Photosynthesis Research 25: 213-224, 1990. (~) 1990 KluwerAcademic Publishers. Printed in the Netherlands. Regular paper

The relationship between CO 2 assimilation and electron transport in leaves Jeremy Harbinson 1'3, Bernard Genty 2 & Neil R. Baker 2 ~John Innes Institute, Colney Lane, Norwich, NR4 7UH, UK; 2Dept. of Biology, University of Essex, Wivenhoe Park, Colchester, Essex, C04 3SQ, UK; 3Current address A T O Agrotechnology, Postbus 17, 6700 A A Wageningen, The Netherlands Received 6 December 1989; accepted in revised form 11 May 1990

Key words: efficiency

Hedera helix, photorespiration, photosystem I, photosystem II, Pisum sativum, quantum

Abstract

The inter-relationships between the quantum efficiencies of photosystems I (thl) and II ((~II) and the quantum yield of CO 2 fixation (thcoz) were investigated in pea (Pisum sativum (L)) leaves with differing rates of photosynthesis using both photorespiratory and non-photorespiratory conditions, and in a leaf of Hedera helix (L) under photorespiratory conditions. The results indicate that under photorespiratory conditions the relationship between thco2 and both thl and thxl is non-linear and variable. The relationship between thi and ~II under these circumstances remains predominantly linear. Under non-photorespiratory conditions, leaves with a low rate of photosynthesis due to sink limitation exhibit a non-linear relationship between I#1 and I#ii, though the relationship between thit and tbc02 remains linear suggesting a close relationship between linear electron flow and CO 2 fixation. Leaves irradiated at the CO 2 compensation point also exhibit a non-linear relationship between &i and 4'i1. These results suggest that for leaves in air linear electron flow is the predominant source of energy for metabolism. The role of cyclic electron transport is considered when the requirement for the products of linear electron transport is depressed.

Abbreviations: qp - the coefficient for photochemical quenching of chlorophyll fluorescence; thexc- the quantum efficiency of excitation energy capture by open PS II traps; ~bn - t h e quantum efficiency for electron transport by PS II, thi- the quantum efficiency (for electron transport) by PS I; ~bco2- t h e quantum yield for CO 2 fixation (obtained as the gross rate of CO 2 fixation divided by the irradiance); A/XH÷ -trans-thylakoid proton potential difference; P A Q F - photosynthetically active quantum flux

Introduction

Numerous metabolic processes are located within the stroma and many of these, of which CO 2 fixation by the Calvin cycle is the most significant, act as sinks for the products of thylakoid electron transport. These reactions regenerate the substrates (ADP, oxidized ferredoxin and NADP) required as acceptors for light-driven electron transport within the thylakoid. The

coordination of the activities of the light-driven electron flux through the thylakoid electron transport chain with the sink reactions of the stroma has recently been the subject of considerable attention. Qualitative studies have shown that a depression of thylakoid activity occurs in response to diminished sink demand (Dietz et al. 1985, Horton 1985). Recently it has become possible to measure the quantum efficiencies of photosystems I ((~I) and II ($II) in vivo (Harbin-

214 son and Woodward 1987, Weis et al. 1987, Genty et al. 1 9 8 9 ) a n d to describe, using these techniques, the inter-relationship between ~bI and ~n in vivo (Harbinson et al. 1989, G e n t y et al. 1990). A predominant role for linear electron flow in thylakoids in vivo has been demonstrated from these studies which have also indicated that in vivo electron flow is limited between photosystems I and II. Concurrent measurements of the quantum yield for CO 2 fixation (~bco2), 4,I and ~bn in vivo have revealed a linear relationship between thco2 and both ~bI and ~bll under non-photorespiratory conditions, whereas under photorespiratory conditions these relationships are non-linear (Weis et al. 1987, G e n t y et al. 1989, 1990, Peterson 1989). In this paper we will examine the effect of changing rates of CO 2 fixation on the interrelationship between ~b~, ~bn and thc% under conditions conducive and prohibitive for photorespiration, thus allowing the effect of changing sink activity on the relationships between ~bn, ~b~ and ~bc% to be evaluated. In the majority of experiments cold grown pea plants are used since they possess a lower rate of CO s fixation at saturating irradiance than do warm grown pea plants (Harbinson and Hedley, in preparation) and therefore the action of photosynthetic carbon reduction as a sink for A T P and N A D P H is diminished. This depressed rate of photosynthesis is stable and is not accompanied by any marked reduction in the intrinsic efficiency of ~bH (the maximum efficiency of ~b~i, which is obtained by dark adapting the leaves, and which is estimated by the dark adapted value of Fv/Fm) or chlorosis except under extreme circumstances. In order to examine the effect of extreme reductions in the rate of CO s fixation on the relationship between ~bl, ~n and thco: cold stressed Hedera helix leaves are used. These leaves have a maximum rate of CO 2 fixation in air of only - 2 - 3 / z m o l m -2 s -~ and it is impossible to obtain pea leaves with such low rates of CO 2 fixation without other changes, such as chlorosis occurring. Light induced absorbance changes at 820 nm are used to estimate the pool of oxidized P700 (the reaction centre of PS I) in leaves from which t~l is calculated (Harbinson and Woodward 1987). The quantum efficiency of PS II (~bn) is

determined using chlorophyll fluorescence changes to obtain an estimate of qp (the fraction of open PS II reaction centres) and the excitation transfer efficiency of photosystem II (~bexc) (the quantum efficiency of photochemical quenching of fluorescence by open PS II traps); ~bn is then calculated as the product of ~bexc and qp ( G e n t y et al. 1989).

Materials and methods

Pisum sativum (L), varieties B C 1 / 9 R R , JI 1345 and JI79", and Hedera helix (L) were used. Plants of Pisum sativum were grown in one of two environments: 'warm grown' plants were grown at a day temperature of 20°C, a night temperature of 15°C and an irradiance of 500/zmol m -2 s -1 photosynthetically active quantum flux (PAQF); 'cold grown' plants were grown at a day temperature of 5°C, a night temperature of 5°C and an irradiance of 350~zmol m - 2 s -1 PAQF. The response of photosynthesis to cold growth conditions is the same for each of the varieties of pea used. All plants were grown in a mixture of 70% of John Innes compost (no. 1) and 30% chicken grit supplemented with low nitrogen fertilizer, and the photoperiod was 16 h. Hedera helix was collected from a shaded site within a patch of woodland on the estate of the University of East Anglia, Norwich, U.K. in November 1988 after a period of heavy frost. Stems of the fertile, aerial phase of H. helix were cut carefully under water to maintain xylem continuity, a practice which experience has shown to allow normal stomatal opening to occur reliably with this species. Measurements of leaf gas exchange, chlorophyll fluorescence and the light induced absorbance change at 820 nm (AA820 nm) were made concurrently using equipment and techniques described previously ( G e n t y et al. 1989, Harbinson and Hedley 1989, Harbinson et al. 1989). Chlorophyll fluorescence was analyzed as described by Genty et al. (1989) to provide estimates of * Varieties JI 1345 and JI 79 are germplasm accessionsin the John Innes Inst. Pisum Germplasm Collection, seeds of var. BC1/9RR were a kind gift of Dr C.L. Hedley, John Innes Inst. Norwich, U.K.

215 qp, ~bexc and ~btt, and the AA820 nm was analyzed as described by Harbinson et al. (1989) to provide an estimate of ~bI. When measured in the dark adapted state ~b~x~ is the same as Fv/F m, when measured in the presence of non-photochemical quenching of chlorophyll fluorescence greater than that found in the dark adapted state th~x~ is equal to F'v/F'. The quantum efficiency of CO 2 fixation (~bco2) was calculated by dividing the rate of CO 2 fixation of the irradiated area of leaf tissue by irradiance. All measurements were made at 20°C. The irradiance responses of ~bI, qp, ~b~x~ and ~bli and CO 2 fixation were obtained by subjecting a leaf to progressively higher irradiances produced by an array of light emitting diodes (H-3K (Stanley, Tokyo, Japan), peak emission 660 nm). A series of decreasing irradiances from the maximum irradiance could have been used. This produces similar results to a series of increasing irradiances provided no slowly reversible decrease in ~bi~ is produced by the initial high irradiances. Following each irradiance increase the leaf was allowed to reach steady state before recording the fluorescence and absorbance changes necessary for the calculation of the photochemical activities. The time necessary to arrive at a new steady state was variable depend1.0

l

A

ing on the environment and history of the leaf but normally took about 20 min. From the dependence of ~bi, ~bll, ~b.... qp and the rate of CO 2 fixation on irradiance the relationships between (~II' ~ .... qp and ~bi; and tkco2, ~b~ and ~II were determined. The irradiance response will not be shown here but the data corresponding to the values of thi and ~bi~at 520 ~ m o l m -2 s -1 PAQF will be indicated on each figure to provide an indication of the irradiance sensitivity of th~ and ~bn.

Results

The relationships between ~I and &u, ~bexcand qp in a mature leaf of pea var. B C 1 / 9 R R in an atmosphere that will largely abolish photorespiration (350ppm CO2, 2% oxygen, remainder N2) are shown in Fig. la. The quantum efficiencies of P S I (~bt) and PS II (~bn) are apparently linearly related over the greater part of the range shown. Only at the highest values of ~I and ~bli (which occur at low irradiances) does the relationship become non-linear with (~II falling relatively more rapidly than ~b~.The rapid drop in ~II at low irradiance is not due to a sudden decrease in ~b~xc but is due largely to a decline in qp in this 0.05

B

¢co, 0.04

0.8

O

~

~

0,03

qp

0.6

~)II

0.4

0.02

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0.6

i 0.8 I~)1

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i 0.6

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Fig. 1. (A) T h e inter-relationship between ~bI and qe ( O ) , ~bexc (&) and q~l~ ( 0 ) for a m a t u r e w a r m grown leaf of pea variety B C 1 / 9 R R over a range of irradiances. During the m e a s u r e m e n t s a gaseous phase comprised of 350 p p m in a 2% 0 2 and ~ 98% N 2 was m a i n t a i n e d a r o u n d the leaf to minimize photorespiration. T h e d a t u m m a r k e d ( i ) indicates the sample m e a s u r e d at an irradiance of 520/~mol m 2 s - i P A Q F , the values obtained at this irradiance are ~bn = 0.37, ~ I = 0.71. T h e dark adapted Fv/F m is 0.72. (B) T h e inter-relationship between ~b~ (11) and &u ( 0 ) , and the q u a n t u m efficiency for CO~ fixation (~bco2) obtained concurrently from the s a m e leaf.

216 region as previously described by Harbinson et al. (1989). The relationship between ~bex~ and 4)1 appears to be linear over the entire range of the data. Figure lb shows that a linear relationship exists between the quantum efficiencies of both photosystems and the quantum efficiency of CO 2 fixation under these conditions. When data are obtained from a young pea leaf of var. B C 1 / 9 R R in an atmosphere of 350ppm CO2, 2% 0 2, remainder N2, the relationships between ~bI and qe, ~b~x~ and ~bn, and between ~bco2 and both ~bI and ~b~i are similar to those shown in Fig. 1 except in a number of important respects (Fig. 2). The major difference is the non-linearity between ~bco2 and both ~b~ and ~bii at high values of thi and (~II" At lower values of ~bi, tkii the relationship between ~bco2 and both ~b~ and ~bn becomes linear. This is in contrast not only to the data obtained from the mature leaf (Fig. lb), but also to previously reported relationships between (~I o r (~II and ~bco: (Weis et al. 1987, Genty et al. 1990). The relationship between 4,I and tb~x~is non-linear, like the relationship between qp and thi but whereas the ~ e x c becomes less sensitive to changes in ~b~ a s t~l

decreases, qp becomes more sensitive. The decline of ~ I I , relative to 4~i, at high values of ~bI is less marked than in the mature leaf (Fig. la), and this is paralleled by a smaller decline of qp at high values of ~bI. Following a change of the gaseous phase to one where photorespiration can occur (350 ppm CO 2, 20% 02, remainder N2) the relationships between &i, and qp, ~bexc and (~II (fig. 3) are largely similar to those under non-photorespiratory conditions (Fig. 2a). The major change in the relationships is in the size of the non-linear portion of the relationship between ~b~ and ~bi~at high values of these parameters, which is smaller than that recorded under non-photorespitatory conditions (compare Figs. 2a and 3a), and a smaller fall in qe at high values of ~b~is observed. In addition, the relationship between ~bI and ~bexc is again non-linear except that ~bexc becomes relatively more sensitive to changes in (~I a s ~ i decreases. The values for ~b~ and ~ I I a t 520/~mol m - 2 S -1 show that both th~ and ~b~i are higher than under non-photorespiratory conditions (Figs. 2a and 3a). The relationships between ~b~i, ~b~ and ~bco2 under photorespiratory conditions 0.06

B

0.05

1.0

A 0.04 ,

0.8

qp 0.03

4~)exc 0.6

~)C02

1~II

0.4

0.02

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0

|

w

i

i

0.2

0.4

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O.g

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i 0.6

I O.g

i 1.0

Fig. 2. (A) T h e inter-relationship between ~b, a n d qp (©), ~bexc ( A ) and ~bit ( O ) for a young, warm grown leaf of pea variety B C 1 / 9 R R over a range of irradiances from 64 to 1250/zmol m -2 s -x PAQF. During the course of the m e a s u r e m e n t s a gaseous phase comprising of 350 p p m C O : , 2% 0 2 and ~ 98% N 2 was maintained around the leaf to minimize photorespiration. T h e d a t u m m a r k e d (A) indicates the sample m e a s u r e d at an irradiance of 520/~mol m -z s -1 P A Q F , the values obtained at this irradiance are ~b,~= 0.37, ~b~= 0.71. T h e dark adapted Fv/F m is 0.79. (B) T h e inter-relationship between ~bt (11) and ~b~ (O), and the q u a n t u m efficiency for C O z fixation (~bcoz) obtained concurrently from the same leaf.

217 1.0

A 0.04

0.8

qp

B 0.03

Cexc 0.6 co2 0.4

0.02

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i

0

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i

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0.8

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0.4

¢I

0.6 ¢I,

0.8

i

1.0

¢II

Fig. 3. (A) The inter-relationship between ~bI and qp ( O ) , ~bexc ( & ) and SH (Q) for a young, warm grown leaf of pea variety BC1/9RR over a range of irradiances from 64 to 1250/xmol m -2 s -1 PAQF. During the course of the measurements the leaf (which was also used in the experiment described in Fig. 2) was maintained in a gaseous phase comprising of 350 ppm CO2, 20% 0 2 and ~ 80% N 2. The datum marked ( t ) indicates the sample obtained at an irradiance of 520/xmol m -2 s -1 PAOF where ~b~x= 0.51 and th~ 0.73. The dark adapted Fv/Fm is 0.77. (B) The inter-relationship between tbi ( I ) and thxi(O), and the quantum efficiency for CO 2 fixation (~bco2) obtained concurrently from the same leaf.

(Fig. 3b) s h o w t h a t thco2 is s m a l l e r for all v a l u e s o f e i t h e r ~)I or (~ii t h a n u n d e r n o n - p h o t o r e s p i r a t o r y c o n d i t i o n s ( F i g . 2b). T h e r e l a t i o n s h i p b e t w e e n ~bc% a n d b o t h ~bI a n d ~bu a p p e a r s to b e n o n - l i n e a r at h i g h v a l u e s o f ~bI a n d (~II a n d t h e n as ~i a n d ~bli d e c l i n e t h e r e l a t i o n s h i p w i t h 4~co2 becomes linear. Figure 4 shows the relationship b e t w e e n t h e v a l u e s o f 4~co2 o b t a i n e d f r o m t h e y o u n g p e a l e a f u n d e r n o n - p h o t o r e s p i r a t o r y (Fig. 2b) a n d p h o t o r e s p i r a t o r y c o n d i t i o n s ( F i g . 3b). C l e a r l y t h e q u a n t u m y i e l d o f C O 2 fixation for

this l e a f u n d e r p h o t o r e s p i r a t o r y c o n d i t i o n s is l i n e a r l y r e l a t e d to t h e y i e l d u n d e r n o n - p h o t o respiratory conditions over the greater part of a r a n g e o f v a l u e s o b t a i n e d . O n l y at t w o p o i n t s ( o b t a i n e d at 54 a n d 120/.~mol m -2 s -1 P A Q F ) does the linear relationship break down. W h e n a y o u n g p e a l e a f is o p e r a t i n g c l o s e to t h e c o m p e n s a t i o n p o i n t for C O 2 fixation in an atmosphere containing 20% 0 2 and no CO 2 (Fig. 5), t h e r e l a t i o n s h i p s a r e d i f f e r e n t t o t h o s e o b t a i n e d f r o m c o m p a r a b l e l e a v e s in a g a s e o u s

0.04 QQ

0.03

co2 0.02 (20%02) O.Ol

0.02

0.04 t ~ CO2

0.06 (2% 02)

Fig. 4. The relationship between the quantum yields of CO 2 fixation by a young, warm grown leaf of pea variety BC1/9RR irradiated from 64 to 1250 p.mol m -2 S -1 PAQF in either 20% 02, 350ppm CO2, remainder N 2 or 2% 02, 350ppm CO2, remainder N 2. These data were included in Figs. 2b and 3b.

218 1.0

0.8

qp 0.6

d:n 0.4

0.2

0 0

0.2

0.4

0.6

0.8

1.0

Fig. 5. The relationship b e t w e e n ~b~and qp (©), ~

(&) and ~bu ( O ) for a warm grown leaf pea variety of B C 1 / 9 R R over a range of irradiances from 64 to 1 2 5 0 / z m o l m -2 s -~ PAQF. Du rin g the course of the m e a s u r e m e n t s the leaf was maintained in a gaseous phase comprising 20% O~ and 80% N2, no C O 2 was ad ded except by leaf respiration and photo respiration so the leaf was acting close to the compensation point for CO z fixation. The m a r k e d d a t u m ( ,t ) indicates the sample o btained at an irradiance of 5 2 0 / x m o l m 2 s-~ (~bj = 0.49, ~bn = 0.18). The dark a d a p t e d F J F m is 0.79.

phase containing CO 2 at an atmospheric concentration (Figs. 1-3). The relationship between ~bexc and ~b~ is non-linear and the value of ~bexc appears to be reaching a minimum. The quantum efficiency of PS II has a biphasic relationship with ~bx; there is a sudden fall in ~bji at low irradiances and high values of ~bI (again largely due to a marked drop in qp), then ~bi~ displays a curvilinear relationship with respect to ~bi, with ~b[i falling rapidly at moderately high values of

grown pea leaf and a leaf of H. helix are shown in Fig. 6. The gross rate of CO 2 fixation of the cold grown pea is about 25% lower than that of the warm grown pea at the highest irradiance employed (1240/zmol m -2 s -1 PAQF) and the H. helix leaf had a light saturated rate of CO 2 fixation of 2 . 8 / z m o l m -2 s -~ which is 12% of the highest rate obtained from the warm grown pea leaf. The relationships between qp, ~bex c and ~bn, and ~bI for a cold grown leaf measured in air (i.e., under photorespiratory conditions) are shown in Fig. 7a. They are qualitatively similar to those described for young, warm grown leaves of B C 1 / 9 R R (Figs. 2a-3a). However, at 520/zmol m -2 s -~ both (~I and ~b~iare lower than for a young, warm grown pea leaf photosynthesizing in air (compare Figs. 3a and 7a). The dark adapted Fv/F m is 0.73 which is lower than for young warm grown peas (FJFrn (dark adapted) = 0.79, Fig. 2a) indicating that the efficiency of PS II is intrinsically lower than for the

16

12 --4

t~

8

g The effect of decreased rates of CO 2 assimilation on the relationship between ~b., qSi and qSco2 under photorespiratory conditions were investigated using a pea leaf grown under cold conditions and a leaf of H. helix. It was necessary to use a leaf of H. helix instead of the pea leaf because a low rate of photosynthesis was required for the study and pea leaves with a maximum rate of CO~ fixation of between 2 - 3 tzmol - 2 -1 m s would display considerable secondary damage, such as chlorosis, which would complicate the interpretation of the results. The relationships between the gross rate of CO e fixation and irradiance of a warm grown pea leaf, a cold

4

0

r 200

i 400

v 600

'l 800

7 1000

1200

i 1400

irradiance(lamol m-2 s-l)

Fig. 6. The de pe nde nc y of the gross rate of CO 2 fixation upon irradiance for warm grown ( A ) and cold grown ( 0 ) pea leaves, and for a leaf of Hedera helix (field collected) (V). All m e a s u r e m e n t s were ma de at 20°C in an a t mos phe re of 20% 02, 350 ppm CO2, r e m a i n d e r N 2.

219 1.0

qp

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016

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Fig. 7. (A) T h e inter-relationship, over a range of irradiances from 64 to 1250/zmol m -2 s -1 P A Q F , between ¢b~ and qp ( O ) , ~boxc ( A ) and ~bi~ ( 0 ) for a leaf of pea phase comprising of 20% 0 2 , 3 5 0 sample obtained at an irradiance inter-relationship b e t w e e n ~b~ ( 1 ) leaf.

variety JI 1345 grown u n d e r cold conditions. D u r i n g the course of the m e a s u r e m e n t s a gaseous p p m CO2, r e m a i n d e r N 2 was maintained around the leaf. T h e d a t u m m a r k e d ( l ) indicates the of 5 2 0 / z m o l m 2 s-~ P A Q F (q~i = 0.63, ~bii = 0.40). T h e dark adapted F J F m is 0.73. (B) T h e and ~I1 (0), and the q u a n t u m efficiency for C O 2 fixation (~bco2) obtained concurrently from the

young warm grown control plants. The relationship, for the cold grown pea leaf, between photochemistry and the quantum efficiency of CO 2 fixation showed a strong curvilinear relationship (compare Figs. 3b and 7b). This is associated with a generally lower rate of CO 2 fixation 1.0

measured from the cold grown pea leaf relative to that of the warm grown pea leaf under identical measurement conditions (Fig. 6). Although the rate of photosynthesis recorded from a leaf of H. helix is low (Fig. 6) and the quantum efficiency at 520 ~ m o l m -2 s-1 is lower

j

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qp

~exc 0.6

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0, Fig. 8. The inter-relationship, over a range of irradiances from 54 to 620/~mol m -2 s -1 P A Q F , b e t w e e n ~b~and qp (©), ~bexc ( A ) and ~bn ( 0 ) for a leaf of Hedera helix (collected from the field). D u r i n g the course of the m e a s u r e m e n t s the leaf was m a i n t a i n e d in a gaseous phase comprising of 350 p p m CO2, 20% O z and ~ 80% N 2. T h e d a t u m m a r k e d ( t ) indicates the sample obtained at an irradiance of 520 tzmol m -z s -~ (~i = 0.41, qbn = 0.26). T h e dark adapted Fv/F m is 0.754. (B) T h e inter-relationship b e t w e e n ~b~ (11) and ~bn ( O ) , and the q u a n t u m efficiency for C O 2 fixation (~bco2) obtained concurrently from the s a m e leaf. T h e relationship b e t w e e n ~bu and the q u a n t u m efficiency for C O z fixation for a leaf of H. helix u n d e r non-photorespiratory conditions is also s h o w n

(A).

220 for both P S I and PS II, the relationships between the photochemical parameters of the leaf (Fig. 8a) are similar to those from comparable pea leaf examples (Figs. la, 2a, 3a and 7a). The relationships in the H. helix leaf between the quantum efficiency of CO 2 fixation in air and ~i and 4~ir are non-linear (Fig. 8b). At high values of (~I or ~II there is a marked drop in ~co2 with only a comparatively small fall in either ~bI or thii. This is followed by a phase with ~/h and ~II declining relatively more sharply than thco:. The transition between these two phases corresponds approximately to the transition from the light-limited to the light-saturated regions of the photosynthesis-irradiance curve (Fig. 6). Though, however, the relationships between ~i and ~II and thco2 are non-linear under photorespiratory conditions, for a similar H. helix leaf under non-photorespiratory conditions this relationship between 4h or t~H and 4~co2 is linear (Fig. 8b, only data for ~ll shown). It is evident that under photorespiratory conditions leaves with a low rate of photosynthesis retain a largely linear relationship between th~ and ~II but have a non-linear relationship between ~bco2 and t~i or (~ii. The consequences for the relationships between the photochemical parameters and thco: following the elimination of

1.0

photorespiration in a cold grown pea leaf with a low rate of CO 2 fixation are shown in Fig. 9. At saturating irradiance the gross rate of CO 2 fixation of this leaf was 6.25/.tmol m -2 s -1, approximately 25% of the rate displayed by warm grown pea leaf (23.5/xmol m -2 s-l). The relationships between thli, ~bx and ~bexc are non-linear (Fig. 9a) with thexc tending to minimum value. This leaf had a dark adapted Fv/F m (0.67) which was lower than for the warm grown pea leaves. The relationship between ~)II and thco2 remained linear (Fig. 9b) whereas that for ~i and 4~co2 became non-linear paralleling the relationship between thl and &ii.

Discussion

Relationships between CO 2 fixation and photochemical efficiencies of P S I and PS H Under conditions where photorespiration is largely abolished, 4~co2 has been shown to be linearly related to 4h and (~II (Weis et al. 1987, Genty et al. 1989, 1990) and the data for a mature pea leaf presented in Fig. lb are in agreement with this. Although linear relation-

0.05

A 0.8

0.04

qp

t~co2

(~exe 0.6

0.03

~n 0.4

OO

0.02

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0 0

, 0.2

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0 0.8

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, 02

, 0'

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1.0

Fig. 9. (A) T h e relationship, over a range of irradiances b e t w e e n 64 and 1 2 5 0 / x m o l m 2 s-1 P A Q F , b e t w e e n 4h and qp (C)), ~bxc ( & ) and ~bu ( 0 ) for a cold g r o w n leaf of pea variety J179. The leaf was m a i n t a i n e d in a gaseous p h a s e comprising 350 p p m CO2, 2 % 0 2 and r e m a i n d e r N 2. T h e d a t u m m a r k e d ( 1 ) indicates the sample obtained at an irradiance of 5 2 0 / x m o l m 2 s-~ P A Q F (~b~ = 0.57, ~bll = 0.11). T h e dark adapted F v / F m is 0.67. ( B ) The relationship b e t w e e n ~b~ ( I ) and ~b~ ( O ) , and the q u a n t u m efficiency for C O 2 fixation (4'co2) obtained concurrently f r o m the s a m e leaf.

221 ships between either (~I o r ~bi~ and qbco2 are expected under non-photorespiratory conditions, the data shown in Fig. 2b conflict with this generalization. In this instance a very young leaf was used to obtain a high rate of CO 2 fixation, consequently it is possible that the non-linearity between either ~b~or ~bi~is due to competition for reductants and ATP between CO 2 fixation and the numerous other processes known to be located in the chloroplast (e.g., nitrite reduction, sulphate reduction protein synthesis (Anderson 1981)). Such processes are likely to be more active in younger leaves and thus their effect as a competitive sink for the products of electron transport would be more evident. An alternative explanation could be that the level of 'dark' respiration occurring in parallel with photosynthesis is either over-estimated at the lowest irradiances, resulting in an over-estimate of ~bco2, or under-estimated at moderate to high irradiances causing an under-estimate of ~bco2. Besides possibly producing the non-linearity between ~bco2 and photochemical efficiency in Fig. 2b, consistent error in the estimation of the dark respiration occurring in parallel with photosynthesis might also explain the failure of the extrapolation of both t h e ~)CO2--(DII a n d ~)CO2--(~I relationships to intercept the origin in Figs. 2b, 3b and 7b. The values of ~bco2, relative to ~)I o r ~)II are lower than under non-photorespiratory conditions (Genty et al. 1990). This is a consequence of the competition between O 2 and CO 2 for the active site of RuBisCo and the subsequent loss of CO 2 via photorespiratory cycle following an oxygenation of ribulose bisphosphate coupled with the need for ATP and N A D P H to regenerate ribulose bisphosphate from the products of the oxygenation reaction (Edwards and Walker 1983). Contrary to the previously reported relationships between ~bco2 and 4h o r ~)ii for barley leaves under photorespiratory conditions (Genty et al. 1990) the relationships shown in Fig. 3b are no more curvilinear than those for the same leaf under non-photorespiratory conditions (Figs. 2b and 4). These data and the more curvilinear relationships between ~bco2 and both ~bi and (~I| obtained from a cold grown pea leaf (Fig. 7b) and a H. helix leaf (Fig. 8b), reveal the potential flexibility in the relationship between the quan-

tum efficiency for electron transport and carbon dioxide fixation in air. This implies a flexibility between the relative rates of carboxylation and oxygenation which may be due to stomatal modulation of the intercellular CO 2 pressure and the related intra-cellular oxygen: carbon dioxide ratio (Peterson 1989) and possibly also reflects competition between the Mehler reaction and the Calvin cycle for electrons from the thylakoid electron transport chain. These results also demonstrate the capacity of photorespiration to act as a sink for the products of thylakoid electron transport. The data for H . helix (Fig. 8a) illustrates an extreme response from a leaf exhibiting a very low rate of CO2 fixation at saturating irradiance, and with a high intrinsic ~b~ and no evident chlorosis. In this case when ~bI = 0.5, ~bco2 = 0.0065, whereas if ~bco2 and ~bI had been linearly related (as in Fig. lb) qbco2 would have been 0.0165. It is apparent from the values of ~bI or ~bii at 520/xmol m -2 s -~ PAQF (Figs. 3a, 7a and 8a) that the depressed rates of photosynthesis (Fig. 6) are also paralleled by reduced values of ~b~and ~bl~.Thus the decline in the rates of CO 2 fixation under photorespiratory conditions, and the decline of ~bco2 that this represents, can be attributed to two factors. Firstly, there is a change in the irradiance response of ~b7 and ~bii such that these parameters decline more sharply with increasing irradiance; this will result in a fall in the efficiency of electron transport and the flux of reducing equivalents through the electron transport chain. Secondly, the relationship between either ~b~ or ~bi~ and ~bco2 changes, possibly due to changes in the relative rates of the oxygenation and carboxylation of ribulose bisphosphate or of the Mehler reaction, such that ~bco~ declines relative to either ~bj or ~bn . It is also evident that the presence of photorespiration, with its potentially variable contribution to the utilization of N A D P H and ATP, will prevent the use of measurements of either ~bI or ~bu alone to provide a quantitative estimate of ~bco2 in C3 plants. The parallel changes in the irradiance response of ~ and ~bu (as indicated by the values of ~bI and ff)ll at 520/zmol m -2 s -1 in Figs. 3a, 7a and 8a) will, however, allow a qualitative estimate of the stress-induced restriction of photosynthetic CO 2 fixation to be made using measurements of ~bI and ~bii.

222

Relationships between PS H and PS I a) Predominantly linear relationships The linear relationship between ~bI and ~II shown in Figs. la, 2a, 3a, 7a and 8a are consistent with a predominant role for linear electron flow from water to ferredoxin relative to any cyclical electron transfer processes. Under conditions of linear electron flow the fluxes through both photosystems must be equal under steady state conditions. In this paper we describe changes in quantum efficiencies of the photosystem, not electron fluxes; nonetheless, at the steady state for linear electron flow a change in the quantum efficiency of one photosystem must be balanced by a proportional change in the quantum efficiency of the other in order to maintain an equivalent flux through both photosystems. With increasing irradiance the rate of linear electron flow will become progressively limited by the reaction between plastoquinol and the Reiske FeS centre of the cytochrome b6/f complex (Siggel 1976, Tikhonov et al. 1984). This will result in a progressive, balanced loss of ~besii and (DPSI" A small non-linear phase in the relationship between ~bI and ~bn is consistently observed at low irradiances and high values of 4>l and ~bn and, as reported previously (Harbinson et al. 1989), can be attributed principally to changes in qa. This decline of ~b~ relative to ~bI is greater under conditions where photorespiration is abolished (compare Figs. 2a and 3a, see also Genty et al. 1990) so it is not possible to explain the decline in ~bn relative to ~b~ solely in terms of the stoichiometry and absorbance cross sections of PS I and PS II (see Harbinson et al. 1989). This is similar to results reported by Genty et al. (1990). It is possible that this decline and shift of the balance between ~b~ and t~i I is due to a consequence of regulatory processes influencing the photochemical efficiency following alterations in the demand for ATP and N A D P H as photorespiration is eliminated. The more marked loss of ~i~ relative to ~b~in these circumstances (via a decline of qp) could be due to an increase in cyclic flow around PS I, though without an explicit demonstration of cyclic flux this must be considered a speculation. In leaves of cold grown pea and H. helix the rate of photosynthesis is considerably reduced

relative to that recorded for the warm grown pea leaf (Fig. 6). Though the relationships between ~bI and ~brl were apparently linear, as for the warm grown pea example, the relationships between ~b~, ~b~ and irradiance were different. As the rate of CO 2 fixation declined then so the values of ~bI and ~bli at 520/xmol m -2 s -a also declined. This implies a lower rate of photosynthetic electron transport and a larger pool of P700 + at a given irradiance following a decline in the rate of CO 2 fixation in the presence of 20% 0 2. However the relationship between the decline in the rate of CO 2 fixation and the decline of ~b~ or ~bl~ is not quantitative under photorespiratory conditions because of changes that occur in the relationship between both ~bi and ~bn and 4~c%.

b) Non-linear relationships In a cold grown pea leaf with a depressed rate of CO 2 fixation and a high dark adapted Fv/F m (i.e., the intrinsic efficiency of PS II is high) the relationship between ~bi~and qbco2 remains linear under non-photorespiratory conditions (Fig. 9) even though values of ~bco2 and ~II are both generally low relative to those of warm grown pea leaves (Figs. lb and 2b), whereas the relationship between ~bI and ~bc% was curvilinear. In these circumstances where ~b~ no longer correlates with the quantum efficiency of linear electron flow, ~bH is still a reliable means with which to estimate the quantum efficiency of linear electron flow. The curvilinear relationship between (~1 and ~b~i, and the relatively sharp decrease in qp with decreasing ~b~ would be consistent with a cyclic flux of electrons around PS I. As cyclic electron flow increases in proportion to linear electron flow then 4'~ decrease relative to ~bI. This can be seen more clearly in Fig. 10 which shows the relationship between the index for electron flux through PSII, J~i, which is the product of ~II and irradiance, and the index for electron flux through PS I, Jp which is the product of ~b~ and irradiance. In the case of young warm grown pea leaves in air or under nonphotorespiratory conditions the relationship between J~ and J~i is linear though that for the non-photorespiratory example indicates a relatively greater flux through P S I than PS II. This is a reflection of the relatively greater fall of ~II

223 400

JIl 2oo

0

w

w

200

~

i

600

400

JI

relationship between the index of electron flow through PSI (J0 (given by the product of ~ and irradiance), and the index of electron flux through PS II (JlI) (given by product of dSi and irradiance) for young, warm grown leaves of BC1/9RR in 350ppm CO2, 2% 0 2, and -98% N2 (©); or 350 ppm CO2, 20% 02 and 80% N2 (O); or in 20% 0 2, and 80% N2 (T); and a leaf of pea variety JI79 (cold grown) in 350 ppm CO2, 2% O2 and 98% N2 (ll). These data were calculated from data used in Figs. 2, 3, 7 and 8, respectively. Fig. 10. T h e

relative to ~b~ that occurs at high values of ~II and ~I (and low irradiances) under non-photorespiratory conditions compared to photorespiratory conditions. The curvilinear relationships between thl and ~II obtained from a warm grown pea leaf at the C O 2 compensation point and a cold grown pea leaf under non-photorespiratory conditions both result in non-linear relationships between JI and J~ with JII saturating with respect t o J i . In these cases the maximum indices for electron flux through both photosystems are considerably less than those estimated for warm grown pea leaves in air or under non-photorespiratory conditions even though the maximum irradiance employed was the same in all cases. The predominantly non-linear relationships between J~ and Jn represent a qualitatively different relationship between photosystems I and II to that described previously (Harbinson et al. 1989, G e n t y et al. 1990) and which is consistent with a predominant role for linear electron flow. It appears that for leaves photosynthesizing in air, or actively photosynthesizing in non-photorespiratory conditions a balanced flux through both photosystems can be maintained, and electron flow is predominantly linear. In such a situation the total d e m a n d for A T P and reductants can be balanced against the combined A ~ + and reduced ferredoxin generating capaci-

ty of linear electron flow, and the A/.q~+ generating capacity of pseudocyclic electron flow to O 2. Cyclic electron flow may occur in this situation, however it must represent only a small proportion or a fixed proportion of the total electron flux from plastoquinol to PS I. If cyclic electron flow were to vary as a proportion of total electron flow the relationship between Ses i and SPs n would be non-linear throughout the range. U n d e r more extreme physiological conditions the relationship between ~I and ~II is predominantly non-linear which is inconsistent with a predominant role for linear electron flow and may indicate a substantial cyclic electron flux about P S I . This may be a response to an extreme imbalance between the demand for reducing equivalents and the potential supply of reducing equivalents by the thylakoid electron transport.

Conclusions

The following major conclusions can be drawn from the results presented here. i) In air (i.e., photorespiratory conditions) ~)I and ~II are linearly related implying a predominant role for linear electron flow. The overall quantum yield of CO z fixation is determined by photochemical efficiency of the thylakoids a n d to a variable degree, by competition between CO 2 and 0 2 (both via photorespiration or the Mehler reaction) for the products of thylakoid electron transport. ii) When photorespiration is prevented the relationship between I#i and (~II will be predominantly linear. U n d e r these conditions the rate of linear electron transport will be directly related to the rate of CO 2 fixation and the quantum yield of CO 2 fixation will be linearly related to both (~I and ~II" iii) A non-linear relationship between ~bI and (~II may develop when a marked imbalance exists between the potential rate of electron flow and the metabolic sink activity of the stroma and the Mehler reaction for reducing equivalents. This non-linear relationship may involve a cyclic electron flux around P S I which would result in a depression of (~II and 4~co2 relative to thl.

224

Acknowledgements J . H . was s u p p o r t e d by the P e r r y F o u n d a t i o n , B o r e h a m , Essex, U . K . T h e O r g a n i z a t i o n for Economic Cooperation and D e v e l o p m e n t (Project o n F o o d P r o d u c t i o n a n d P r e s e r v a t i o n ) prov i d e d s u p p o r t for B G . T h e w o r k was also supp o r t e d b y a g r a n t f r o m the U . K . A g r i c u l t u r a l a n d F o o d R e s e a r c h C o u n c i l (no. A G 8 4 / 4 ) . J . H . w o u l d like to t h a n k D r C.L. H e d l e y for s u p p o r t a n d g u i d a n c e d u r i n g this w o r k a n d also to ack n o w l e d g e the help of Messrs K. B u r c h a m , D. W i l t o n a n d D. W o o d s in c o n s t r u c t i n g the e q u i p m e n t u s e d in this work.

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