Light and CO2 Response of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Arabidopsis Leaves Received for Publication August 22, 1985

Plant Physiol. (1986) 80, 655-659 0032-0889/86/80/0655/05/$01.00/0

Light and CO2 Response of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Arabidopsis Leaves Received for publication August 22, 1985

MICHAEL E. SALVUCCI*I, ARCHIE R. PORTIS, JR., AND WILLIAM L. OGREN United States Department ofAgriculture, Agricultural Research Service, Urbana, Illinois 61801

ABSTRACT in the chloroplast stroma upon illumination (18, 19). However, the response of rubisco activation to light intensity and CO2 The requirements for activation of ribulose 1,5-bisphosphate carbox- concentration is inconsistent with this suggested mechanism. ylase/oxygenase (rubisco) were investigated in leaves ofArabidopsis wild- The activation state of rubisco in vivo is a function of light type and a mutant incapable of light activating rubisco in vivo. Upon intensity (11, 16), but the level of light required to saturate illumination with saturating light intensities, the activation state of rub- rubisco activation is considerably higher than that necessary for isco increased 2-fold in the wild-type and decreased in the mutant. saturation of stromal alkalinization and Mg2e efflux (4). Fur- Activation of fructose 1,6-bisphosphate phosphatase was unaffected by thermore, under saturating light, rubisco extracted from leaves the mutation. Under low light, rubisco deactivated in both the wild-type is almost completely activated even though the CO2 concentra- and the mutant. Deactivation of rubisco in the mutant under high and tion under ambient conditions is several-fold less than the low light led to the accumulation of high concentrations of ribulose 1,5- KI,Q(CO2) for activation of the isolated enzyme (7, 12). Under bisphosphate. Inhibiting photosynthesis with methyl viologen prevented sub-saturating light, rubisco in leaves exists largely in an inactive ribulose 1,5-bisphosphate accumulation but was ineffective in restoring form but is insensitive to CO2 concentration (15, 21). These rubisco activation to the mutant. Net photosynthesis and the rubisco observations demonstrate that the requirements for light activa- activation level were closely correlated and saturated at a lower light tion of rubisco in vivo differ substantially from those observed intensity in the mutant than in wild-type. At CO2 concentrations between for the isolated enzyme. 100 and 2000 microliters per liter, the activation state was a function of Somerville et al. (24) presented genetic evidence for the in- the CO2 concentration in the dark but was independent of CO2 concen- volvement of a heritable factor in the light activation of rubisco. tration in the light. High CO2 concentration (1%) suppressed activation These investigators isolated a mutant ofArabidopsis (designated in the wild-type and deactivation in the mutant. These results support the rca mutant) which was incapable of activating rubisco upon the concept that rubisco activation in vivo is not a spontaneous process illumination. Rubisco isolated from leaves of the mutant and but is catalyzed by a specific protein. The absence of this protein, rubisco wild-type plants, however, had identical activation kinetics and activase, is responsible for the altered characteristics ofrubisco activation isoelectric characteristics. Thus, the lesion in the rca mutant did in the mutant. not affect rubisco itself. We have recently shown that the rca mutation affects the expression of a soluble chloroplast protein, rubisco activase, which promotes light activation of rubisco in a reconstituted light activation system (20). These data indicate that rubisco activation in vivo is not a spontaneous process but is catalyzed by a mechanism that is energetically coupled to the light reactions ofphotosynthesis in the thylakoid membrane. The Ribulose- 1 ,5,-bisphosphate carboxylase/oxygenase catalyzes present study compares the response of rubisco activation to the first reactions of the photosynthetic carbon reduction and light and CO2 in Arabidopsis wild-type and the rca mutant. photorespiratory carbon oxidation pathways (14). Catalytic com- These investigations demonstrate that the properties of rubisco petence of the enzyme requires prior activation by carbamate activation in leaves are consistent with the concept that the formation on a lysine of the large subunit followed by the activation process in vivo is an enzyme-catalyzed reaction. addition of Mg2" (9, 12). Activation of rubisco2 can be readily achieved in vitro by incubating the enzyme with CO2 and Mg2' prior to the initiation of catalysis with RuBP, but complete MATERIALS AND METHODS activation of rubisco by this spontaneous process requires CO2 Plant Material. Arabidopsis 'Columbia' wild-type and mutant concentrations much greater than occur physiologically (7, 10, strains were grown at 1% CO2 as described (23), except that a 14 12). h light/1O h dark photoperiod (200-300 gmol photons/M2.s) The activation state of rubisco increases upon illumination in was used instead of continuous illumination. Isolation of the isolated chloroplasts (2, 10, 26) and in leaves (1 1, 15, 16, 24). By mutant line CS207 1 carrying the rca mutation has been described analogy to the in vitro requirements, "light-activation" ofrubisco (24). has been attributed to increases in pH and Mg2' (5) which occur Labeling Studies. Intact plants were labeled with '4C02 and labeled products were separated into basic and nonbasic fractions 'Present address: USDA-ARS, Agronomy Department, University of by ion-exchange chromatography (23). The nonbasic fraction Kentucky, N222, ASC-N, Lexington, KY 40546. was further fractionated by HPLC (24). 2Abbreviations: rubisco, ribulose 1,5-bisphosphate carboxylase/oxy- Photosynthesis, Rubisco Activation, RuBP Levels and FBPase genase; RuBP, ribulose 1,5-bisphosphate; FBP, fructose 1,6-bisphos- Activation. IR gas analysis was performed on intact plants at phate; SBP, sedoheptulose 1,7-bisphosphate; PGA, 3-phosphoglyceric 25°C (23). Light levels (PAR) were varied with neutral density acid; FBPase, fructose 1,6-bisphosphate phosphatase; SBPase, sedohep- filters. For measurements of rubisco activation and RuBP con- tulose 1,7-bisphosphate phosphatase. centration (22) intact plants were flushed at 25°C with the desired 655 656 SALVWCCI ET AL. Plant Physiol. Vol. 80, 1986 humidified gas stream, and at the designated time entire plants were rapidly plunged into liquid N2. While frozen in N2, roots were removed and the remainder ofthe plant was transferred to NU 10~~~~~~~~~00-I~ a Ten Broeck glass homogenizer at 4°C containing 100 mM N-(2 120 Bsoo ~[ hydroxy-l ,l-bis[hydroxymethyl]ethyl)-glycine (Tricine)-NaOH N 600[7- and 10 mM at pH 8.1. of from two to MgCl2 Samples consisting - four plants were homogenized for 30 s and aliquots were assayed ioo ~004 immediately for activation level (initial RuBP carboxylase activity) or RuBP concentration. Total rubisco activity in wild-type 80 40 80 120 0 40 80 120 and mutant Arabidopsis, measured after incubating the extract M sMinutes for 10 min with 10 mm NaHCO3 and 10 mM MgCl2 at 25C, was M ~~~WT not affected by light, dark, or low light treatment of the plants. Rubisco activity was determined in an assay system containing I ~~~~~~~~~~~~~~~~~~rca 100 mm Tricine, 10 mM MgCl2, 0.4 mM RuBP, and 1.0 mM NaH'4C03 at pH 8.1. Replicate assays were conducted at 25C for 30 s and mean values were reported for each sample. For ~20O measurements of FBPase activity, plants were incubated under the conditions described in the text, frozen in liquid N2, and then rapidly (30 s) extracted and assayed as described previously 0 40 80 120 0 40 80 120 (8). Minutes Methyl Viologen Treatments. Leaves were treated by immers- FIG. 1. Time course of rubisco activation level (initial activity) in ing plants for two 3-min intervals in 0.05% Triton X-100 con- Arabidopsis wild-type (, O[, 0) and rca mutant (0, O, 0). Plants were taining 0.2 mm methyl viologen prior to incubation in the dark. flushed with 350 ,d C02/L in 2% 02, bal N2 for 2 h in the dark prior to This treatment caused nearly complete inhibition of net photo- the onset of illumination (zero time) at 350 (O1, 0) or 30 (0, 0) ,mol synthesis as measured by IR gas analysis. Triton X-100 alone at photons/M2.s or at 30 smol photons/m2.s after pretreatment with this concentration had no effect on activation or net photosyn- methyl viologen (U, 0). Inset. Time course of RuBP levels in the rca thesis. mutant following the onset of illumination (zero time) at 350 (0) or 30 Chlorophyll Determination. Chl concentration was determined (0) jsmol photons/M2 *s or at 30 ;&mol photons/m2 _ s after pretreatment after extraction in 80% acetone (1). with methyl viologen (0). light-induced changes in pH and Mg2e concentration. Under low RESULTS light, FBPase activity in the wild-type decreased by 50%. The Arabidopsis rca mutant required a very high CO2 concen- The time course of rubisco activation level in Arabidopsis tration to saturate photosynthesis, accumulated a large percent- wild-type and mutant was determined during illumination with age of photosynthetically fixed '4C02 into RuBP, and exhibited high (near-saturating) and low light (Fig. 1). To examine activa- a decrease in rubisco activity upon exposure to saturating light tion under conditions where CO2 fixation but not electron trans- at air levels of CO2 (24). However, since rubisco activation is port was inhibited, the activation level was also followed for dependent on light intensity (11, 16) we examined the effects of plants that were illuminated with low light after pretreatment light intensity on the in vivo activation level in Arabidopsis. with methyl viologen (Fig. 1). In wild-type plants, rubisco acti- Pretreatment of the wild-type with saturating light at air levels vation state rapidly increased under high light and slowly de- ofCO2 (350 ,l/L) increased the activation state ofrubisco 2-fold creased under low light. Slow deactivation occurred under both overthe dark level (Table I). In contrast, this same light treatment of these conditions in the mutant. The activation state in wild- caused deactivation below the dark level in the mutant. Under type leaves treated with methyl viologen rapidly increased under low light (30 gmol photons/mi2 s) the activation state of rubisco low light, but methyl viologen treatment was not effective in decreased below the dark level in both wild-type and the mutant, stimulating activation ofrubisco in the mutant. However, methyl and the decrease was greater in the mutant. To determine viologen did reduce the extent of deactivation. whether deactivation of rubisco in the rca mutant under high Mutant plants exposed to high light in the absence of methyl light might be caused by changes in the stromal pH or Mg2" viologen accumulated very high levels ofRuBP, up to nearly 850 concentration, light activation ofFBPase was examined. In both nmol/mg Chl (Fig. 1, inset). RuBP levels also increased under the dark and the light, the activation level ofFBPase was similar low light, reaching 400 nmol/mg Chl after 2 h. Treatment of in mutant and wild-type leaves (Table I). Thus, the lack ofrubisco mutant leaves with methyl viologen caused RuBP levels to activation in the rca mutant was not caused by the absence of remain at a constant low level, below 100 nmol/mg Chl. In the initial characterization of the rca mutant, short-term Table I. Rubisco and FBPase Activation Levels in Leaves of labeling experiments showed that 14C and 32P accumulated in Arabidopsis Wild-type and rca Mutant in the Dark and at Two Light RuBP upon illumination of leaves or chloroplasts (24). When Intensities wild-type plants were preilluminated for various intervals up to Plants were flushed continuously with 350 IAI CO2/L, 2% 02, balance 2 h and then labeled for 10 min with 14C02 at air levels of C02, N2 for 60 min prior to sampling. the percentage oflabel in RuBP remained at a constant and low Rubisco FBPase level (Fig. 2). In contrast, the rca mutant under identical conditions incorporated an increasingly greater percentage ofthe total Treatment rca rca '4C fixed into RuBP. After 2 h in the light, RuBP accounted for Wild-type mutant Wild-type rca over 42% of the 14CO2 fixed by the mutant during the 10 min period. For the rca mutant, insoluble and neutral compounds jumol/mg Chl- h were reduced as a percentage of the total '4C-labeled products. Dark 54 37 50 49 Throughout the entire time course, PGA accounted for a reduced Light percentage of the '4C label in the mutant compared with wild- 350 umol photons/m2_s 135 29 152 172 type, while the percentages of triose-phosphate + sugar- 30 usmol photons/m2r* s 15 6 76 monophosphates, SBP, and FBP were similar in the mutant and RUBISCO ACTIVATION IN ARABIDOPSIS 657

lo N. NEUTRAL" e 100 ~~~~~BASICsZlC:OINSOL

40 - /P_

30 _ 200 300 /Lmol Photons/rn2 .s 20 4 FIG. 3. Response of net photosynthesis and rubisco activation level TP TP (initial activity) to light intensity in Arabidopsis wild-type (0) and rca 10 MPe mutant (0). Photosynthesis and activation were measured at 350 ,d C02/ RuBP L in 2% 02, bal N2 after I h at the indicated light intensities. Plants were ~~~SBP+FBP preincubated for 30 min in the dark prior to the onset ofillumination. PFBPSep#MEWiPGA 0 40 80 120 0 40 80 120 mutant and rapidly activated in the wild-type. RuBP concentra- Minutes tion at 1% CO2 was considerably higher in the rca mutant than FIG. 2. Distribution of '4C among the photosynthetic intermediates in the wild-type (Fig. 4). At air levels of C02, a large increase in in Arabidopsis wild-type and rca mutant. Plants were flushed with 350 RuBP concentration occurred in the mutant. A comparatively ,ul C02/L in 2% 02, bal N2 and labeled for 10 min at various times after minor increase in RuBP level occurred in the wild-type (Fig. 4). the onset of illumination (zero time) at 350,gmol photonS/in2_ S. Incor- After 1 h in the dark, RuBP concentration and rubisco activation poration is expressed as a percentage ofthe total 14C fixed. Photosynthetic level were similar in the mutant and the wild-type. rates in wild-type were constant over the 2 h time courseI130 iml C02/ These effects of 1% CO2 prompted additional measurements mg Chl-h) but declined from 88 to 48 gmol C02/mg Chl-h in the rca of the response of rubisco activation state to the external CO2 mutant. concentration. In the dark, the activation state of rubisco was similar in the mutant and wild-type plants, increasing as a function of CO2 concentration over the range of 0 to 2000 gl the wild-type (Fig. 2). The time course of the increase in labeled C02/L (Fig. 5). A similar response ofthe activation state to CO2 RuBP was inversely correlated with the time course of rubisco concentration in the dark was observed previously (21). In light, deactivation in the mutant (Fig. 1). rubisco activation state in both the mutant and the wild-type The light response ofphotosynthesis and rubisco activation in was relatively insensitive to changes in the external CO2 concen- leaves of both wild-type Arabidopsis and the rca mutant dem- tration between 100 and 2000 1l C02/L. The extent of light onstrated that the activation state of rubisco in leaves, measured activation, however, differed substantially between the two after 1 h, closely matched the steady state photosynthetic rate of strains. While the wild-type activation state in the light was the plants (Fig. 3). A correlation between rubisco activation and considerably higher than in the dark at CO2 concentrations below photosynthetic rate has been reported by others (16). The acti- 2000 ,ul C02/L and in C02-free air, the activation state of the vation level in leaves from wild-type plants at light intensities mutant was lower in the light than in the dark except in C02- below 150 Mmol photons/m2 s was lower than found in the dark, free air, where activation states in the light and dark were similar while in the mutant the activation level in light was always below (Fig. 5). At 2000 Ul C02/L, rubisco activation level in the mutant the dark level regardless of the light intensity. The deactivation in the light was only 30% ofwild-type. ofrubisco which occurred at low light intensities followed a slow time course, requiring 60 min for complete deactivation (Fig. 1). In contrast, the lower photosynthetic rate that accompanied a DISCUSSION reduction in light intensity occurred rapidly, usually reaching The results presented here support the conclusion of Somer- steady state within a few min (data not shown). Similar results ville et al. (24) that the rca mutation specifically affected the light have been-reported by others (13). activation of rubisco. The changes in rubisco activation in the The rca mutant requires high CO2 (1%) for survival and mutant at high light intensities resembled those which occurred becomes chlorotic in air (24). The effects ofhigh CO2 and air on in the wild-type at low light intensities. However, this similarlity the rubisco activation level were therefore examined in situ at a was specific for light activation of rubisco since activation of constant light intensity of 150 Amol photons/m2 s in wild-type FBPase was not affected by the rca mutation. Portis et al. (18) and the mutant (Fig. 4). High CO2 (zero time point) suppressed and Purczeld et al. (19) showed that the activation states of light deactivation in the rca mutant and inhibited light activation FBPase and SBPase were affected by changes in the stromal pH of rubisco in wild-type plants. This effect on wild-type plants and Mg2' concentration, so it is unlikely that the rca mutation may be due to a suppression ofstromal alkalinization (27). Upon affects rubisco activation in such a manner. This conclusion is transfer to air levels of CO2, rubisco slowly deactivated in the also supported by labeling data which demonstrated that RuBP 658 SALVUCCI ET AL. Plant Physiol. Vol. 80, 1986

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0 20 40 60 80 100 120 Minutes 0 400 800 1200 1600 2000 FIG. 4. Time course of rubisco activation level (initial activity) and I CO2/t RuBP levels in Arabidopsis wild-type (0, *) and rca mutant (0, 0) FIG. 5. Response of rubisco activation level (initial activity) in the following transfer from 1% CO2 to air. Samples were taken from plants light and dark to external CO2 concentration in Arabidopsis wild-type in a growth cabinet at 1% CO2 (zero time) and at various times following (0, *) and rca mutant (0, 0). Activation was measured at the indicated transfer to air. After 60 min in the dark, plants were again illuminated CO2 concentration in 2% 02, bal N2 following 60 min incubation. Plants (1). were preincubated for 60 min in the dark prior to the onset of illumination with 350 Amol photon/m2.s. and PGA were the primary Calvin cycle intermediates affected by the mutation. Labeling patterns similar to those of the rca example, several investigations have demonstrated that the acti- mutant have been obtained with chloroplasts following brief vation state of rubisco in vivo is a function of light intensity (1 1, exposure to high temperatures (25), a treatment which specifi- 15, 16) and can limit photosynthesis under steady state condi- cally inhibited light activation of rubisco (26). tions (16). The altered response of photosynthesis to light inten- One possible explanation for the absence of rubisco activation sity in the mutant and its close correlation with activation state in the rca mutant is that RuBP levels, which are increased by the supports this latter point. The mechanism by which light inten- mutation, might inhibit rubisco activation in vivo (24). This sity controls the activation state of rubisco is not known, but explanation is suggested by data from studies with isolated rub- activation state of rubisco may be modulated by photon flux via isco which demonstrated that RuBP can bind tightly to inactive rubisco activase. Studies with a reconstituted light activation rubisco and thus act as a potent inhibitor of activation (6). system have shown that light (20), and specifically ApH (17) are However, when RuBP accumulation was prevented by treating required for in vivo activation of rubisco. Two PSI acceptors, leaves with methyl viologen, light activation was not restored in methyl viologen and pyocyanine, promote the rubisco activation the mutant. From these results we conclude that the lack of process both in vivo (Fig. 2) and in a reconstituted system (20). rubisco activation is the cause, not the effect, ofhigh RuBP levels These compounds inhibit light activation ofthose photosynthetic in the mutant. enzymes which are activated via the ferredoxin-thioredoxin sys- The nuclear mutation which is responsible for the rca pheno- tem (3), thus implying that light activation ofrubisco is mechan- type (24) affects the expression of two stromal polypeptides in istically distinct from other photosynthetic enzymes. The inabil- Arabidopsis (20). We have presented genetic and biochemical ity of methyl viologen to restore rubisco activation in the rca evidence indicating that these polypeptides are subunits of a mutant suggests that the effect of these acceptors on rubisco stromal protein which catalyzes rubisco activation in vivo (20). activation is indirect and that rubisco activation is mediated by The revelation that rubisco activation is catalyzed by rubisco the activase. activase, a protein not expressed in the rca mutant, provides a Measurements of leaf RuBP indicate that under steady state mechanism to explain the lack of rubisco light activation in the conditions in the light, RuBP concentration is generally greater mutant. than rubisco binding site concentration regardless of the light The occurrence of rubisco activase also provides a means to intensity or CO2 concentration (13, 15, 16). Since RuBP forms explain some characteristics ofthe in vivo activation process. For a tight complex with inactive rubisco (6), spontaneous activation RUBISCO ACTIVATION IN ARABIDOPSIS 659 of inactive rubisco by CO2 and Mg2+ would largely be inhibited. 9. LORIMER GH, HM MIZIORKO 1980 Carbamate formation on the E-amino Results the group ofa lysyl residue as the basis for the activation ofribulosebisphosphate from previous study indicated that rubisco activase carboxylase by CO2 and Mg2'. Biochemistry 19: 5321-5328 promotes activation ofthe inactive E-RuBP form ofthe enzyme 10. LORIMER GH, MR BADGER, HW HELDT 1983 The activation of ribulose 1,5- (20). Thus, we suggest that inactive rubisco complexes with RuBP bisphosphate carboxylase/oxygenase. In HW Siegelman, G Hind, eds, Pho- so that rubisco activation is determined primarily by the rate at tosynthetic Carbon Assimilation. Plenum, New York, pp 283-306 which activase converts E-RuBP to active enzyme. The response 1 1. MACHLER F, J NOSBERGER 1980 Regulation of ribulose bisphosphate carboxylase activity in intact wheat leaves by light, CO2 and temperature. J Exp of rubisco activation state to CO2 concentration observed here Bot 31: 1485-1491 and in other studies (15, 21) supports this conclusion since, 12. MIZIORKo HM, GH LORIMER 1983 Ribulose 1,5-bisphosphate carboxylase/ unlike the isolated enzyme, the activation state in the light (but oxygenase. Annu Rev Biochem 52: 507-535 not in the 13. MoTT KA, RG JENSEN, JW O'LEARY, JA BERRY 1984 Photosynthesis and dark, when RuBP levels are low) was largely inde- ribulose 1,5-bisphosphate concentrations in intact leaves of Xanthium stru- pendent of CO2 concentration. In the case of the rca mutant, marium L. Plant Physiol 76: 968-971 formation of the inactive E-RuBP complex in the absence of 14. OGREN WL 1984 Photorespiration: pathways, regulation and modification. rubisco activase could be the cause of the low activation state in Annu Rev Plant Physiol 35: 415-442 the light and might explain why activation was not increased 15. PERCHOROWICZ JT, RG JENSEN 1983 Photosynthesis and activation ofribulose by bisphosphate carboxylase in wheat seedlings. Regulation by CO2 and 02. C02. Plant Physiol 71: 955-960 The presence of rubisco activase in plants provides a mecha- 16. PERCHOROWICZ JT, DA RAYNES, RG JENSEN 1981 Light limitation of photo- nism to explain the coordinate regulation of photon flux rate synthesis and activation of ribulose bisphosphate carboxylase in wheat through the electron transport chain with seedlings. Proc Natl Acad Sci USA 78: 2985-2989 the activity ofrubisco, 17. PORTIs, AR JR 1985 Energetics of light activation of rubisco in isolated which catalyzes the initial reaction of photosynthetic carbon chloroplasts. Plant Physiol 77: S-108 reduction. Rubisco activation in leaves was stimulated by in- 18. PORTIs AR JR, CJ CHON, A MOSBACH, HW HELDT 1977 Fructose- and creasing light intensity but not by increasing CO2 concentration. sedoheptulose bisphosphatase. The sites ofa possible control ofCO2 fixation by light-dependent changes of the stromal Mg2' concentration. Biochim Thus, rubisco activation in vivo is controlled primarily by light Biophys Acta 461: 313-325 intensity and not CO2 concentration. By modulating the first 19. PURCZELD P, CJ CHON, AR PORTIS JR, HW HELDT, U HEBER 1978 The reaction in carbon reduction, rubisco activase ultimately controls mechanism of the control of carbon fixation by the pH in the chloroplast the balance between the utilization and regeneration of RuBP stroma. Studies with nitrite-mediated proton transfer across the envelope. and hence the rate net Biochim Biophys Acta 501: 488-498 of photosynthesis under steady state 20. SALVUCCI ME, AR PORTIS, WL OGREN 1985 A soluble chloroplast protein conditions. catalyzes activation of ribulosebisphosphate carboxylase/oxygenase in vivo. Photosynth Res 7: 193-201 LITERATURE CITED 2 1. SCHNYDER H, F MACHLER, J N6SBERGER 1984 Influence of temperature and 02 concentration on photosynthesis and light activation of ribulosebisphos- 1. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase phate carboxylase oxygenase in intact leaves ofwhite clover (Trifolium repens in Beta vulgaris. Plant Physiol 24: 1-15 L.). J Exp Bot 35: 147-156 2. BAHR JT, RG JENSEN 1978 Activation of ribulose bisphosphate carboxylase in 22. SICHER RC, JT BAHR, RG JENSEN 1979 Measurement of ribulose 1,5-bisphos- intact chloroplasts by C02 and light. Arch Biochem Biophys 185: 38-48 phate from spinach chloroplasts. Plant Physiol 64: 876-879 3. BUCHANAN BB 1980 Role of light in the regulation of chloroplast enzymes. 23. SOMERVILLE CR, WL OGREN 1982 Isolation of photorespiratory mutants of Annu Rev Plant Physiol 31: 341-374 Arabidopsis thaliana. In M Edelman, RB Hallick, NH Chua, eds, Methods 4. HEBER U, U TAKAHAMA, S NEIMANIS, M SHIMIzu-TAKAHAMA 1982 Transport in Chloroplast Molecular Biology, Elsevier, Amsterdam, pp 129-138 as the basis of the Kok effect. Levels of some photosynthetic intermediates 24. SOMERVILLE CR, AR PORTIS, WL OGREN 1982 A mutant of Arabidopsis and activation of light regulated enzymes during photosynthesis of chloro- thaliana which lacks activation of RuBP carboxylase in vivo. Plant Physiol plasts and green leaf protoplasts. Biochim Biophys Acta 679: 289-299 70: 381-387 5. JENSEN RG, JT BAHR 1977 Ribulose 1,5-bisphosphate carboxylase-oxygenase. 25. WEIs E 1981 Reversible heat-inactivation of the Calvin Cycle: A possible Annu Rev Plant Physiol 28: 379-400 mechanism ofthe temperature regulation ofphotosynthesis. Planta 151: 33- 6. JORDAN DB, R CHOLLET 1983 Inhibition of ribulose bisphosphate carboxylase 39 by substrate ribulose 1,5-bisphosphate. J Biol Chem 258: 13752-13758 26. WEIs E 1981 The temperature-sensitivity of dark-inactivation and light- 7. JORDAN DB, WL OGREN 1981 A sensitive assay procedure for simultaneous activation of the ribulose-1,5-bisphosphate carboxylase in spinach chloro- determination ofribulose 1 ,5-bisphosphate carboxylase and oxygenase activ- plasts. FEBS Lett 129: 197-200 ities. Plant Physiol 67: 237-245 27. WERDEN K, HW HELDT, M MILOVANCEV 1975 The role ofpH in the regulation 8. LEEGOOD RC, DA WALKER 1982 Regulation of fructose-1,6-bisphosphatase of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the activity in leaves. Planta 156: 449-456 light and dark. Biochim Biophys Acta 396: 276-292