Nimmo, H.G. (1987) Changes in the kinetic properties and phosphorylation state of phosphoenolpyruvate carboxylase in Zea mays leaves in response to light and dark. FEBS Lett. 213, 18 22 Perchorowicz, J.T., Raynes, D.A., Jensen, R.G. (1981) Light limitation of photosynthesis and activation of ribulose bisphosphate carboxylase in wheat seedlings. Proc. Natl. Acad. Sci. USA 78, 2985~2989

Prinsley, R.T., Dieth, K.-J., Leegood, R.C. (1986) Regulation of photosynthetic carbon assimilation after a decrease in irradiance. Biochim. Biophys. Acta 849, 254-263 Sage, R.F., Pearcy, R.W., Seemann, J.R. (1987) The nitrogen use efficiency of C3 and C4 plants. III. Leaf nitrogen effects on the activity of carboxylating enzymes in Chenopodium album (L.) and Amaranthus retrojTexus (L.). Plant Physiol. 85, 355-359 Seemann, J.R., Berry, J.A., Freas, S.M., Krump, M.A. (1985) Regulation of ribulose bisphosphate carboxylase activity by a light modulated inhibitor of catalysis. Proc. Natl. Acad. Sci. USA 82, 8024-8028 Servaites, J.C., Parry, M.A.J., Gutteridge, S., Keys, A.J. (1986) Species variation in the predawn inhibition of ribulose-l,5bisphosphate carboxylase/oxygenase. Plant Physiol. 82, 1161-1163 Slack, C.R., Hatch, M.D., Goodchild, D.J. (1969) Distribution of enzymes in mesophyll and parenchyma sheath chloroplasts of Z. mays leaves in relation to the C4 dicarboxylic acid pathway of photosynthesis. Biochem. J. 114, 489-498 Stitt, M., Heldt, H.W. (1985a) Control of photosynthetic sucrose synthesis by fructose-2,6-bisphosphate. Intercellular metabolite distribution and properties of the cytosolic fructose bisphosphatase in leaves of Zea rnays L. Planta 164, 179-188 Stitt, M., Heldt, H.W. (1985b) Generation and maintenance of concentration gradients between the mesophyll and bundle-sheath in Z. mays leaves. Biochim. Biophys. Acta 808, 400-414 Terry, N., Farquhar, G.D. (1984) Photochemical capacity and photosynthesis. In: Control of crop productivity, pp. 43-57, Pearson, C.J., ed. Academic Press, Sydney Usuda, H. (1985) The activation state of ribulose-l,5-bisphosphate carboxylase in maize leaves in dark and light. Plant Cell Physiol. 26, 1455 1463 Usuda, H. (1987a) Changes in levels of intermediates of the C4 cycle and reductive pentose phosphate pathway under various concentrations of COz in maize leaves. Plant Physiol. 83, 29-32 Usuda, H. (1987b) Changes in levels of intermediates of the C4 cycle and reductive pentose phosphate pathway under various light intensities in maize leaves. Plant Physiol. 84, 549-554 Usuda, H. (1988) Non-aqueous purification of maize mesophyll chloroplasts. Plant Physiol. 87, 427-430 Usuda, H., Kalt-Torres, W., Kerr, P.S., Huber, S.C. (1987) Diurnal changes in maize leaf photosynthesis. II. Levels of metabolic intermediates of sucrose synthesis and the regulatory metabolite fructose-2,6-bisphosphate. Plant Physiol. 83, 289~93 von Caemmerer, S., Edmonson, D.L. (1986) The relation between steady-state gas exchange, in vivo ribulose bisphosphate carboxylase activity and some carbon reduction cycle intermediates in Raphanus sativus. Aust. J. Plant Physiol. 13, 669 688 Vu, J.C.V., Allen, L.H., Bowes, G. (1984) Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories. Plant Physiol. 76, 843-845 Wong, S.-C., Cowan, I.R., Farquhar, G.D. (1985) Leaf conductance in relation to rate of CO2 assimilation. I. Influence of nitrogen nutrition, phosphorus nutrition, photon flux density, and ambient partial pressure of CO2 during ontogeny. Plant Physiol. 78, 821-825 Received 27 October; accepted 20 December 1988