Deactivation Of Rubisco Research Articles

Effect of activase level and isoform on the thermotolerance of photosynthesis in Arabidopsis

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation decreases under moderate heat stress. This decrease is caused by an impairment of activase function, which is exacerbated by faster rates of Rubisco deactivation at elevated temperatures. To determine if stromal oxidation causes inhibition of activase, transgenic Arabidopsis plants expressing suboptimal amounts of either the redox-regulated 46 kDa alpha- or non-redox regulated 43 kDa beta-isoform of activase were examined. Photosynthesis, as measured by gas exchange and chlorophyll fluorescence, and Rubisco activation were inhibited to a much greater extent by moderately high temperatures in the two transgenic lines expressing suboptimal levels of the individual isoforms of activase compared with wild-type plants or transgenic plants expressing levels of the beta-isoform sufficient for wild-type rates of photosynthesis. Net photosynthesis and Rubisco activation in transgenic plants expressing suboptimal amounts of the beta-isoform of activase from the Antarctic hairgrass were even more sensitive to inhibition by moderate heat stress than in the transgenic plants containing Arabidopsis activase. The results demonstrate that photosynthesis exhibits a similar sensitivity to inhibition by moderately high temperature in plants expressing either of the two different isoforms of activase. Thus, impairment of activase function under heat stress is not caused by oxidation of the redox-sensitive sulphydryls of the alpha-isoform of activase. Instead, the results are consistent with thermal denaturation of activase under moderate heat stress, the effects of which on Rubisco activation would be enhanced when activase levels are suboptimal for photosynthesis.

Response of Rice to p(CO2) Enrichment

SUMMARY Rice (Oryza sativa L.) is grown in many different agro-ecological zones and it assimilates CO2 directly through C3 photosynthesis. At current CO2 partial pressure [p(CO2)], rice is not photosynthetically saturated but immediately after exposure to 100 p(CO2), photosynthetic rates are increased by 48 percent on an average. However, this increase is not sustained during prolonged exposure and is associated with reduced amounts of ribulose-1-5-bisphospate carboxylase/oxygenase (Rubisco) content and its maximum potential activity (Vcmax). Rubisco content and Vcmax were reduced by 33 and 22%, respectively, at elevated p(CO2) and this has an additive effect on leaf photosynthesis. Activation state of the Rubisco is also decreased by elevated p(CO2) but no deactivation of Rubisco occurs in rbc S antisense rice with 40% wild type Rubisco suggesting that deactivation is an optimize response. Large amounts of Rubisco (about 80–90%) is synthesized prior to full expansion of leaf when sugar accumulation is minimal. After the leaf has fully expanded, very little Rubisco (about 10-20%) is synthesized and a large amount of sugar accumulates during this period. Therefore, it is unlikely that downregulation of Rubisco at elevated p(CO2) is caused by decreasing the mRNA transcripts for Rubisco small subunit (rbcS) and Rubisco large subunit (rbcL) through soluble sugars. It is more likely that elevated p(CO2) mediated decline in Rubisco is due to rapid degradation of protein and lower nitrogen (N) allocation to Rubisco due to reduced N afflux. Leaf N content was reduced by 16% in plants grown at elevated p(CO2) though leaf N to Rubisco ratio remained consistently unchanged regardless of growth p(CO2) suggesting that N assimilation, distribution and remobilization at the whole plant level is tightly controlled at elevated p(CO2). Further, changes in N and C metabolisms at elevated p(CO2) are likely to reduce the critical N concentration by 30% at elevated p(CO2).

Effects of moderate heat stress on photosynthesis: importance of thylakoid reactions, rubisco deactivation, reactive oxygen species, and thermotolerance provided by isoprene

ABSTRACTPhotosynthesis is particularly sensitive to heat stress and recent results provide important new insights into the mechanisms by which moderate heat stress reduces photosynthetic capacity. Perhaps most surprising is that there is little or no damage to photosystem II as a result of moderate heat stress even though moderate heat stress can reduce the photosynthetic rate to near zero. Moderate heat stress can stimulate dark reduction of plastoquinone and cyclic electron flow in the light. In addition, moderate heat stress may increase thylakoid leakiness. At the same time, rubisco deactivates at moderately high temperature. Relationships between effects of moderate heat on rubisco activation and thylakoid reactions are not yet clear. Reactive oxygen species such as H2O2 may also be important during moderate heat stress. Rubisco can make hydrogen peroxide as a result of oxygenase side reactions and H2O2 production by rubisco was recently shown to increase substantially with temperature. The ability to withstand moderately high temperature can be improved by altering thylakoid lipid composition or by supplying isoprene. In my opinion this indicates that thylakoid reactions are important during moderate heat stress. The deactivation of rubisco at moderately high temperature could be a parallel deleterious effect or a regulatory response to limit damage to thylakoid reactions.

Mechanism for deactivation of Rubisco under moderate heat stress

Photosynthesis is particularly sensitive to direct inhibition by heat stress. This inhibition is closely associated with the inactivation of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco). To develop a more complete understanding of the mechanism of inactivation of Rubisco under moderate heat stress, various aspects of the process were examined both in vivo and in vitro. Experiments with isolated Rubisco revealed that the rate of synthesis of the catalytic misfire product, xylulose‐1,5‐bisphosphate, increased with temperature. Activated Rubisco, produced by reaction with activase at a control temperature of 25°C or by incubation with high CO2, deactivated when the temperature of the reaction exceeded temperatures that were equivalent to the optimum for activase adenosine triphosphatase (ATPase) activity. Measurements of the activation state of Rubisco in cotton and tobacco leaves showed that Rubisco inactivated within 7 s of imposing a heat stress. Thus, elevated temperature had an opposite effect on the two processes that ultimately determine the activation state of Rubisco, decreasing activase activity but stimulating the catalytic misfire reaction that inactivates Rubisco. These data support a mechanism for the inactivation of Rubisco at high temperature involving an inability of activase to overcome the inherently faster rates of Rubisco inactivation. That the net effect of elevated temperatures on Rubisco activation is similar both in vivo and under controlled conditions in vitro argues for a direct effect of temperature on the activation of Rubisco by activase and against the proposal that the deactivation of Rubisco under moderate heat stress is a secondary consequence of perturbations in the thylakoid membrane.

Inhibition of photosynthesis by heat stress: the activation state of Rubisco as a limiting factor in photosynthesis.

Although the catalytic activity of Rubisco increases with temperature, the low affinity of the enzyme for CO(2) and its dual nature as an oxygenase limit the possible increase in net photosynthesis with temperature. For cotton, comparisons of measured rates of net photosynthesis with predicted rates that take into account limitations imposed by the kinetic properties of Rubisco indicate that direct inhibition of photosynthesis occurs at temperatures higher than about 30 degrees C. Inhibition of photosynthesis by moderate heat stress (i.e. 30-42 degrees C) is generally attributed to reduced rates of RuBP regeneration caused by disruption of electron transport activity, and specifically inactivation of the oxygen evolving enzymes of photosystem II. However, measurements of chlorophyll fluorescence and metabolite levels at air-levels of CO(2) indicate that electron transport activity is not limiting at temperatures that inhibit CO(2) fixation. Instead, recent evidence shows that inhibition of net photosynthesis correlates with a decrease in the activation state of Rubisco in both C(3) and C(4) plants and that this decrease in the amount of active Rubisco can fully account for the temperature response of net photosynthesis. Biochemically, the decrease in Rubisco activation can be attributed to: (1) more rapid de-activation of Rubisco caused by a faster rate of dead-end product formation; and (2) slower re-activation of Rubisco by activase. The net result is that as temperature increases activase becomes less effective in keeping Rubisco catalytically competent. In this opinionated review, we discuss how these processes limit photosynthetic performance under moderate heat stress.

The activation state of Rubisco directly limits photosynthesis at low CO(2) and low O(2) partial pressures.

Using gas exchange, enzyme assays, and theoretical modeling of photosynthetic responses to light and CO(2), we investigated whether decarbamylation of the active site of Rubisco at low CO(2) and low light leads to a condition where the activation state of Rubisco directly limits the rate of net CO(2) assimilation. Photosynthetic limitation by a reduction in the activation state of Rubisco would be indicated as a decline in the initial slope of the photosynthetic CO(2) response relative to what is predicted using theoretical models. In bean (Phaseolus vulgaris) and oat (Avena sativa), we saw no discrepancy between predicted and observed initial slope values at 200 and 400 mbar O(2), indicating no limitation by the carbamylation state of Rubisco. At 30 mbar O(2) and light saturation, we also saw no discrepancy between predicted and observed initial slope values; however, at subsaturating light intensity, our observed initial slope values were less than the modeled initial slope values that corresponded to an RuBP regeneration limitation. Moreover, significant reduction of the Rubisco activation state occurred in both species at 30 mbar O(2) and 30 mubar CO(2). When the model was reprogrammed to account for observed levels of Rubisco deactivation, the predicted and measured initial slope values at low O(2) and low PPFD were similar, indicating the reduction in carbamylation state accounted for the discrepancy. We interpret this as evidence for a direct limitation of the carbamylation state of Rubisco, probably because of a CO(2) limitation for carbamate formation. This limitation was only observed at intercellular CO(2) levels below what is encountered in vivo. At physiologically relevant CO(2) levels in situ, the leaves maintained sufficient Rubisco activity to avoid cabamylation state limitations in the steady state.

Exceptional Sensitivity of Rubisco Activase to Thermal Denaturation in Vitro and in Vivo

Heat stress inhibits photosynthesis by reducing the activation of Rubisco by Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vitro and in vivo and compared with the stabilities of two other soluble chloroplast proteins. Isolated activase exhibited a temperature optimum for ATP hydrolysis of 44°C compared with ≥60°C for carboxylation by Rubisco. Light scattering showed that unfolding/aggregation occurred at 45°C and 37°C for activase in the presence and absence of ATPγS, respectively, and at 65°C for Rubisco. Addition of chemically denatured rhodanese to heat-treated activase trapped partially folded activase in an insoluble complex at treatment temperatures that were similar to those that caused increased light scattering and loss of activity. To examine thermal stability in vivo, heat-treated tobacco (Nicotiana rustica cv Pulmila) protoplasts and chloroplasts were lysed with detergent in the presence of rhodanese and the amount of target protein that aggregated was determined by immunoblotting. The results of these experiments showed that thermal denaturation of activase in vivo occurred at temperatures similar to those that denatured isolated activase and far below those required to denature Rubisco or phosphoribulokinase. Edman degradation analysis of aggregated proteins from tobacco and pea (Pisum sativum cv “Little Marvel”) chloroplasts showed that activase was the major protein that denatured in response to heat stress. Thus, loss of activase activity during heat stress is caused by an exceptional sensitivity of the protein to thermal denaturation and is responsible, in part, for deactivation of Rubisco.

Exceptional Sensitivity of Rubisco Activase to Thermal Denaturation in Vitro and in Vivo

Heat stress inhibits photosynthesis by reducing the activation of Rubisco by Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vitro and in vivo and compared with the stabilities of two other soluble chloroplast proteins. Isolated activase exhibited a temperature optimum for ATP hydrolysis of 44 degrees C compared with > or =60 degrees C for carboxylation by Rubisco. Light scattering showed that unfolding/aggregation occurred at 45 degrees C and 37 degrees C for activase in the presence and absence of ATPgammaS, respectively, and at 65 degrees C for Rubisco. Addition of chemically denatured rhodanese to heat-treated activase trapped partially folded activase in an insoluble complex at treatment temperatures that were similar to those that caused increased light scattering and loss of activity. To examine thermal stability in vivo, heat-treated tobacco (Nicotiana rustica cv Pulmila) protoplasts and chloroplasts were lysed with detergent in the presence of rhodanese and the amount of target protein that aggregated was determined by immunoblotting. The results of these experiments showed that thermal denaturation of activase in vivo occurred at temperatures similar to those that denatured isolated activase and far below those required to denature Rubisco or phosphoribulokinase. Edman degradation analysis of aggregated proteins from tobacco and pea (Pisum sativum cv "Little Marvel") chloroplasts showed that activase was the major protein that denatured in response to heat stress. Thus, loss of activase activity during heat stress is caused by an exceptional sensitivity of the protein to thermal denaturation and is responsible, in part, for deactivation of Rubisco.

Increased heat sensitivity of photosynthesis in tobacco plants with reduced Rubisco activase.

High temperature inhibits photosynthesis by several mechanisms including deactivation of Rubisco. The inhibition of photosynthesis by high temperature and its relationship to Rubisco deactivation was studied using tobacco (Nicotiana tabaccum L. cv W38) transformed with a Rubisco activase gene inserted in the antisense orientation and untransformed controls. High temperature (42 degrees C) reduced photosynthesis in both lines of plants. However, photosynthesis recovered nearly completely in wild-type plants and very little in plants lacking Rubisco activase. The F(0)' level of chlorophyll fluorescence decreased and q(N) increased in the control plants during heating. In the antisense plants, q(N) was always high and F(0)' increased slightly during heat stress. NADP-malate dehydrogenase activation was unaffected by heat stress in control plants but was increased in the transgenic plants, consistent with a high redox status in the chloroplast. In wild-type plants, the inhibition of photosynthesis could be explained by a reversible decarbamylation of Rubisco and an acceptor-side limitation imposed on photosynthetic electron transport. However, in the anti-activase plants, carbamylation was low and constant and could not explain how photosynthesis was reduced at high temperature. Because ribulose bisphosphate was saturating at high temperature, the reduction in photosynthesis must have been caused by some impairment of Rubisco function not reflected in measurements of activation state or carbamylation status. This in vivo Rubisco impairment was not relieved upon return to lower temperature. We speculate that the reversible decarbamylation of Rubisco at moderately high temperature may be a protective mechanism by which the plant avoids more serious effects on Rubisco and the rest of the photosynthetic apparatus.

Heterogeneous inhibition of photosynthesis over the leaf surface of rosa rubiginosa L. during water stress and abscisic acid treatment: induction of a metabolic component by limitation of CO(2) diffusion

The contribution of changes in stomatal conductance and metabolism in determining heterogeneous photosynthesis inhibition during dehydration and abscisic acid (ABA) feeding was investigated using detached leaves of Rosa rubiginosa L. The steady-state and maximal rates of electron transport under a transient high CO(2) concentration were monitored using chlorophyll fluorescence imaging. The decrease in electron transport rate induced by dehydration and ABA treatment almost reverted to the control rate under transient high CO(2) availability. Therefore, inhibition of photosynthesis was mainly mediated through stomatal closure. However, since reversion was not complete, a metabolic inhibition was also identified as a decrease in the maximal electron transport rate driven by carboxylation. Under dehydration or ABA feeding, as under low ambient CO(2) treatment, in 21% or 0.4% O(2), the lower the steady-state electron transport was, the lower was the maximal electron transport rate during transient high CO(2) availability. We conclude that low CO(2) availability reduced the capacity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) to drive electron transport. The potential contribution of Rubisco deactivation mediated by stomatal closure is discussed.

Effects of growth-light quantity, growth-light quality and CO2 concentration on Rubisco deactivation during low PFD or darkness

The effects of CO2 concentration and the effects of growth-light conditions on Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) deactivation were examined for Spinacea oleracea (spinach). Rubisco deactivation kinetics and the degree that Rubisco activation limited the rise in photosynthesis following an increase in photon flux density (PFD) were determined from gas-exchange time courses. There were no significant differences in the apparent relaxation time for Rubisco deactivation among leaves exposed to high or low CO2 (50 or 1000 μmol mol-1) and low PFD (170 μmol m-2 s-1) or darkness. However, when PFD was increased to 1700 μmol m-2 s-1 following a period of low PFD or darkness, leaves exposed to low CO2 × low PFD showed a lower contribution to the photosynthetic induction process by the activation of Rubisco than leaves exposed to the other treatments. For the growth-light experiments, spinach was grown under high PFD × high red:far-red ratio (R:FR), low PFD × high R:FR, or low PFD × low R:FR light environments. Leaves that matured under the low PFD × low R:FR treatment showed a lower percent change in photosynthesis due to Rubisco activation than leaves exposed to the other growth-light treatments. However, there were no significant differences among the growth-light treatments in the maximum contribution of Rubisco activation to the induction response or in the apparent relaxation time for Rubisco deactivation during shade events.

Combined effects of chronic ozone and elevated CO2 on Rubisco activity and leaf components in soybean (Glycine max)

per unit Rubisco content of plants in grown CF-AA, and increased LMA by 27-74% of the leaf mass Content and activity of Rubisco and concentrations of per unit area in CF-AA because of a 23-148% increase leaf nitrogen, chlorophyll and total non-structural car- in starch. However, the data suggest that, at elevated bohydrates (TNC) were determined at regular intervals (CO 2 ), increases in starch and sucrose are not directly during the 1993 and 1994 growing seasons to under- responsible for the deactivation of Rubisco. Also, there stand the effects and interactions of (O 3 ) and elevated was little evidence of an adjustment of Rubisco activity (CO 2 ) on biochemical limitations to photosynthesis in response to starch and sucrose metabolism. during ontogeny. Soybean (Glycine max var. Essex) was Significant interactions between elevated (CO 2 ) and grown in open-top field chambers in either charcoal- (O

The Relationship between Rubisco Activity and Photosynthesis in Apple Leaves with Different Nitrogen Content

Based on the curvilinear relationship between carboxylation efficiency and leaf N in apple leaves, we hypothesized that deactivation of Rubisco accounts for the lack of response of photosynthesis to increasing leaf N under high N supply. A wide range of leaf N content (from 1.0 to 5.0 g·m–2) was achieved by fertigating bench-grafted Fuji/M26 apple trees for 6 weeks with different N concentrations using a modified Hoagland solution. Analysis of photosynthesis in response to intercellular CO2 under both 21% and 2% O2 indicated that photosynthesis at ambient CO2 was mainly determined by the activity of Rubisco. Measurements of Rubisco activity revealed that initial Rubisco activity increased with leaf N up to 3.0 g·m–2, then leveled off with further rise in leaf N, whereas total Rubisco activity increased linearly with increasing leaf N throughout the leaf N range. As a result, Rubisco activation state decreased with increasing leaf N. Photosynthesis at ambient CO2 and carboxylation efficiency were both linearly correlated with initial Rubisco activity, but showed curvilinear relationships with total Rubisco activity and leaf N. As leaf N increased, photosynthetic nitrogen use efficiency declined with decreasing Rubisco activation state.

Responses of Rubisco activation and deactivation rates to variations in growth-light conditions

Basil (Ocimum basilicum) and impatiens (Impatiens wallerana) were grown in sun, shade, or fluctuating light (15 min sun, 15 min shade) to examine the effects of growth-light conditions on the rates of light-induced Rubisco activation and deactivation. Rubisco activation and deactivation rates were determined from gas-exchange measurements of photosynthesis following a step increase in PFD. Rubisco deactivation rates were also determined from biochemical analyses of leaf extracts. There were no significant differences in Rubisco activation rate among the growth conditions or between the two species. However, there were significant differences in Rubisco deactivation rate among the growth conditions in basil and between the two species. In basil, Rubisco deactivated more slowly following a decrease in PFD in sun- and fluctuating-light grown plants than in shade grown plants. Slower rates of Rubisco deactivation during periods at low PFD resulted in higher activation states at the onset of increased PFD. Thus, the contribution of Rubisco activation to the induction process was less for basil plants grown under sun and fluctuating light than for those grown under shade. Impatiens deactivated Rubisco more rapidly than in basil, but there was no substantial effect of the three growth-light conditions on Rubisco deactivation rates in impatiens.

Activation and deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in three marine microalgae

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was examined in three marine microalgae: the chlorophyte t Dunaliella tertiolecta and the chromophytes t Pavlova lutheri and t Thalassiosira pseudonana. The three species differed in the sensitivity of Rubisco activity in crude extracts to magnesium ion concentration, the presence of protease inhibitors, the duration of the incubation on activity, and the potential for full activation of Rubisco with 20 mM magnesium chloride and 20 mM bicarbonate t in vitro. t D. tertiolecta had responses that were similar to those described in vascular plants: regulation of initial activity on a gradient of irradiances; maximum initial activities that were 80– 90% of light-saturated photosynthesis; total activities that exceeded light-saturated photosynthesis by 30–100%; and deactivation of Rubisco in darkness. Both initial and total activity declined in darkness and increased on a return to growth irradiance. First-order time constants were about 9 min for deactivation and 3 min for reactivation of initial activity. The decline in total activity after a transition into darkness could not be reversed t in vitro but could be reversed by exposing t D. tertiolecta to light, a characteristic of regulation by CA1P. The responses of t T. pseudonana were qualitatively similar, except that recovery of initial activity was low and could only account for 30–40% of light-saturated photosynthesis. Rubisco from t T. pseudonana exposed to low irradiance could be activated t in vitro but at growth irradiance and higher, total activity was lower than initial activity. The time constants for deactivation and reactivation of initial activity after reciprocal switches between growth irradiance and darkness were 12–18 min and 3 min in t T. pseudonana. t P. lutheri showed no regulation of Rubisco activity in response to changes in irradiance or light-dark transitions. This may have been an artifact of the conditions chosen to measure activity.

Regulation of Rubisco activity and its potential effect on photosynthesis during mixing in a turbid estuary

The recent demonstration that Rubisco activity in phytoplankton is regulated in response to changes in irradiance allows development of a mechanistic model of the photosynthetic response to fluctuating irradiance. The model was used to predict photosynthetic responses during mixing in estuarine systems, the only environments in which the rate of change of irradiance is likely to occur on time scales comparable to induction of photosynthesis. Non-steady-state rates of photosynthesis were calculated based on the lags associated with activation and deactivation of Rubisco in response to an increase or decrease in Irradiance. Non-steady-state rates were compared with steady-state rates, calculated assuming an instantaneous change in photosynthetic response to a change in irradiance. Simulation~ were run for a deep, relatively clear estuary and a shallow, turbid estuary, using input parameters measured in Delaware Bay (DE, USA) and San Antonio Bay (TX, USA), respectively. When estimates of production are based on steady-state rates of photosynthesis, the model predicts average overestimates of 14%) in the former and 22':', In the latter Sensitivity analyses show that, within the range of reported values, the model is more sensitive to changes in turbidity than to changes in the depth of the mixed layer, incident irradiance or diffusivity. When compared with published data, the model tended to overestimate the reduction in photosynthesis in the deep bay condition, possibly because of a compensating increase in photosynthesis due to alleviation of feedback limitation by mixing. In contrast, the model provided reasonably accurate estimates in the shallow bay condition. This suggests that whlle induction IS unlikely to impose a constraint on production in relatively deep and clear estuaries such as Delaware Bay, it may impose a severe constraint in the shallow, turbid estuaries typified by San Antonio Bay.

Regulation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activity in Response to Reduced Light Intensity in C4 Plants.

The light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in 16 species of C4 plants representing all three biochemical subtypes and a variety of taxonomic groups. Rubisco regulation was assessed by measuring (a) the ratio of initial to total Rubisco activity, which reflects primarily the carbamylation state of the enzyme, and (b) total Rubisco activity per mol of Rubisco catalytic sites, which declines when 2-carboxyarabinitol 1-phosphate (CA1P) binds to carbamylated Rubisco. In all species examined, the activity ratio of Rubisco declined with a reduction in light intensity, although substantial variation was apparent between species in the degree of Rubisco deactivation. No relationship existed between the degree of Rubisco deactivation and C4 subtype. Dicots generally deactivated Rubisco to a greater degree than monocots. The total activity of Rubisco per catalytic site was generally independent of light intensity, indicating that CA1P and other inhibitors are not major contributors to the light-dependent regulation of Rubisco activity in C4 plants. The light response of the activity ratio of Rubisco was measured in detail in Amaranthus retroflexus, Brachiaria texana, and Zea mays. In A. retroflexus and B. texana, the activity ratio declined dramatically below a light intensity of 400 to 500 [mu]mol of photons m-2 s-1. In Z. mays, the activity ratio of Rubisco was relatively insensitive to light intensity compared with the other species. In A. retroflexus, the pool size of ribulose bisphosphate (RuBP) declined with reduced light intensity except between 50 and 500 [mu]mol m-2 s-1, when the activity ratio of Rubisco was light dependent. In Z. mays, by contrast, the pool size of RuBP was light dependent only below 350 [mu]mol m-2 s-1. These results indicate that, in response to changes in light intensity, most C4 species regulate Rubisco by reversible carbamylation of catalytic sites, as commonly observed in C3 plants. In a few species, notably Z. mays, Rubisco is not extensively regulated in response to changes in light intensity, possibly because the activity of the CO2 pump may become limiting for photosynthesis at subsaturating light intensity.

PHOTOSYNTHETIC INHIBITION IN STRAWBERRY GROWN UNDER TWICE AMBIENT CO2

`Midway' and `Raritan' strawberry cultivars were grown for six weeks under ambient (350) or twice ambient (700 μmol mol-1) CO2 Photosynthetic inhibition (40-50%) was observed in both cultivars as indicated by reductions (P < 0.001) in CO2 assimilation, carboxylation efficiency, stomatal conductance, and apparent quantum yield. Growth under high CO2 did not affect Rubisco activity in either cultivar, whereas Rubisco activation state (%) was reduced (72.9 to 67.6) in `Midway' and increased (70.8 to 83.3) in `Raritan' compared to ambient CO2 grown plants. Soluble and Rubisco protein decreased 10-15% in `Midway' and 40-45% in `Raritan' when grown under high CO2 Thus, `Raritan' reduced the amount of protein partitioned into photosynthetic enzymes under conditions of photosynthetic inhibition, whereas `Midway' experiences Rubisco deactivation. The reduction in Rubisco protein appears to be under the regulation of chloroplast genes.

Rate Limitation of Non-Steady-State Photosynthesis by Ribulose-1,5-Bisphosphate Carboxylase in Spinach

A mathematical framework was developed to analyse rate limitation of non-steady-state photosynthesis following an increase in photon flux density (PFD). This analysis was employed to resolve an exponential phase of the photosynthetic response of Spinacia oleracea L. to a step increase from darkness to moderate PFD. This phase had a relaxation time of approximately 5 min, similar to the relaxation time for the activation of ribulose-1,5-bisphosphate carboxylase (Rubisco) as determined by freeze-clamp experiments following the same change in PFD. Furthermore, as the time in darkness prior to illumination was increased, the exponential phase contributed more to the overall trajectory of photosynthesis following the increase in light. The relaxation time for the increase in the contribution of this phase was 24 min. Freeze-clamp studies showed a relaxation time of 28 min for Rubisco deactivation in the dark. These results, together with measurements of RuP2 levels, suggest that the exponential phase resolved from gas exchange experiments was limited by activation of Rubisco and that Rubisco deactivation in the dark was the reason that this phase contributed more to the overall photosynthetic trajectory as time in darkness increased. A quantitative expression for the amount of extra photosynthesis that could have been obtained had Rubisco activated instantly following an increase in PFD was derived and discussed in relation to optimal functioning of the system under different environmental conditions.

Purification and Assay of Rubisco Activase from Leaves

Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase protein was purified from spinach leaves by ammonium sulfate precipitation and ion exchange fast protein liquid chromatography. This resulted in 48-fold purification with 70% recovery of activity and yielded up to 18 milligrams of rubisco activase protein from 100 grams of leaves. Based on these figures, the protein comprised approximately 2% by weight of soluble protein in spinach (Spinacia oleracea L.) leaves. The preparations were at least 95% pure and were stable when frozen in liquid nitrogen. Addition of ATP during purification and storage was necessary to maintain activity. Assay of rubisco activase was based on its ability to promote activation of rubisco in the presence of ribulose-1,5-bisphosphate. There was an absolute requirement for ATP which could not be replaced by other nucleoside phosphates. The initial rate of increase of rubisco activity and the final rubisco specific activity achieved were both dependent on the concentration of rubisco activase. The initial rate was directly proportional to the rubisco activase concentration and was used as the basis of activity. The rate of activation of rubisco was also dependent on the rubisco concentration, suggesting that the activation process is a second order reaction dependent on the concentrations of both rubisco and rubisco activase. It is suggested that deactivation of rubisco occurs simultaneously with rubisco activase-mediated activation, and that rubisco activation state represents a dynamic equilibrium between these two processes.