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Influence of glycerate on photosynthesis by wheat chloroplasts
OF BIOCHEMISTRY AND BIOPHYSICS 231, No. 1, May 15, pp. 124-135, 1934
ARCHIVES
Vol.
Influence of Glycerate on Photosynthesis GERALD
E. EDWARDS’
AND
by Wheat Chloroplasts’
DAVID
A. WALKER
Department of Botany, ARC Research Group on Photosynthesis, University of Sh&ti, Sh&k.!d ,910 %TN, United Kingdom Received
November
1, 1983, and in revised
form
January
19, 1934
Glycerate was found to effect photosynthetic O2 evolution in wheat chloroplasts by its conversion to triose phosphate and by influencing the rate of photosynthesis through the reductive pentose phosphate pathway. In the absence of bicarbonate, the photosynthetic Oz evolution with glycerate was low (10 to 25 pmol mg chlorophyll-’ h-l), and only about 15% of the rate of bicarbonate-dependent O2 evolution under optimum conditions. This corresponds to a rate of glycerate conversion to triose phosphate of 20 to 50 pmol mg chlorophyll-’ h-l, which appears sufficient to accommodate flux through the glycolate pathway in viva. Pi was required for this glycerate-dependent Oz evolution; rates remained relatively constant between 0.1 and 40 mM Pi, and proceeded with little lag upon illumination (less than 0.5 min). Evidence for Oz evolution due to glycerate conversion to triose phosphate could be conclusively demonstrated by addition of glycolaldehyde, an inhibitor of the regenerative phase of photosynthesis, which prevents COz fixation. The effect of glycerate on photosynthesis in the presence of bicarbonate was determined by measuring both photosynthetic O2 evolution and 14COzfixation at varying Pi concentrations. Low concentrations of glycerate (micro- to millimolar levels) prevented inhibition of photosynthesis by Pia With 1 mM bicarbonate and pH 8.2, which is favorable for glycolate synthesis, maximum rates of photosynthesis were obtained at low Pi (25 PM), whereas strong inhibition of photosynthesis occurred at only 0.2 mM Pi. Addition of glycerate relieved the inhibition of photosynthesis by Pi, indicating the possible importance of glycerate metabolism in the chloroplast under photorespiratory conditions. The initiation of photosynthesis by glycerate at inhibitory Pi levels occurred with little reduction in the ratio of COz fixed/O2 evolved, and the main effect of glycerate was on carbon assimilation. While the basis for the beneficial effect of glycerate on COZ assimilation under moderate to high Pi levels is uncertain, it may increase the concentration of 3-phosphoglycerate (PGA) in the chloroplast, and thus make conditions more favorable for induction of photosynthesis and reduction of PGA to triose phosphate.
During photorespiration in plants, glycerate is synthesized outside the chloroplast
as a product of the glycolate pathway. There is considerable evidence which suggests that glycerate enters the chloroplast and is converted to organic phosphates. Recent studies on a number of species indicate glycerate kinase may be exclusively located in the chloroplast (1, 2). Robinson (3, 4) has evidence for a light-dependent uptake of glycerate into the chloroplast on a specific glycerate translocator. Glyceratedependent Oz evolution occurs with isolated chloroplasts (5-Q, although it has been considered insufficient to accommodate
r G.E.E. is indebted to the Royal Society for a Guest Research Fellowship, and to the Science and Education Administration for support under Grant 83CRCR-1-1321 from the Competitive Research Grants Office. The authors appreciate helpful discussions with Bob Furbank, Richard Leegood, and Simon Robinson, and other assistance by Jackie Rowe11 and Christine Foyer during the course of the work. ‘To whom correspondence should be addressed: Botany Department, Washington State University, Pullman, Wash. 99164-4230. 0003-9861134 Copyright All rights
$3.00
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
124
INFLUENCE
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photorespiration (5). The purpose of the present study with wheat chloroplasts was to determine the rate of glycerate conversion to triose-P3 and the influence of glycerate on CO, assimilation under varying levels of Pi and bicarbonate. MATERIALS
AND
METHODS
Plant materiaL Wheat (TrScum w&&urn, var. Timmo) was grown in vermiculite in a greenhouse with sunlight supplemented by 400-W Wotan lamps. Day temperatures were normally 2’i-33°C and night temperature about 19°C. Isolation of protop~ts and chJuropl&s. Plants were used 6 to 7 days after planting with a plant height of 7 to 8 cm. The upper 4 cm of the plants was harvested and mesophyll protoplasts were prepared essentially as previously described (8). Leaf segments approximately 0.7 mm in width were prepared by stacking and cutting about 70 to 90 leaves at a time with a single-edge razor blade. Cutting small segments with frequent changes of blades is essential for obtaining good yields of protoplasts. Approximately 8 g of leaf tissue was prepared and incubated in 75 ml of digestion medium (see Ref. (8) for details). Following purification of the protoplasts, they were suspended in 0.5 M sorbitol, 0.05% BSA, centrifuged for 2 min at 25Og, and resuspended in 3 or 4 ml of the chloroplast isolation, resuspension, and assay medium, which included ‘25 mM Tricine-KOH, pH 8.2, 10 mM EDTA, and 0.4 ivt sorbitol. Chloroplasts were isolated by passage of the protoplasts (approximately 500 pg Chl/ml) through a 20-pm nylon net (8). Chl was determined in 96% ethanol according to Wintermans and De Mots (9). Assay of photosynthesis. 0, evolution was followed polarographically at 20°C using a twin Clark-type electrode system (10) purchased from Hansatech Ltd., Norfolk, U. K. The assay medium contained 0.4 M sorbitol, 10 mM EDTA, 25 mM Tricine-KOH, pH 8.2, 200 units of catalase in a total volume of 0.6 ml, and additions of bicarbonate, Pi, and glycerate as indicated. The pH Iof the stock solution for assay containing buffer, E:DTA, and sorbitol was adjusted with fresh pellets of KOH. This solution was normally boiled to remove CO*, and stored sealed with parafilm in a 50-ml test tube prior to use for experiments with-
3 Abbreviations used: Chl, chlorophyll; DHAP, dihydroxyacetone phosphate; PGA, 3-phosphoglycerate; pyridoxal-P, pyridoxal phosphate; RuBP, ribulose 1,5bisphosphate; triose-P, triose phosphate; BSA, bovine serum albumin; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyll)ethyl~lycine; TCA, trichloroacetic acid.
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out bicarbonate, or with 1 mM bicarbonate in the assay. COz fixation was determined simultaneously with Oz evolution (11) using [14C]bicarbonate. Determination of glycerate and glycerate kinase. In experiments where glycerate was assayed, the reaction mixture was the same as the assay medium for O2 evolution studies, plus other additions were made as indicated in Table I. After 9 min of incubation in the light a 0.5-ml aliquot was killed with 30 pl of 12 N perchloric acid. Following storage on ice for 30 min, samples were centrifuged at 4OOg for 2 min and then neutralized by addition of 60 pl of 5 N K.&O3 (R. Leegood, personal communication). After centrifugation at 4009 for 2 min, the supernatant was analyzed for glycerate using glycerate kinase, PGA kinase, and NAD triose-P dehydrogenase as previously described (12). Glycerate kinase activity extracted from chloroplasts was determined by coupling to PGA kinase and NAD triose-P dehydrogenase (2). Infomnation on certain chemicals and enzymes. The isomers of glycerate were obtained in the form of hemi-calcium salts and DHAP was obtained in the form of lithium salt, all from Sigma. The calcium and lithium were removed by exchange on Dowex 50W hydrogen form cation-exchange resin. PGA was obtained as the trisodium salt from Sigma. Glycolaldehyde was purchased as the dimer (2,5-dihydroxy1,4-dioxane) from Aldrich Chemical Company and is converted to the monomer in solution. PGA kinase and NAD-triose-P dehydrogenase were obtained as a mixture from Boehringer-Mannheim, while glycerate kinase (from rye) was provided by Mark Schmitt.
RESULTS
The influence of Pi on rates of photosynthesis and on the induction period upon illumination of wheat chloroplasts in the presence of 10 mM bicarbonate (Figs. 1A and B) was similar to that previously reported (8). At high levels of Pi, the rates of photosynthesis decreased and the lag period increased up to 5 to 6 min, typical of C3-type chloroplasts. The optimum Pi level was around 0.2 mM and there was little photosynthesis without addition of Pi. Addition of 2 mM DL-glycerate had a strong influence on photosynthetic O2 evolution, preventing the inhibition of photosynthesis by Pi concentrations between 0.4 and 2 mM, and decreasing the induction period over the entire Pi range from 0 to 2 rnM. At 1 mM bicarbonate (Figs. 1C and D), the Pi optimum was very low (around 25 PM), and substantial photosynthesis oc-
126
EDWARDS I A. pi 6.2 10 mM btcarbonate
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AND
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WALKER
I
1
6
I
B. pH 62 10 mM bxarbonate
OC d o-
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2 [Pl]mM I -
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D. pH 6.2 1 mM bourbonate
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,
[Pi]mM I
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F. pii 8.2 No bocarbonats
No bicarbonate 1.5 -
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IA
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.
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m-glycerate I 0.75
A I 1.0
FIG. 1. Effect of 2 mM DL-glycerate on the rate and induction period of light-dependent Oa evolution by wheat chloroplasts under varying levels of Pi. (A) Rate of O2 evolution and (B) induction period in the presence of 10 mre bicarbonate (assay contained 38 pg ChlI0.6 ml). (C) Rate of O2 evolution and (D) induction period in the presence of 1 mM bicarbonate (assay contained 20 ng ChlI0.6 ml). (E) Rate of Oa evolution and (F) induction period without bicarbonate (assay contained 2’7 pg Chl/ 0.6 ml). The results shown are at pH 8.2. Similar data were obtained at pH 7.6 in experiments with 10 mM bicarbonate and without bicarbonate.
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curred without addition of Pi as previously reported (13). Whereas 0.2 mM Pi was strongly inhibitory to photosynthesis, the addition of 41 mM DL-glycerate alleviated the Pi inhibition of photosynthesis between 50 PM and 1 mM Pi. At 10~ levels of Pi (0 to 25 PM), addition of glycerate caused an inhibition of photosynthesis (Fig. 1C) and, with time, rates progressively curved off more rapidly in the presence of glycerate than in its a.bsence (not shown). The induction period was dramatically decreased by addition of glycerate, being less than 1 Inin Up to Pi levels Of 1 mM. Photosynthetic O2 evolution in the presence of 2 mMDL-glycerate, without addition of bicarbonate (Figs. 1E and F), was largely dependent on additions of Pi, and the induction period was 0.2 min or less up to 1 mM Pi. Rates of glycerate-dependent O2 evolution without bicarbonate were typically between 15 and 25 /*mol O2 evolved mg Chl-l h-l,, which are about five- to sevenfold lower than the maximum rates obtained under optimum conditions with bicarbonate as substrate. Photosynthesis in the presence of 2 mM DL-glycerate ‘was inhibited with increasing levels of Pi up to 20 mM (Fig. 2); however, even at 40 m&I Pi the rate of photosynthetic O2 evolution in the presence of 2 mM DLglycerate was about half the maximum rate at lower Pi. Even at 40 mM Pi with 2 mM DL-glycerate, the rate in the presence of bicarbonate was about twofold the rate without bicalrbonate. Addition of pyridoxal-P, an inhibitor of the phosphate translocator (14), prevented inhibition of photosynthesis at high Pi in the presence of glycerate. ‘The rate of photosynthetic O2 evolution in the presence of glycerate, and without bicarbonate, was relatively constant between 1 and 40 mM Pi. Without bicarbonate, the concentration of DL-glyceralte required for half-maximum rates of photosynthetic O2 evolution was about 0.5 m&I with both 0.2 and 2 mM Pi (Fig. 3). With 10 mM bicarbonate and 2 mM Pi, increasing concentrations of DL-glycerate initiated photosynthesis and reduced the induction period, with half-maximum effectiveness at 0.1 mM DL-glycerate (Fig. 3).
IN
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20 [Pe]mM
30
127
40
FIG. 2. Influence of 2 mM DL-glycerate on the rate of photosynthetic O2 evolution by wheat chloroplasts under high levels of Pi. Assays contained 27 pg Chl/ 0.6 ml. Pyridoxal-P was added to the assay medium with chloroplasts 1 min prior to illumination. Pi was added immediately before illumination. Similar results were obtained at pH 7.6.
Eflect Of DL, D, and L i.xme?-s of glycerate on photosynthesis. The D isomer was more effective than the L isomer of glycerate as a substrate to induce photosynthetic O2 evolution, either in the absence of bicarbonate or in the presence of bicarbonate with high Pi concentrations. Based on measurements on glycerate-dependent OS evolution in the absence of bicarbonate, the D isomer gave twofold higher rates than the L isomer at saturating concentrations (5 mM), while one-half of the maximum rate occurred at 0.3 mM D-glycerate and 1.5 mM L-glycerate. At saturating concentrations, the D isomer and DL mixture of isomers of glycerate had the same effect (results not shown). In subsequent experiments the D isomer was used.
Cmparism of the eflects of o-glycerate, DHAP, and PGA on photosynthetic 0, eve lution. D-Glycerate and DHAP were found to be very similar in their ability to prevent of photosynthesis with 10 mM bicarbonate and 3 mM Pi, as seen in Fig. 4. PGA also partially overcame the Pi in-
Pi inhibition
128
EDWARDS
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WALKER
Evaluation of the efect of glycerate on function of the C, cycle. In the presence of
[DL-glvcerate] I pi+ 8.2 1OmM bicarbonate
200 x
I
mM I
I
I
- 10
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-6X F 2 -
4E x !I
I
2 x
40
-“\ 1
x-x-x
2
X
[a-glycerate]
mM
FIG. 3. The
influence of varying concentrations of on photosynthetic O2 evolution and induction period in the absence or presence of 10 mM bicarbonate with wheat chloroplasts. Assays contained 20 pg ChV0.6 ml.
bicarbonate, glycerate could lead to an enhancement of photosynthetic O2 evolution by direct conversion of glycerate to trioseP and, in addition, by making conditions more favorable for the C3 cycle to function. The effect of glycerate on COz fixation was evaluated by making simultaneous measurements of CO2 fixation and O2 evolution. The influence of D-glycerate on rates of O2 evolution, the ratio of CO2 fixed/O2 evolved, and the induction period at 0.2 and 2 mM Pi was determined (Fig. 5). At 0.2 mM Pi, increasing glycerate had little effect on the rates of O2 evolution, while the lag in photosynthesis was reduced from 2 to less than 1 min and the ratio of COB fixed/O2 evolved decreased. At 1 m&i glycerate, the C02/02 ratio decreased to a value of 0.86, suggesting that about 86% of the O2 evolved was due to CO2 fixation and 14% due to glycerate conversion to triose-P. In the presence of 2 mM Pi and 10 mM bicarbonate, low levels of glycerate between 0 and 100 PM caused a large decrease in the induction period and an increase in photosynthetic O2 evolution, while causing little decrease in the ratio of COB fixed/O2 evolved (Fig. 5). At higher glycerate concentrations (up
DL-&CeI’ate
hibition of photosynthesis, but not as well as DHAP or glycerate. DHAP and glycerate were also more effective than PGA in reduction of the lag period (not shown). At this level of Pi (3 mM), the calculated concentrations giving half-maximum reduction of the lag period by DHAP (less than 20 PM) and glycerate (approximately 30 PM) were lower than the concentrations required to give half-maximum stimulation of photosynthetic rates (greater than 0.1 mM). At higher concentrations of Pi (10 mM), the ineffectiveness of PGA, relative to that of D-glycerate or DHAP, in overcoming the Pi inhibition of photosynthesis was even more apparent (results not shown).
loo-
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pH 8.2 10 mM bacarbonsts
I 0.2
1 0.4
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1 0.8
1 1.0
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FIG. 4. A comparison of the effects of D-&Cerate, DHAP, and PGA on photosynthetic 0, evolution by wheat chloroplasts with 10 mM bicarbonate and 3 mM Pi. Assays contained 35 pg ChV0.6 ml. Similar data were obtained in a separate experiment at 2 mM Pi.
INFLUElNCE
I .Floo~~~--_loI 60 -
60-
40 -
OF
GLYCERATE
ON
PHOTOSYNTHESIS
*
-08 6 5 e
- 0.6 ': Q Px -045 I9
FIG. 5. The ratio of COa fixed/O2 evolved and rate of Oz evolution during photosynthesis by wheat chloroplasts in the presence of 10 mM bicarbonate and varying concentrations of D-glyeerate (upper figure with 0.2 mMPi; lower figure with 2 mM Pi). The assays contained 30 pg Chl and 3.4 &i bicarbonate per 0.6 ml. The results are the averages of two experiments with wheat chloroplasts prepared on separate days. At the end of the assay period (5 to 10 min after illumination) duplicate samples of 50 ~1 each were taken, killed with 50 ~1 of 20% TCA and analyzed by scintillation spectroscopy.
to 1 mM) phot’osynthesis reached a maximum and induction a minimal value, while the ratio of CO2 fixed/O2 evolved decreased to a value of 6.89. Therefore, in these experiments, even at the higher levels of glycerate the major portion of the O2 evolved was associated with CO2 fixation.
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Figure 6 shows a time course of COz fixation and Oz evolution in the absence and presence of glycerate. Without glycerate there was a similar increase in O2 evolution and COz fixation, with a maximum rate of photosynthesis of 100 pmol mg Chl-’ h-l. In the presence of 1 mM D-glycerate and 20 mM Pi, the initial rate of O2 evolution following induction was noticeably faster than the initial rate of CO2 fixation. After reaching a steady-state rate, O2 evolution exceeded COz fixation by 13 ,Fcmol mg Chl-’ h-l, which is indicative of the Oz evolution associated with conversion of glycerate to triose-P. The direct utilization of glycerate under conditions where glycerate overcomes Pi inhibition of photosynthesis was evaluated. In the presence of 200 pM glycerate and 2 mM Pi, photosynthetic O2 evolution increased with increasing CO2 from 0 to 1 to 10 mM bicarbonate (Table I). However, under all conditions only one-half or less of the glycerate added was utilized during the assay period. The Oz evolution without bicarbonate could be largely accounted for if the glycerate utilized was converted to triose-P. However, in the presence of 1 or 10 mM bicarbonate, glycerate utilization could only account for a small percentage of the Oz evolved. Injuence of glycolaldehyde on photosynthetic 0, evolution in the presence of glycerate and bicarbonate. Glycolaldehyde (an
inhibitor of the regenerative phase of photosynthesis, see Ref. (15)) at a concentration of 12 mM inhibits bicarbonate-dependent O2 evolution (Fig. 7, traces a and b), following which Oz evolution is initiated by addition of 3 mM D-glycerate (rate of 23 pmol mg Chll’ h-l). With bicarbonate and glycerate added prior to illumination (Fig. 7, traces c and d), glycolaldehyde only partially inhibited photosynthetic Oz evolution (rate in presence of glycolaldehyde of 27 lmol mg Chll’ h-l). Glycolaldehyde had little effect on glycerate-dependent O2 evolution without bicarbonate (Fig. 7, traces e and f); addition of bicarbonate during the course of the assay caused a large stimulation of Oz evolution in the absence of glycolaldehyde but was without effect in the presence of glycolaldehyde.
130
EDWARDS
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3WJzw5 g200-
$ z 150$ 100-
50-
O0
I 2
I 4
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FIG. 6. Time course of CO, fixation (solid circles) and under optimum conditions without glycerate and in levels of Pi. Assays contained 30 pg Chl and 5.3 &i evolution was corrected for the low Or consumption
As shown in Fig. 8, glycolaldehyde prevented CO2 fixation and glycerate, at a concentration of 3 mM, directly contributed to O2 evolution at a significant rate separate from the functioning of the C3 cycle. The rate of glycerate-dependent O2 evolution in this experiment was 22 pmol mg Chl-’ h-’ after correction for O2 evolution, which would be associated with the low rate of COz fixation (3 pmol mg Chl-’ h-l). This would correspond to a rate of glycerate TABLE
Bicarbonate added None 1 mM 10 mM
O2 evolved (PM) 50 285 482
Glycerate (PM) 83 104 75
used
, 12
I 14
I 16
, 16
I 20
Or evolution (solid lines) by wheat chloroplasts the presence of 1 mM D-&CeK& with high [%jbicarbonate per 0.6 ml. The trace for Oa by the Ox electrode (2.5 pM min-I).
conversion to triose-P of 44 pmol mg Chl-’ h-l in the intact chloroplast. By comparison, the activity of glycerate kinase in chloroplast extracts of wheat was 148 pmol mg Chl-’ h-l. DISCUSSION
Glycerate is converted to triose-P in the light by wheat chloroplasts at significant rates independent of the C3 cycle and, under I
Glycerate-dependent Or evolutiona (PM) 42 52 38
Maximum rate Or evolution (pm01 mg Chl-’ h-i) 9 45 96
Note. Each treatment had 200 pM glycerate, 2 rnr+i Pi, and concentrations of bicarbonate, as indicated. The chloroplasts were incubated in the light for 9 min in the Or electrode curvette prior to killing with perchloric acid and assaying for glycerate as indicated under Materials and Methods. A control was run with chloroplasts in the dark with 296 /.tM glycerate, and complete recovery of glycerate was obtained following the killing and centrifugation procedures (104% of expected recovery). a Maximum value if all glycerate utilized is converted to triose-P.
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a 1OmM bicarbonate 0.2 mM Pi
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CHLOROPLASTS
2mMPt 3 mM
250 -
o-glycerate
(I”)
(99)
I
IOmM btcarbonate 2mMPi . ..~ . 5 200 I” 3. ;c 2 150-
I
f 2mMPI 3 mh4 D-aI”C.rat. mM gly&&dehyde /
12
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1 I l
b 1OmM btcarbooate I
0
2
4
6
8
1 10
1 12
I 14
1 16
FIG. 7. Efiect of glycolaldehyde on bicarbonate-dependent by wheat chloroplasts. Assay contained 30 pg ChV0.6
certain conditions, glycerate dramatically influences the rate of CO2 assimilation.
Eflect of D ve?w.&s L isomers
of &p%?-ate.
At low concentrations, D-glycerate was much more eflective than L-glycerate in its influence on photosynthetic O2 evolution, whether in thle presence or absence of bi-
FIG. 8. Influence of glycolaldehyde on photosynthetic 0, evolution and CO, fixation in the presence of bicarbonate and glycerate. The trace for O2 evolution was corrected for a slight O2 consumption by the 0, electrode (2.5 pM min-I). The assay contained 30 pg Chl and 3.4 &i [l*C]bicarbonate per 0.6 ml. This preparation of chloropllasts had rates of photosynthesis in the presence of bicarbonate and 0.2 mM Pi of 129 pmol mg Chl-’ h-’ (see Fig. 7a).
I 18
1 20
4 22
I 24
and glycerate-dependent
1 26
1 28
30
O2 evolution
ml.
carbonate. At saturating concentrations, a mixture of DL-glycerate had the same effectiveness as D-glycerate, which indicates that L-glycerate had neither a negative nor additive effect on utilization of D-glycerate. The results with chloroplasts are consistent with the properties of glycerate kinase from rye and pea chloroplasts (2) which have a much lower Km for D-glycerate (about 200 PM) than for L-glycerate (1.6 mM), and a V,,, for D-glycerate about twofold higher than for L-glycerate. In addition, Robinson (3) reported a light-activated glycerate transporter having a Km for DL-glycerate of 340 PM, with D-glycerate being taken up more effectively than Lglycerate. D-Glycerate is the expected substrate for glycerate kinase in vivo, since it is the isomer formed through hydroxypyruvate reductase. The fact that L-glycerate at high concentrations induces some photosynthetic O2evolution in the absence of bicarbonate and stimulates photosynthesis under inhibitory levels of Pi suggests that L-glycerate is not only recognized by glycerate kinase but that the L isomers of the substrates for PGA kinase and NADPtriose-P dehydrogenase are metabolized as well.
Glgcerate-dependent
0, evolution
i&e-
pendent of the C, cycle. Rates of glyceratedependent O2 evolution without bicarbon-
132
EDWARDS
ate of 10 to 25 pmol Oa evolved mg Chll’ h-’ were observed during this study. Maximum rates were dependent on Pi, and rates remained relatively constant between 0.1 and 40 mM Pi. In the absence of Pi, the conversion of glycerate to triose-P in the chloroplast may cause the internal pool of Pi, and thus the synthesis of ATP, to become limiting for photosynthesis. In addition, if the capacity for metabolism of the triose-P to starch in the chloroplast is limited, as it may be for wheat chloroplasts (8), then exchange of triose-P out of the chloroplast for Pi may be required. High Pi would not be expected to inhibit glycerate-dependent O2 evolution, as it does bicarbonate-dependent 0, evolution, since metabolism of glycerate by the isolated chloroplast does not depend on utilizing internal triose-P for regeneration of RuBP. The rate of glycerate-dependent O2 evolution without addition of bicarbonate may not be totally associated with the rate of conversion of glycerate to triose-P. Even though the major part of the volume of the assay medium (mixture containing sorbitol, Tricine buffer, and EDTA) was boiled to remove COa, COz has not been totally excluded. Thus, in part, glyceratedependent O2 evolution, without addition of bicarbonate, might reflect glycerate induction of COB assimilation at the low levels of COZ which would be present. This might partly account for the variation in rates of glycerate-dependent O2 evolution without bicarbonate addition which has been previously observed (see Ref. (5), 15 pmol mg Chl-’ h-’ with spinach chloroplasm; see Ref. (6), 91 rmol mg Chl-’ h-l with spinach chloroplasts; see Ref. (‘7), greater than 50 pmol mg Chl-’ h-l with wheat chloroplasts). In addition, under COa-limiting conditions the triose-P formed from glycerate may be metabolized to RuBP and react with RuBP oxygenase, leading to O2 consumption and, thus, underestimation of glycerate metabolism. Direct evidence for rates of glycerate conversion to triose-P in the absence of COB fixation is provided by experiments with glycolaldehyde, which inhibits bicarbonate-dependent Oa evolution but not glycerate-dependent Oa evolution (Figs. 7
AND
WALKER
and 8). Therefore, Oz evolution associated with glycerate conversion to triose-P was calculated at 22 pmol mg Chl-l h-’ at 3 mM glycerate from the data of Fig. 8, which corresponds to a rate of glycerate conversion to triose-P of 44 prnol mg Chl-l h-l (2 glycerate converted to triose-P/O2 evolved). This is about 30% of the maximum activity of glycerate kinase measured in chloroplast extracts. Since the activity of glycerate kinase varied by as much as eightfold among different species (2), it is possible that considerable variation will exist in the rates of glycerate metabolism by chloroplasts from various sources. DL-Glyceraldehyde, an inhibitor of the regenerative phase of photosynthesis (16), at a concentration of 8 mM more strongly inhibited bicarbonate-dependent O2 evolution than glycerate-dependent O2 evolution. However, with this inhibitor, the glycerate-dependent 0, evolution curved off with time and eventually ceased (not shown), as does PGA-dependent Oa evolution (16). Either the nature of the inhibition in the regenerative phase by DLglyceraldehyde may cause an eventual effect on glycerate-dependent O2 evolution, or this compound may have a direct secondary inhibitory effect on conversion of glycerate to triose-P. One means of estimating the rate of glycerate conversion to triose-P in the presence of bicarbonate is to determine the effect of glycerate on the ratio of CO, fixed/ O2 evolved. At 1 mM glycerate, the ratio of COa fixed/O2 evolved was 0.86 to 0.89 with 10 mM bicarbonate and 0.2 and 2 mM Pi (Fig. 5). Thus, in this case where the rate of photosynthesis was about 100 pmol mg Chill h-l, the rate of O2 evolution due to conversion of glycerate to triose-P can be estimated at 11 to 14 pmol Oa evolved mg Chl-’ h-l. At high Pi (20 mM) with 10 mM bicarbonate and 1 mM glycerate, the difference in the final rate of O2 evolution and COB fixation suggests a rate of glyceratedependent O2 evolution due to its conversion to triose-P of 13 pmol mg Chl-’ h-l (Fig. 6). Thus, in the presence of bicarbonate the rate of glycerate conversion to triose-P (resulting in 11 to 14 pm01 O2 evolved mg Chl-’ h-l, Figs. 5 and 6) would
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ON
PHOTOSYNTHESIS
appear similar to that measured in the absence of bicarbonate (resulting in 14 to 17 prnol O2 evolved mg Chl-l h-‘, Figs. 1E and 3). These are minimal estimates of glycerate utilization, since not all of the glycerate converted to PGA may be reduced to trioseP, particularly if some PGA is exported. The glycerate kinase reaction might be expected to favorably compete for ATP, since the AF’ for t.he reaction, like that of phosphoribulokinase, would be about -5.2 kcal, whereas that for PGA kinase is about 4.5 kcal, and is susceptible to inhibition by ADP. The rate of flux of carbon through the glycolate pathway and synthesis of glycerate in vivo will depend on the rate of photosynthesis abnd the ratio of RuBP carboxylase/RuBP oxygenase. A reasonable estimate under atmospheric conditions which could account for O2 inhibition of photosynthesis is 7 CO/2 O2 (17). This would lead to a fixa.tion of 7 COZ per 2 glycolate formed or 7 COZ fixed per glycerate synthesized (since 2 glycolate are converted to 1 glycerate in the glycolate pathway). Accordingly, the rate of COe fixation by the chloroplast would be about 7 times the rate of glycerate metabolism to triose-P, or 14 times the rate of O2 evolution due to glycerate conversion to triose-P. The average rate of COZ assimilation by the isolated chloropllasts in the present study was about 100 pm01 mg Chl-’ h-l (maximum rate of 240), with rates of glycerate-dependent O2 evolution of 10 to 25 pmol mg Chl-’ h-l. This results in a ratio of bicarbonate-dependent to glycerate-dependent O2 evolution of 4 to 10/l. Thus, the rate of glycerate utilization would appear sufficient to accommodate the required flux of carbon through the glycolate pathway. The injluence of glycerate on CO&xatim by the isolated chloroplast. In addition to analyzing the rate at which glycerate is converted to triose-P in the chloroplast as discussed above, there is the separate consideration of lhow the availability of glycerate to the chlloroplast influences the rate of COZ fixation. At very low levels of Pi or in the absence of Pi, glycerate inhibits photosynthetic O2 evolution with. 1 mM bicarbonate (Fig. 1C).
IN
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133
This may be due to development of a Pi deficiency when glycerate is converted to organic phosphate. In this respect, it is of interest that Cockburn et al. (18) found that ribosed phosphate inhibited photosynthesis of spinach chloroplasts at low Pi. When Pi becomes limiting in the chloroplast, the ATP/ADP ratio may fall to a value which is no longer adequate for driving the PGA kinase reaction (19). At moderate to high levels of Pi, glycerate reduces the induction period and makes photosynthetic O2 evolution relatively insensitive to the external Pi concentration. Even at concentrations of 20 to 40 mM Pi, glycerate allowed photosynthesis to continue at a significant rate (Figs. 2, 6). It is evident that glycerate allows COZ assimilation to occur at otherwise inhibitory levels of Pi. Glycerate causes a large stimulation of photosynthetic O2 evolution much above that which would be associated with conversion of glycerate to triose-P (Table I, Fig. 5). Since, under photorespiring conditions when glycolate is synthesized, photosynthesis by the isolated chloroplast is very sensitive to Pi, the prevention of this inhibition by glycerate may underscore the importance of glycerate returning to the chloroplast. It has been shown in a number of ways that when the glycolate pathway is blocked in vivo, photosynthesis is inhibited (20-22). While there may be other reasons for this effect on photosynthesis, the results of Figs. 1B and C would support the view that prevention of the return of glycerate to the chloroplast under normal levels of Pi could lead to inhibition of photosynthesis. In previous studies Robinson and Gibbs (23) found photosynthesis by isolated spinach chloroplasts was strongly inhibited by O2 at low levels of bicarbonate, and that this could be largely overcome by addition of low concentrations of ribose 5phosphate and fructose 1,6-bisphosphate. It may be that these two metabolites reduce the sensitivity of photosynthesis to O2 by preventing excessive export of carbon (by the organic phosphates interacting with the phosphate translocator, see Ref. (17)), which would otherwise occur when both glycolate and triose-P were exported.
134
EDWARDS
AND
Thus, O2 inhibition of photosynthesis by isolated chloroplasts may consist of at least two components, one due to the competitive interaction of O2 and COa on RuBP carboxylase/oxygenase, and the other due to excessive export of carbon in the form of glycolate when Pi is present outside the chloroplast. Prevention of the Pi inhibition of photosynthesis by addition of glycerate (Figs. 1A and C) may allow the effect of O2 on photosynthesis of chloroplasts to be determined without interference from excessive export of carbon. It is evident that the presence of glycerate outside the chloroplast, like DHAP, can greatly shorten the induction period and allow a high rate of photosynthesis under high levels of Pi. At pH 8.2, PGA is less effective than glycerate or DHAP. Perhaps PGAw3, the dominant form at higher pH, is poorly transported. With spinach chloroplasts, PGA-dependent 0s evolution ismuch greater at pH 7.6 than pH 8.8, which could be explained if PGA-’ was the form being transported (24). Under steady-state photosynthesis in the light, when the DHAP/PGA ratio is considered high in the cytoplasm, glycerate and DHAP may be the predominant metabolites in the cytoplasm which counteract the rate of export of triose-P from the chloroplast on the phosphate translocator (2526). The extent to which glycerate, PGA, and DHAP may influence the induction period in viva is uncertain. In the dark, the level of DHAP is low relative to PGA (26), and the extent of effect of the cytoplasmic pool of PGA on induction may be controlled by the cytoplasmic pH. If glycerate is present in darkened leaves, it may contribute to the initiation of photosynthesis in the light. It is not clear how glycerate in low concentrations prevents inhibition of photosynthesis by extrachloroplastic Pi. Glycerate might have its effect by conversion to DHAP and its export from the chloroplast, since addition of DHAP directly has an effect similar to addition of glycerate (Fig. 4). However, glycerate is as effective as DHAP over a range of concentrations (Fig. 4), even though glycerate will only be slowly converted to DHAP during the assay. For example, from the experiment of
WALKER
Fig. 5, at 2 mM Pi and 1 mM glycerate the calculated rate of conversion of glycerate to triose-P is only about 0.02 mM min-’ (taking rate of O2evolution due to glycerate conversion to triose-P of 11 pmol mg Chll’ h-l and 30 pg ChV0.6 ml assay). Therefore, glycerate likely has its effect by directly increasing the concentration of PGA in the chloroplast. Due to the unfavorable AF’ of PGA kinase, a high ratio of [ATPIPGA]/ [ADP] is needed for conversion of PGA to glycerate 1,3-bisphosphate and in order for photosynthesis to be initiated (19). During induction with isolated chloroplasts, the presence of glycerate may allow PGA to build up more rapidly than by autocatalysis alone. During steady-state photosynthesis the presence of glycerate may help sustain the chloroplastic pool of triose-P and prevent inhibition by excessive export of carbon (i.e., when glycolate is a product or under high Pi concentration).
REFERENCES 1. USUDA, H., AND EDWARDS, Physiol. 65,1017-1022.
G. E. (1980)
Plant
2. SCHMITT, M. R., AND EDWARDS, G. E. (1983) A&. B&hem Biqphys. 224,3.32-341. 3. ROBINSON, S. P. (1982) Plant Physd 70, 10321038. 4. ROBINSON, Cong.
S. P. (1984) Photosynthesis,
5. HEBER, U., SCHAFER, 6. KOBAYASHI, K., AND 7. USUDA,
M.
G. (1974)
Proceedings, 6th Brussels, in press.
R.,
GIMMLER,
Planta
AND
Physiol
EDWARDS,
Int.
H.,
AND
120, 31-46.
Y., INOUE, Y., FURUYA, HEBER, U. (1979) Planta
H.,
Plant
KIRK,
in
F.,
SHIBATA,
147,69-%X Amt.
G. E. (1980)
J.
7, 655-662.
8. EDWARDS, G. E., ROBINSON, S. P., TYLER, N. J. C., AND WALKER, D. A. (1978) Plant PhysioL 62,
313-319. 9. WINTERMANS,
J. F. G. M., AND DE MOTS,
Biochim. 10.
DELIEU,
Biophys.
Acta
T., AND WALKER,
A. (1965)
109,448-453. D. A. (1972)
New
Phytd
71,201-225. 11.
WALKER, D. A., BALDRY, W. (1968) Plant Physiol
12.
SCHMI~,
M. R., ANDEDWARDS,
Physiol 13.
USUDA,
PhysioL
C. W., AND 43,1419-1422.
COCKBURN,
G. E. (1983)
Plant
72, 728-734. H.,
AND
EDWARDS,
69, 469-473.
G. E. (1982)
Plant
INFLUENCE
OF
GLYCERATE
ON
PHOTOSYNTHESIS
14. FLUGGE, U., AND HELDT, H. W. (1977) FEBS L&t. 82, 29-33. 15. &HER, R. C. (1984) in Proceedings, 6th Int. Cong. on Photosynthesis, Brussels, in press. 16. STOKES, D. M., AND WALKER, D. A. (1972) Bioehem J. 128. 1147-1157. 17. EDWARDS, G. E., AND WALKER, D. A. (1983) C&: Mechanisms, and Cellular and Environmental Regulation of Photosynthesis, p. 542, Blackwell, Oxford. 18. COCKBURN, W., BALDRY, C. W., AND WALKER, D. A. (1967) B&him. Biophys Acta 143, 614624. 19. ROBINSON, S. P., AND WALKER, D. A. (1979) Biochim Biophys. Acta 545, 523-536.
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CHLOROPLASTS
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20. SERVAITES, J. C., AND OGREN, W. L. (1977) Plant Physid 60.461-466. 21. USUDA, H., ARRON, G. P., AND EDWARDS, G. E. (1980) J. Eq. Boii 31,1477-1483. 22. SOMMERVILE, C. R., AND OGREN, W. L. (1982) TIBS 7, 171-174. 23. ROBINSON, J. M., AND GIBBS, M. (1974) Plant Physid 53, 790-797. 24. WALKER, D. A., AND ROBINSON, S. P. (1978) Ber. Deutsch Bot Ges. Bd 91, 513-526. 25. GIERSCH, C., HEBER, U., KAISER, G., WALKER, D. A., AND ROBINSON, S. P. (1980) Arch. Biochem Biophys. 205,246-259. 26. STITT, M., WIRTZ, W., AND HELDT, H. W. (1980) Biochim. Biophys. Acta 593, 85-102.
ARCHIVES
Vol.
Influence of Glycerate on Photosynthesis GERALD
E. EDWARDS’
AND
by Wheat Chloroplasts’
DAVID
A. WALKER
Department of Botany, ARC Research Group on Photosynthesis, University of Sh&ti, Sh&k.!d ,910 %TN, United Kingdom Received
November
1, 1983, and in revised
form
January
19, 1934
Glycerate was found to effect photosynthetic O2 evolution in wheat chloroplasts by its conversion to triose phosphate and by influencing the rate of photosynthesis through the reductive pentose phosphate pathway. In the absence of bicarbonate, the photosynthetic Oz evolution with glycerate was low (10 to 25 pmol mg chlorophyll-’ h-l), and only about 15% of the rate of bicarbonate-dependent O2 evolution under optimum conditions. This corresponds to a rate of glycerate conversion to triose phosphate of 20 to 50 pmol mg chlorophyll-’ h-l, which appears sufficient to accommodate flux through the glycolate pathway in viva. Pi was required for this glycerate-dependent Oz evolution; rates remained relatively constant between 0.1 and 40 mM Pi, and proceeded with little lag upon illumination (less than 0.5 min). Evidence for Oz evolution due to glycerate conversion to triose phosphate could be conclusively demonstrated by addition of glycolaldehyde, an inhibitor of the regenerative phase of photosynthesis, which prevents COz fixation. The effect of glycerate on photosynthesis in the presence of bicarbonate was determined by measuring both photosynthetic O2 evolution and 14COzfixation at varying Pi concentrations. Low concentrations of glycerate (micro- to millimolar levels) prevented inhibition of photosynthesis by Pia With 1 mM bicarbonate and pH 8.2, which is favorable for glycolate synthesis, maximum rates of photosynthesis were obtained at low Pi (25 PM), whereas strong inhibition of photosynthesis occurred at only 0.2 mM Pi. Addition of glycerate relieved the inhibition of photosynthesis by Pi, indicating the possible importance of glycerate metabolism in the chloroplast under photorespiratory conditions. The initiation of photosynthesis by glycerate at inhibitory Pi levels occurred with little reduction in the ratio of COz fixed/O2 evolved, and the main effect of glycerate was on carbon assimilation. While the basis for the beneficial effect of glycerate on COZ assimilation under moderate to high Pi levels is uncertain, it may increase the concentration of 3-phosphoglycerate (PGA) in the chloroplast, and thus make conditions more favorable for induction of photosynthesis and reduction of PGA to triose phosphate.
During photorespiration in plants, glycerate is synthesized outside the chloroplast
as a product of the glycolate pathway. There is considerable evidence which suggests that glycerate enters the chloroplast and is converted to organic phosphates. Recent studies on a number of species indicate glycerate kinase may be exclusively located in the chloroplast (1, 2). Robinson (3, 4) has evidence for a light-dependent uptake of glycerate into the chloroplast on a specific glycerate translocator. Glyceratedependent Oz evolution occurs with isolated chloroplasts (5-Q, although it has been considered insufficient to accommodate
r G.E.E. is indebted to the Royal Society for a Guest Research Fellowship, and to the Science and Education Administration for support under Grant 83CRCR-1-1321 from the Competitive Research Grants Office. The authors appreciate helpful discussions with Bob Furbank, Richard Leegood, and Simon Robinson, and other assistance by Jackie Rowe11 and Christine Foyer during the course of the work. ‘To whom correspondence should be addressed: Botany Department, Washington State University, Pullman, Wash. 99164-4230. 0003-9861134 Copyright All rights
$3.00
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
124
INFLUENCE
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photorespiration (5). The purpose of the present study with wheat chloroplasts was to determine the rate of glycerate conversion to triose-P3 and the influence of glycerate on CO, assimilation under varying levels of Pi and bicarbonate. MATERIALS
AND
METHODS
Plant materiaL Wheat (TrScum w&&urn, var. Timmo) was grown in vermiculite in a greenhouse with sunlight supplemented by 400-W Wotan lamps. Day temperatures were normally 2’i-33°C and night temperature about 19°C. Isolation of protop~ts and chJuropl&s. Plants were used 6 to 7 days after planting with a plant height of 7 to 8 cm. The upper 4 cm of the plants was harvested and mesophyll protoplasts were prepared essentially as previously described (8). Leaf segments approximately 0.7 mm in width were prepared by stacking and cutting about 70 to 90 leaves at a time with a single-edge razor blade. Cutting small segments with frequent changes of blades is essential for obtaining good yields of protoplasts. Approximately 8 g of leaf tissue was prepared and incubated in 75 ml of digestion medium (see Ref. (8) for details). Following purification of the protoplasts, they were suspended in 0.5 M sorbitol, 0.05% BSA, centrifuged for 2 min at 25Og, and resuspended in 3 or 4 ml of the chloroplast isolation, resuspension, and assay medium, which included ‘25 mM Tricine-KOH, pH 8.2, 10 mM EDTA, and 0.4 ivt sorbitol. Chloroplasts were isolated by passage of the protoplasts (approximately 500 pg Chl/ml) through a 20-pm nylon net (8). Chl was determined in 96% ethanol according to Wintermans and De Mots (9). Assay of photosynthesis. 0, evolution was followed polarographically at 20°C using a twin Clark-type electrode system (10) purchased from Hansatech Ltd., Norfolk, U. K. The assay medium contained 0.4 M sorbitol, 10 mM EDTA, 25 mM Tricine-KOH, pH 8.2, 200 units of catalase in a total volume of 0.6 ml, and additions of bicarbonate, Pi, and glycerate as indicated. The pH Iof the stock solution for assay containing buffer, E:DTA, and sorbitol was adjusted with fresh pellets of KOH. This solution was normally boiled to remove CO*, and stored sealed with parafilm in a 50-ml test tube prior to use for experiments with-
3 Abbreviations used: Chl, chlorophyll; DHAP, dihydroxyacetone phosphate; PGA, 3-phosphoglycerate; pyridoxal-P, pyridoxal phosphate; RuBP, ribulose 1,5bisphosphate; triose-P, triose phosphate; BSA, bovine serum albumin; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyll)ethyl~lycine; TCA, trichloroacetic acid.
IN
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125
out bicarbonate, or with 1 mM bicarbonate in the assay. COz fixation was determined simultaneously with Oz evolution (11) using [14C]bicarbonate. Determination of glycerate and glycerate kinase. In experiments where glycerate was assayed, the reaction mixture was the same as the assay medium for O2 evolution studies, plus other additions were made as indicated in Table I. After 9 min of incubation in the light a 0.5-ml aliquot was killed with 30 pl of 12 N perchloric acid. Following storage on ice for 30 min, samples were centrifuged at 4OOg for 2 min and then neutralized by addition of 60 pl of 5 N K.&O3 (R. Leegood, personal communication). After centrifugation at 4009 for 2 min, the supernatant was analyzed for glycerate using glycerate kinase, PGA kinase, and NAD triose-P dehydrogenase as previously described (12). Glycerate kinase activity extracted from chloroplasts was determined by coupling to PGA kinase and NAD triose-P dehydrogenase (2). Infomnation on certain chemicals and enzymes. The isomers of glycerate were obtained in the form of hemi-calcium salts and DHAP was obtained in the form of lithium salt, all from Sigma. The calcium and lithium were removed by exchange on Dowex 50W hydrogen form cation-exchange resin. PGA was obtained as the trisodium salt from Sigma. Glycolaldehyde was purchased as the dimer (2,5-dihydroxy1,4-dioxane) from Aldrich Chemical Company and is converted to the monomer in solution. PGA kinase and NAD-triose-P dehydrogenase were obtained as a mixture from Boehringer-Mannheim, while glycerate kinase (from rye) was provided by Mark Schmitt.
RESULTS
The influence of Pi on rates of photosynthesis and on the induction period upon illumination of wheat chloroplasts in the presence of 10 mM bicarbonate (Figs. 1A and B) was similar to that previously reported (8). At high levels of Pi, the rates of photosynthesis decreased and the lag period increased up to 5 to 6 min, typical of C3-type chloroplasts. The optimum Pi level was around 0.2 mM and there was little photosynthesis without addition of Pi. Addition of 2 mM DL-glycerate had a strong influence on photosynthetic O2 evolution, preventing the inhibition of photosynthesis by Pi concentrations between 0.4 and 2 mM, and decreasing the induction period over the entire Pi range from 0 to 2 rnM. At 1 mM bicarbonate (Figs. 1C and D), the Pi optimum was very low (around 25 PM), and substantial photosynthesis oc-
126
EDWARDS I A. pi 6.2 10 mM btcarbonate
I
I 0.5
I 1.0 [Pt]mM
I 1.5
I
I
AND
I
WALKER
I
1
6
I
B. pH 62 10 mM bxarbonate
OC d o-
o-
I c. pH 6.2 1 mM bicarbonate
2 [Pl]mM I -
I
6
I
D. pH 6.2 1 mM bourbonate
I
I
I
I .
&@J+2 mM DL-glycerate
,
[Pi]mM I
I
F. pii 8.2 No bocarbonats
No bicarbonate 1.5 -
85 z 5
T 5 g 1.0 -
20 +2mM
6
IA
DL-glvcerate
.
.
x : 0.5-
I
E 4 10 I/ O+%iA
m” [Pa]mM
1.0
O t==*
A 0.25
I 0.5 [Po]mM
+2mM
m-glycerate I 0.75
A I 1.0
FIG. 1. Effect of 2 mM DL-glycerate on the rate and induction period of light-dependent Oa evolution by wheat chloroplasts under varying levels of Pi. (A) Rate of O2 evolution and (B) induction period in the presence of 10 mre bicarbonate (assay contained 38 pg ChlI0.6 ml). (C) Rate of O2 evolution and (D) induction period in the presence of 1 mM bicarbonate (assay contained 20 ng ChlI0.6 ml). (E) Rate of Oa evolution and (F) induction period without bicarbonate (assay contained 2’7 pg Chl/ 0.6 ml). The results shown are at pH 8.2. Similar data were obtained at pH 7.6 in experiments with 10 mM bicarbonate and without bicarbonate.
1NFLUE:NCE
OF
GLYCERATE
ON
PHOTOSYNTHESIS
curred without addition of Pi as previously reported (13). Whereas 0.2 mM Pi was strongly inhibitory to photosynthesis, the addition of 41 mM DL-glycerate alleviated the Pi inhibition of photosynthesis between 50 PM and 1 mM Pi. At 10~ levels of Pi (0 to 25 PM), addition of glycerate caused an inhibition of photosynthesis (Fig. 1C) and, with time, rates progressively curved off more rapidly in the presence of glycerate than in its a.bsence (not shown). The induction period was dramatically decreased by addition of glycerate, being less than 1 Inin Up to Pi levels Of 1 mM. Photosynthetic O2 evolution in the presence of 2 mMDL-glycerate, without addition of bicarbonate (Figs. 1E and F), was largely dependent on additions of Pi, and the induction period was 0.2 min or less up to 1 mM Pi. Rates of glycerate-dependent O2 evolution without bicarbonate were typically between 15 and 25 /*mol O2 evolved mg Chl-l h-l,, which are about five- to sevenfold lower than the maximum rates obtained under optimum conditions with bicarbonate as substrate. Photosynthesis in the presence of 2 mM DL-glycerate ‘was inhibited with increasing levels of Pi up to 20 mM (Fig. 2); however, even at 40 m&I Pi the rate of photosynthetic O2 evolution in the presence of 2 mM DLglycerate was about half the maximum rate at lower Pi. Even at 40 mM Pi with 2 mM DL-glycerate, the rate in the presence of bicarbonate was about twofold the rate without bicalrbonate. Addition of pyridoxal-P, an inhibitor of the phosphate translocator (14), prevented inhibition of photosynthesis at high Pi in the presence of glycerate. ‘The rate of photosynthetic O2 evolution in the presence of glycerate, and without bicarbonate, was relatively constant between 1 and 40 mM Pi. Without bicarbonate, the concentration of DL-glyceralte required for half-maximum rates of photosynthetic O2 evolution was about 0.5 m&I with both 0.2 and 2 mM Pi (Fig. 3). With 10 mM bicarbonate and 2 mM Pi, increasing concentrations of DL-glycerate initiated photosynthesis and reduced the induction period, with half-maximum effectiveness at 0.1 mM DL-glycerate (Fig. 3).
IN
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10
CHLOROPLASTS
20 [Pe]mM
30
127
40
FIG. 2. Influence of 2 mM DL-glycerate on the rate of photosynthetic O2 evolution by wheat chloroplasts under high levels of Pi. Assays contained 27 pg Chl/ 0.6 ml. Pyridoxal-P was added to the assay medium with chloroplasts 1 min prior to illumination. Pi was added immediately before illumination. Similar results were obtained at pH 7.6.
Eflect Of DL, D, and L i.xme?-s of glycerate on photosynthesis. The D isomer was more effective than the L isomer of glycerate as a substrate to induce photosynthetic O2 evolution, either in the absence of bicarbonate or in the presence of bicarbonate with high Pi concentrations. Based on measurements on glycerate-dependent OS evolution in the absence of bicarbonate, the D isomer gave twofold higher rates than the L isomer at saturating concentrations (5 mM), while one-half of the maximum rate occurred at 0.3 mM D-glycerate and 1.5 mM L-glycerate. At saturating concentrations, the D isomer and DL mixture of isomers of glycerate had the same effect (results not shown). In subsequent experiments the D isomer was used.
Cmparism of the eflects of o-glycerate, DHAP, and PGA on photosynthetic 0, eve lution. D-Glycerate and DHAP were found to be very similar in their ability to prevent of photosynthesis with 10 mM bicarbonate and 3 mM Pi, as seen in Fig. 4. PGA also partially overcame the Pi in-
Pi inhibition
128
EDWARDS
AND
WALKER
Evaluation of the efect of glycerate on function of the C, cycle. In the presence of
[DL-glvcerate] I pi+ 8.2 1OmM bicarbonate
200 x
I
mM I
I
I
- 10
-0
-6X F 2 -
4E x !I
I
2 x
40
-“\ 1
x-x-x
2
X
[a-glycerate]
mM
FIG. 3. The
influence of varying concentrations of on photosynthetic O2 evolution and induction period in the absence or presence of 10 mM bicarbonate with wheat chloroplasts. Assays contained 20 pg ChV0.6 ml.
bicarbonate, glycerate could lead to an enhancement of photosynthetic O2 evolution by direct conversion of glycerate to trioseP and, in addition, by making conditions more favorable for the C3 cycle to function. The effect of glycerate on COz fixation was evaluated by making simultaneous measurements of CO2 fixation and O2 evolution. The influence of D-glycerate on rates of O2 evolution, the ratio of CO2 fixed/O2 evolved, and the induction period at 0.2 and 2 mM Pi was determined (Fig. 5). At 0.2 mM Pi, increasing glycerate had little effect on the rates of O2 evolution, while the lag in photosynthesis was reduced from 2 to less than 1 min and the ratio of COB fixed/O2 evolved decreased. At 1 m&i glycerate, the C02/02 ratio decreased to a value of 0.86, suggesting that about 86% of the O2 evolved was due to CO2 fixation and 14% due to glycerate conversion to triose-P. In the presence of 2 mM Pi and 10 mM bicarbonate, low levels of glycerate between 0 and 100 PM caused a large decrease in the induction period and an increase in photosynthetic O2 evolution, while causing little decrease in the ratio of COB fixed/O2 evolved (Fig. 5). At higher glycerate concentrations (up
DL-&CeI’ate
hibition of photosynthesis, but not as well as DHAP or glycerate. DHAP and glycerate were also more effective than PGA in reduction of the lag period (not shown). At this level of Pi (3 mM), the calculated concentrations giving half-maximum reduction of the lag period by DHAP (less than 20 PM) and glycerate (approximately 30 PM) were lower than the concentrations required to give half-maximum stimulation of photosynthetic rates (greater than 0.1 mM). At higher concentrations of Pi (10 mM), the ineffectiveness of PGA, relative to that of D-glycerate or DHAP, in overcoming the Pi inhibition of photosynthesis was even more apparent (results not shown).
loo-
OO
pH 8.2 10 mM bacarbonsts
I 0.2
1 0.4
I 0.6
1 0.8
1 1.0
nlM
FIG. 4. A comparison of the effects of D-&Cerate, DHAP, and PGA on photosynthetic 0, evolution by wheat chloroplasts with 10 mM bicarbonate and 3 mM Pi. Assays contained 35 pg ChV0.6 ml. Similar data were obtained in a separate experiment at 2 mM Pi.
INFLUElNCE
I .Floo~~~--_loI 60 -
60-
40 -
OF
GLYCERATE
ON
PHOTOSYNTHESIS
*
-08 6 5 e
- 0.6 ': Q Px -045 I9
FIG. 5. The ratio of COa fixed/O2 evolved and rate of Oz evolution during photosynthesis by wheat chloroplasts in the presence of 10 mM bicarbonate and varying concentrations of D-glyeerate (upper figure with 0.2 mMPi; lower figure with 2 mM Pi). The assays contained 30 pg Chl and 3.4 &i bicarbonate per 0.6 ml. The results are the averages of two experiments with wheat chloroplasts prepared on separate days. At the end of the assay period (5 to 10 min after illumination) duplicate samples of 50 ~1 each were taken, killed with 50 ~1 of 20% TCA and analyzed by scintillation spectroscopy.
to 1 mM) phot’osynthesis reached a maximum and induction a minimal value, while the ratio of CO2 fixed/O2 evolved decreased to a value of 6.89. Therefore, in these experiments, even at the higher levels of glycerate the major portion of the O2 evolved was associated with CO2 fixation.
IN
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CHLOROPLASTS
129
Figure 6 shows a time course of COz fixation and Oz evolution in the absence and presence of glycerate. Without glycerate there was a similar increase in O2 evolution and COz fixation, with a maximum rate of photosynthesis of 100 pmol mg Chl-’ h-l. In the presence of 1 mM D-glycerate and 20 mM Pi, the initial rate of O2 evolution following induction was noticeably faster than the initial rate of CO2 fixation. After reaching a steady-state rate, O2 evolution exceeded COz fixation by 13 ,Fcmol mg Chl-’ h-l, which is indicative of the Oz evolution associated with conversion of glycerate to triose-P. The direct utilization of glycerate under conditions where glycerate overcomes Pi inhibition of photosynthesis was evaluated. In the presence of 200 pM glycerate and 2 mM Pi, photosynthetic O2 evolution increased with increasing CO2 from 0 to 1 to 10 mM bicarbonate (Table I). However, under all conditions only one-half or less of the glycerate added was utilized during the assay period. The Oz evolution without bicarbonate could be largely accounted for if the glycerate utilized was converted to triose-P. However, in the presence of 1 or 10 mM bicarbonate, glycerate utilization could only account for a small percentage of the Oz evolved. Injuence of glycolaldehyde on photosynthetic 0, evolution in the presence of glycerate and bicarbonate. Glycolaldehyde (an
inhibitor of the regenerative phase of photosynthesis, see Ref. (15)) at a concentration of 12 mM inhibits bicarbonate-dependent O2 evolution (Fig. 7, traces a and b), following which Oz evolution is initiated by addition of 3 mM D-glycerate (rate of 23 pmol mg Chll’ h-l). With bicarbonate and glycerate added prior to illumination (Fig. 7, traces c and d), glycolaldehyde only partially inhibited photosynthetic Oz evolution (rate in presence of glycolaldehyde of 27 lmol mg Chll’ h-l). Glycolaldehyde had little effect on glycerate-dependent O2 evolution without bicarbonate (Fig. 7, traces e and f); addition of bicarbonate during the course of the assay caused a large stimulation of Oz evolution in the absence of glycolaldehyde but was without effect in the presence of glycolaldehyde.
130
EDWARDS
AND
WALKER
3WJzw5 g200-
$ z 150$ 100-
50-
O0
I 2
I 4
I 6
I 6
1 10 Ml”“lF&
FIG. 6. Time course of CO, fixation (solid circles) and under optimum conditions without glycerate and in levels of Pi. Assays contained 30 pg Chl and 5.3 &i evolution was corrected for the low Or consumption
As shown in Fig. 8, glycolaldehyde prevented CO2 fixation and glycerate, at a concentration of 3 mM, directly contributed to O2 evolution at a significant rate separate from the functioning of the C3 cycle. The rate of glycerate-dependent O2 evolution in this experiment was 22 pmol mg Chl-’ h-’ after correction for O2 evolution, which would be associated with the low rate of COz fixation (3 pmol mg Chl-’ h-l). This would correspond to a rate of glycerate TABLE
Bicarbonate added None 1 mM 10 mM
O2 evolved (PM) 50 285 482
Glycerate (PM) 83 104 75
used
, 12
I 14
I 16
, 16
I 20
Or evolution (solid lines) by wheat chloroplasts the presence of 1 mM D-&CeK& with high [%jbicarbonate per 0.6 ml. The trace for Oa by the Ox electrode (2.5 pM min-I).
conversion to triose-P of 44 pmol mg Chl-’ h-l in the intact chloroplast. By comparison, the activity of glycerate kinase in chloroplast extracts of wheat was 148 pmol mg Chl-’ h-l. DISCUSSION
Glycerate is converted to triose-P in the light by wheat chloroplasts at significant rates independent of the C3 cycle and, under I
Glycerate-dependent Or evolutiona (PM) 42 52 38
Maximum rate Or evolution (pm01 mg Chl-’ h-i) 9 45 96
Note. Each treatment had 200 pM glycerate, 2 rnr+i Pi, and concentrations of bicarbonate, as indicated. The chloroplasts were incubated in the light for 9 min in the Or electrode curvette prior to killing with perchloric acid and assaying for glycerate as indicated under Materials and Methods. A control was run with chloroplasts in the dark with 296 /.tM glycerate, and complete recovery of glycerate was obtained following the killing and centrifugation procedures (104% of expected recovery). a Maximum value if all glycerate utilized is converted to triose-P.
1NFLUE:NCE
OF
GLYCERATE
ON
PHOTOSYNTHESIS
a 1OmM bicarbonate 0.2 mM Pi
IN
WHEAT
131
CHLOROPLASTS
2mMPt 3 mM
250 -
o-glycerate
(I”)
(99)
I
IOmM btcarbonate 2mMPi . ..~ . 5 200 I” 3. ;c 2 150-
I
f 2mMPI 3 mh4 D-aI”C.rat. mM gly&&dehyde /
12
~;:~y~
1 I l
b 1OmM btcarbooate I
0
2
4
6
8
1 10
1 12
I 14
1 16
FIG. 7. Efiect of glycolaldehyde on bicarbonate-dependent by wheat chloroplasts. Assay contained 30 pg ChV0.6
certain conditions, glycerate dramatically influences the rate of CO2 assimilation.
Eflect of D ve?w.&s L isomers
of &p%?-ate.
At low concentrations, D-glycerate was much more eflective than L-glycerate in its influence on photosynthetic O2 evolution, whether in thle presence or absence of bi-
FIG. 8. Influence of glycolaldehyde on photosynthetic 0, evolution and CO, fixation in the presence of bicarbonate and glycerate. The trace for O2 evolution was corrected for a slight O2 consumption by the 0, electrode (2.5 pM min-I). The assay contained 30 pg Chl and 3.4 &i [l*C]bicarbonate per 0.6 ml. This preparation of chloropllasts had rates of photosynthesis in the presence of bicarbonate and 0.2 mM Pi of 129 pmol mg Chl-’ h-’ (see Fig. 7a).
I 18
1 20
4 22
I 24
and glycerate-dependent
1 26
1 28
30
O2 evolution
ml.
carbonate. At saturating concentrations, a mixture of DL-glycerate had the same effectiveness as D-glycerate, which indicates that L-glycerate had neither a negative nor additive effect on utilization of D-glycerate. The results with chloroplasts are consistent with the properties of glycerate kinase from rye and pea chloroplasts (2) which have a much lower Km for D-glycerate (about 200 PM) than for L-glycerate (1.6 mM), and a V,,, for D-glycerate about twofold higher than for L-glycerate. In addition, Robinson (3) reported a light-activated glycerate transporter having a Km for DL-glycerate of 340 PM, with D-glycerate being taken up more effectively than Lglycerate. D-Glycerate is the expected substrate for glycerate kinase in vivo, since it is the isomer formed through hydroxypyruvate reductase. The fact that L-glycerate at high concentrations induces some photosynthetic O2evolution in the absence of bicarbonate and stimulates photosynthesis under inhibitory levels of Pi suggests that L-glycerate is not only recognized by glycerate kinase but that the L isomers of the substrates for PGA kinase and NADPtriose-P dehydrogenase are metabolized as well.
Glgcerate-dependent
0, evolution
i&e-
pendent of the C, cycle. Rates of glyceratedependent O2 evolution without bicarbon-
132
EDWARDS
ate of 10 to 25 pmol Oa evolved mg Chll’ h-’ were observed during this study. Maximum rates were dependent on Pi, and rates remained relatively constant between 0.1 and 40 mM Pi. In the absence of Pi, the conversion of glycerate to triose-P in the chloroplast may cause the internal pool of Pi, and thus the synthesis of ATP, to become limiting for photosynthesis. In addition, if the capacity for metabolism of the triose-P to starch in the chloroplast is limited, as it may be for wheat chloroplasts (8), then exchange of triose-P out of the chloroplast for Pi may be required. High Pi would not be expected to inhibit glycerate-dependent O2 evolution, as it does bicarbonate-dependent 0, evolution, since metabolism of glycerate by the isolated chloroplast does not depend on utilizing internal triose-P for regeneration of RuBP. The rate of glycerate-dependent O2 evolution without addition of bicarbonate may not be totally associated with the rate of conversion of glycerate to triose-P. Even though the major part of the volume of the assay medium (mixture containing sorbitol, Tricine buffer, and EDTA) was boiled to remove COa, COz has not been totally excluded. Thus, in part, glyceratedependent O2 evolution, without addition of bicarbonate, might reflect glycerate induction of COB assimilation at the low levels of COZ which would be present. This might partly account for the variation in rates of glycerate-dependent O2 evolution without bicarbonate addition which has been previously observed (see Ref. (5), 15 pmol mg Chl-’ h-’ with spinach chloroplasm; see Ref. (6), 91 rmol mg Chl-’ h-l with spinach chloroplasts; see Ref. (‘7), greater than 50 pmol mg Chl-’ h-l with wheat chloroplasts). In addition, under COa-limiting conditions the triose-P formed from glycerate may be metabolized to RuBP and react with RuBP oxygenase, leading to O2 consumption and, thus, underestimation of glycerate metabolism. Direct evidence for rates of glycerate conversion to triose-P in the absence of COB fixation is provided by experiments with glycolaldehyde, which inhibits bicarbonate-dependent Oa evolution but not glycerate-dependent Oa evolution (Figs. 7
AND
WALKER
and 8). Therefore, Oz evolution associated with glycerate conversion to triose-P was calculated at 22 pmol mg Chl-l h-’ at 3 mM glycerate from the data of Fig. 8, which corresponds to a rate of glycerate conversion to triose-P of 44 prnol mg Chl-l h-l (2 glycerate converted to triose-P/O2 evolved). This is about 30% of the maximum activity of glycerate kinase measured in chloroplast extracts. Since the activity of glycerate kinase varied by as much as eightfold among different species (2), it is possible that considerable variation will exist in the rates of glycerate metabolism by chloroplasts from various sources. DL-Glyceraldehyde, an inhibitor of the regenerative phase of photosynthesis (16), at a concentration of 8 mM more strongly inhibited bicarbonate-dependent O2 evolution than glycerate-dependent O2 evolution. However, with this inhibitor, the glycerate-dependent 0, evolution curved off with time and eventually ceased (not shown), as does PGA-dependent Oa evolution (16). Either the nature of the inhibition in the regenerative phase by DLglyceraldehyde may cause an eventual effect on glycerate-dependent O2 evolution, or this compound may have a direct secondary inhibitory effect on conversion of glycerate to triose-P. One means of estimating the rate of glycerate conversion to triose-P in the presence of bicarbonate is to determine the effect of glycerate on the ratio of CO, fixed/ O2 evolved. At 1 mM glycerate, the ratio of COa fixed/O2 evolved was 0.86 to 0.89 with 10 mM bicarbonate and 0.2 and 2 mM Pi (Fig. 5). Thus, in this case where the rate of photosynthesis was about 100 pmol mg Chill h-l, the rate of O2 evolution due to conversion of glycerate to triose-P can be estimated at 11 to 14 pmol Oa evolved mg Chl-’ h-l. At high Pi (20 mM) with 10 mM bicarbonate and 1 mM glycerate, the difference in the final rate of O2 evolution and COB fixation suggests a rate of glyceratedependent O2 evolution due to its conversion to triose-P of 13 pmol mg Chl-’ h-l (Fig. 6). Thus, in the presence of bicarbonate the rate of glycerate conversion to triose-P (resulting in 11 to 14 pm01 O2 evolved mg Chl-’ h-l, Figs. 5 and 6) would
INFLUECNCE
OF
GLYCERATE
ON
PHOTOSYNTHESIS
appear similar to that measured in the absence of bicarbonate (resulting in 14 to 17 prnol O2 evolved mg Chl-l h-‘, Figs. 1E and 3). These are minimal estimates of glycerate utilization, since not all of the glycerate converted to PGA may be reduced to trioseP, particularly if some PGA is exported. The glycerate kinase reaction might be expected to favorably compete for ATP, since the AF’ for t.he reaction, like that of phosphoribulokinase, would be about -5.2 kcal, whereas that for PGA kinase is about 4.5 kcal, and is susceptible to inhibition by ADP. The rate of flux of carbon through the glycolate pathway and synthesis of glycerate in vivo will depend on the rate of photosynthesis abnd the ratio of RuBP carboxylase/RuBP oxygenase. A reasonable estimate under atmospheric conditions which could account for O2 inhibition of photosynthesis is 7 CO/2 O2 (17). This would lead to a fixa.tion of 7 COZ per 2 glycolate formed or 7 COZ fixed per glycerate synthesized (since 2 glycolate are converted to 1 glycerate in the glycolate pathway). Accordingly, the rate of COe fixation by the chloroplast would be about 7 times the rate of glycerate metabolism to triose-P, or 14 times the rate of O2 evolution due to glycerate conversion to triose-P. The average rate of COZ assimilation by the isolated chloropllasts in the present study was about 100 pm01 mg Chl-’ h-l (maximum rate of 240), with rates of glycerate-dependent O2 evolution of 10 to 25 pmol mg Chl-’ h-l. This results in a ratio of bicarbonate-dependent to glycerate-dependent O2 evolution of 4 to 10/l. Thus, the rate of glycerate utilization would appear sufficient to accommodate the required flux of carbon through the glycolate pathway. The injluence of glycerate on CO&xatim by the isolated chloroplast. In addition to analyzing the rate at which glycerate is converted to triose-P in the chloroplast as discussed above, there is the separate consideration of lhow the availability of glycerate to the chlloroplast influences the rate of COZ fixation. At very low levels of Pi or in the absence of Pi, glycerate inhibits photosynthetic O2 evolution with. 1 mM bicarbonate (Fig. 1C).
IN
WHEAT
CHLOROPLASTS
133
This may be due to development of a Pi deficiency when glycerate is converted to organic phosphate. In this respect, it is of interest that Cockburn et al. (18) found that ribosed phosphate inhibited photosynthesis of spinach chloroplasts at low Pi. When Pi becomes limiting in the chloroplast, the ATP/ADP ratio may fall to a value which is no longer adequate for driving the PGA kinase reaction (19). At moderate to high levels of Pi, glycerate reduces the induction period and makes photosynthetic O2 evolution relatively insensitive to the external Pi concentration. Even at concentrations of 20 to 40 mM Pi, glycerate allowed photosynthesis to continue at a significant rate (Figs. 2, 6). It is evident that glycerate allows COZ assimilation to occur at otherwise inhibitory levels of Pi. Glycerate causes a large stimulation of photosynthetic O2 evolution much above that which would be associated with conversion of glycerate to triose-P (Table I, Fig. 5). Since, under photorespiring conditions when glycolate is synthesized, photosynthesis by the isolated chloroplast is very sensitive to Pi, the prevention of this inhibition by glycerate may underscore the importance of glycerate returning to the chloroplast. It has been shown in a number of ways that when the glycolate pathway is blocked in vivo, photosynthesis is inhibited (20-22). While there may be other reasons for this effect on photosynthesis, the results of Figs. 1B and C would support the view that prevention of the return of glycerate to the chloroplast under normal levels of Pi could lead to inhibition of photosynthesis. In previous studies Robinson and Gibbs (23) found photosynthesis by isolated spinach chloroplasts was strongly inhibited by O2 at low levels of bicarbonate, and that this could be largely overcome by addition of low concentrations of ribose 5phosphate and fructose 1,6-bisphosphate. It may be that these two metabolites reduce the sensitivity of photosynthesis to O2 by preventing excessive export of carbon (by the organic phosphates interacting with the phosphate translocator, see Ref. (17)), which would otherwise occur when both glycolate and triose-P were exported.
134
EDWARDS
AND
Thus, O2 inhibition of photosynthesis by isolated chloroplasts may consist of at least two components, one due to the competitive interaction of O2 and COa on RuBP carboxylase/oxygenase, and the other due to excessive export of carbon in the form of glycolate when Pi is present outside the chloroplast. Prevention of the Pi inhibition of photosynthesis by addition of glycerate (Figs. 1A and C) may allow the effect of O2 on photosynthesis of chloroplasts to be determined without interference from excessive export of carbon. It is evident that the presence of glycerate outside the chloroplast, like DHAP, can greatly shorten the induction period and allow a high rate of photosynthesis under high levels of Pi. At pH 8.2, PGA is less effective than glycerate or DHAP. Perhaps PGAw3, the dominant form at higher pH, is poorly transported. With spinach chloroplasts, PGA-dependent 0s evolution ismuch greater at pH 7.6 than pH 8.8, which could be explained if PGA-’ was the form being transported (24). Under steady-state photosynthesis in the light, when the DHAP/PGA ratio is considered high in the cytoplasm, glycerate and DHAP may be the predominant metabolites in the cytoplasm which counteract the rate of export of triose-P from the chloroplast on the phosphate translocator (2526). The extent to which glycerate, PGA, and DHAP may influence the induction period in viva is uncertain. In the dark, the level of DHAP is low relative to PGA (26), and the extent of effect of the cytoplasmic pool of PGA on induction may be controlled by the cytoplasmic pH. If glycerate is present in darkened leaves, it may contribute to the initiation of photosynthesis in the light. It is not clear how glycerate in low concentrations prevents inhibition of photosynthesis by extrachloroplastic Pi. Glycerate might have its effect by conversion to DHAP and its export from the chloroplast, since addition of DHAP directly has an effect similar to addition of glycerate (Fig. 4). However, glycerate is as effective as DHAP over a range of concentrations (Fig. 4), even though glycerate will only be slowly converted to DHAP during the assay. For example, from the experiment of
WALKER
Fig. 5, at 2 mM Pi and 1 mM glycerate the calculated rate of conversion of glycerate to triose-P is only about 0.02 mM min-’ (taking rate of O2evolution due to glycerate conversion to triose-P of 11 pmol mg Chll’ h-l and 30 pg ChV0.6 ml assay). Therefore, glycerate likely has its effect by directly increasing the concentration of PGA in the chloroplast. Due to the unfavorable AF’ of PGA kinase, a high ratio of [ATPIPGA]/ [ADP] is needed for conversion of PGA to glycerate 1,3-bisphosphate and in order for photosynthesis to be initiated (19). During induction with isolated chloroplasts, the presence of glycerate may allow PGA to build up more rapidly than by autocatalysis alone. During steady-state photosynthesis the presence of glycerate may help sustain the chloroplastic pool of triose-P and prevent inhibition by excessive export of carbon (i.e., when glycolate is a product or under high Pi concentration).
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WJ$EAT
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