The activation state of Rubisco directly limits photosynthesis at low CO2 and low O2 partial pressures

Using gas exchange, enzyme assays, and theoretical modeling of photosynthetic responses to light and CO₂, we investigated whether decarbamylation of the active site of Rubisco at low CO₂ and low light leads to a condition where the activation state of Rubisco directly limits the rate of net CO₂ 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 CO₂ 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 O₂, indicating no limitation by the carbamylation state of Rubisco. At 30 mbar O₂ 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 O₂ and 30 μbar CO₂. When the model was reprogrammed to account for observed levels of Rubisco deactivation, the predicted and measured initial slope values at low O₂ 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 CO₂ limitation for carbamate formation. This limitation was only observed at intercellular CO₂ levels below what is encountered in vivo. At physiologically relevant CO₂ levels in situ, the leaves maintained sufficient Rubisco activity to avoid cabamylation state limitations in the steady state. This revised version was published online in June 2006 with corrections to the Cover Date.     

The drelationship between CO2-assimilation rate,Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO₂-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r₁ progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO₂ supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO₂-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO₂-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO₂-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P 2-carboxyarabinitol-1-phosphate - P_ip_a intercellular, ambient partial pressure of CO₂ - PGA 3-phospho-glycerate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SSU small subunit of Rubisco     

The single-process biochemical reaction of Rubisco: a unified theory and model with the effects of irradiance, CO₂ and rate-limiting step on the kinetics of C₃ and C₄ photosynthesis from gas exchange

Photosynthesis is the origin of oxygenic life on the planet, and its models are the core of all models of plant biology, agriculture, environmental quality and global climate change. A theory is presented here, based on single process biochemical reactions of Rubisco, recognizing that: In the light, Rubisco activase helps separate Rubisco from the stored ribulose-1,5-bisphosphate (RuBP), activates Rubisco with carbamylation and addition of Mg²⁺, and then produces two products, in two steps: (Step 1) Reaction of Rubisco with RuBP produces a Rubisco-enediol complex, which is the carboxylase-oxygenase enzyme (Enco) and (Step 2) Enco captures CO₂ and/or O₂ and produces intermediate products leading to production and release of 3-phosphoglycerate (PGA) and Rubisco. PGA interactively controls (1) the carboxylation-oxygenation, (2) electron transport, and (3) triosephosphate pathway of the Calvin-Benson cycle that leads to the release of glucose and regeneration of RuBP. Initially, the total enzyme participates in the two steps of the reaction transitionally and its rate follows Michaelis-Menten kinetics. But, for a continuous steady state, Rubisco must be divided into two concurrently active segments for the two steps. This causes a deviation of the steady state from the transitional rate. Kinetic models are developed that integrate the transitional and the steady state reactions. They are tested and successfully validated with verifiable experimental data. The single-process theory is compared to the widely used two-process theory of Farquhar et al. (1980. Planta 149, 78-90), which assumes that the carboxylation rate is either Rubisco-limited at low CO₂ levels such as CO₂ compensation point, or RuBP regeneration-limited at high CO₂. Since the photosynthesis rate cannot increase beyond the two-process theory's Rubisco limit at the CO₂ compensation point, net photosynthesis cannot increase above zero in daylight, and since there is always respiration at night, it leads to progressively negative daily CO₂ fixation with no possibility of oxygenic life on the planet. The Rubisco-limited theory at low CO₂ also contradicts all experimental evidence for low substrate reactions, and for all known enzymes, Rubisco included.     

Proteolysis and transition-state-analogue binding of mutant forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

Trypsin digestion reduces the sizes of both the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from the green alga Chlamydomonas reinhardtii. Incubation of either CO₂/Mg²⁺ -activated or nonactivated enzyme with the transition-state analogue carboxyarabinitol bisphosphate protects a trypsin-sensitive site of the large subunit, but not of the small subunit. Incubation of the nonactivated enzyme with ribulosebisphosphate (RuBP) provided the same degree of protection. Thus, the very tight binding that is a characteristic of the transitionstate analogue is apparently not required for the protection of the trypsin-sensitive site of the large subunit. Mutant enzymes that have reduced CO₂/O₂ specificities failed to bind carboxyarabinitol bisphosphate tightly. However, their large-subunit trypsin-sensitive sites could still be protected. The K _m values for RuBP were not significantly changed for the mutant enzymes, but the V _max values for carboxylation were reduced substantially. These results indicate that the failure of the mutant enzymes to bind the transition-state analogue tightly is primarily the consequence of an impairment in the second irreversible binding step. Thus, in all of the mutant enzymes, defects appear to exist in stabilizing the transition state of the carboxylation step, which is precisely the step proposed to influence the CO₂/O₂ specificity of Rubisco.Abbreviations and Symbols CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K _c K _m for CO₂ - K _o K _m for O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V _c V _max for carboxylation - V _o V _max for oxygenation Paper No. 9313, Journal Series, Nebraska Agricultural Research DivisionThis work was supported by National Science Foundation grant DMB-8703820. We thank Drs. Archie Portis and Raymond Chollet for their helpful comments, and also thank Dr. Chollet for graciously providing CABP and [¹⁴C]CABP.     

Photosynthesis and Activation of Ribulose Bisphosphate Carboxylase in Wheat Seedlings : Regulation by CO(2) and O(2)

Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO₂ to activate the enzyme, changes in CO₂ between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO₂ levels and 21% O₂ or 1% or less O₂, the levels of ribulose bisphosphate were high and not limiting for CO₂ fixation. With high leaf ribulose bisphosphate, the K_act(CO₂) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO₂ and O₂, activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex.

The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg²⁺ and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg²⁺ and pH most likely determine the ratio of inactive enzyme-RuBP to active enzyme-CO₂-Mg²⁺-RuBP forms. Higher irradiances favor more optimal Mg²⁺ and pH, with greater activation of the carboxylase and increased photosynthesis.

     

RuBP Limitation of Photosynthetic Carbon Fixation during NH(3) Assimilation : Interactions between Photosynthesis, Respiration, and Ammonium Assimilation in N-Limited Green Algae

The effects of ammonium assimilation on photosynthetic carbon fixation and O₂ exchange were examined in two species of N-limited green algae, Chlorella pyrenoidosa and Selenastrum minutum. Under light-saturating conditions, ammonium assimilation resulted in a suppression of photosynthetic carbon fixation by S. minutum but not by C. pyrenoidosa. These different responses are due to different relationships between cellular ribulose bisphosphate (RuBP) concentration and the RuBP binding site density of ribulose bisphosphate carboxylase/oxygenase (Rubisco). In both species, ammonium assimilation resulted in a decrease in RuBP concentration. In S. minutum the concentration fell below the RuBP binding site density of Rubisco, indicating RuBP limitation of carboxylation. In contrast, RuBP concentration remained above the binding site density in C. pyrenoidosa. Compromising RuBP regeneration in C. pyrenoidosa with low light resulted in an ammonium-induced decrease in RuBP concentration below the RuBP binding site density of Rubisco. This resulted in a decrease in photosynthetic carbon fixation. In both species, ammonium assimilation resulted in a larger decrease in net O₂ evolution than in carbon fixation. Mass spectrometric analysis shows this to be a result of an increase in the rate of mitochondrial respiration in the light.     

Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state

Experiments were conducted to determine the relative contributions of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activation state vis-à-vis Rubisco activase and metabolite levels to the inhibition of cotton (Gossypium hirsutum L.) photosynthesis by heat stress. Exposure of leaf tissue in the light to temperatures of 40 or 45 °C decreased the activation state of Rubisco to levels that were 65 or 10%, respectively, of the 28 °C control. Ribulose-1,5-bisphosphate (RuBP) levels increased in heat-stressed leaves, whereas the 3-phosphoglyceric acid pool was depleted. Heat stress did not affect Rubisco per se, as full activity could be restored by incubation with CO₂ and Mg²⁺. Inhibition and recovery of Rubisco activation state and carbon dioxide exchange rate (CER) were closely related under moderate heat stress (up to 42.5 °C). Moderate heat stress had negligible effect on Fv/Fm, the maximal quantum yield of photosystem II. In contrast, severe heat stress (45 °C) caused significant and irreversible damage to Rubisco activation, CER, and Fv/Fm. The rate of Rubisco activation after alleviating moderate heat stress was comparable to that of controls, indicating rapid reversibility of the process. However, moderate heat stress decreased both the rate and final extent of CER activation during dark-to-light transition. Treatment of cotton leaves with methyl viologen or an oxygen-enriched atmosphere reduced the effect of heat stress on Rubisco inactivation. Both treatments also reduced tissue RuBP levels, indicating that the amount of RuBP present during heat stress may influence the degree of Rubisco inactivation. Under both photorespiratory and non-photorespiratory conditions, the inhibition of the CER during heat stress could be completely reversed by increasing the internal partial pressure of CO₂ (Ci). However, the inhibition of the CER by nigericin, a K⁺ ionophore, was not reversible when the Ci was increased at ambient or high temperature. Our results indicate that inhibition of photosynthesis by moderate heat stress is not caused by inhibition of the capacity for RuBP regeneration. We conclude that heat stress inhibits Rubisco activation via a rapid and direct effect on Rubisco activase, possibly by perturbing Rubisco activase subunit interactions with each other or with Rubisco. Received: 25 February 2000 / Accepted: 13 May 2000     

The relationship between steady-state gas exchange of bean leaves and the levels of carbon-reduction-cycle intermediates

The relationship between the gas-exchange characteristics of attached leaves of Phaseolus vulgaris L. and the pool sizes of several carbon-reduction-cycle intermediates was examined. After determining the rate of CO₂ assimilation at known intercellular CO₂ pressure, O₂ pressure and light, the leaf was rapidly killed (<0.1 s) and the levels of ribulose-1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate were measured. In 210 mbar O₂, photosynthesis appeared RuBP-saturated at low CO₂ pressure and RuBP-limited at high CO₂ pressure. In 21 mbar (2%) O₂, the level of RuBP always appeared saturating. Very high levels of PGA and other phosphate-containing compounds were found with some conditions, especially under low oxygen.Abbreviations and symbols C₁ intercellular CO₂ pressure - PGA 3-phosphoglyceric acid - RuBP ribulose-1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase     

Ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase activities of photoautotrophic callus of Platycerium coronarium (Koenig ex O.F. Muell.) Desv. under CO2 enrichment

The in vitro activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) were measured in cell-free extracts of Platycerium coronarium callus cultured for up to 42 days under photoautotrophic conditions with CO₂ enrichment. With an increase in CO₂ in the culture environment to 10% (v/v) at low light, the apparent photoautotrophic fixation of CO₂ by Rubisco declined, whereas the non-photoautotrophic CO₂ fixation by PEPC activity was enhanced. Hence, photosynthesis appears to play a lesser role in providing carbon skeletons and energy with prolonged culture in a CO₂-enriched environment. Instead, the anaplerotic supply of C-skeletons by PEPC may be important under such a situation. Short-term H¹⁴CO₃-fixation experiments indicated that photoautotrophic callus cultured for 3 weeks with 10% CO₂ enrichment assimilated less ¹⁴CO₂ than the control (0.03% CO₂). Analyses of ¹⁴C-metabolites indicated that about 50% of the total soluble ¹⁴CO₂ fixed was in the organic acid fraction and 35% in the amino acid fraction. Despite the changes in the in vitro Rubisco/PEPC activity-ratio, no significant change in the ¹⁴C distribution pattern was apparent in response to increasing sucrose or CO₂ concentrations. The suppression of Rubisco activity and total chlorophyll content in high sucrose or elevated CO₂ concentrations suggests an inhibition of the capacity for photoautotrophic callus growth under these conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of growth and measurement light intensities on temperature dependence of CO2 assimilation rate in tobacco leaves

Effects of growth light intensity on the temperature dependence of CO₂ assimilation rate were studied in tobacco (Nicotiana tabacum) because growth light intensity alters nitrogen allocation between photosynthetic components. Leaf nitrogen, ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco) and cytochrome f (cyt f) contents increased with increasing growth light intensity, but the cyt f/Rubisco ratio was unaltered. Mesophyll conductance to CO₂ diffusion (g_m) measured with carbon isotope discrimination increased with growth light intensity but not with measuring light intensity. The responses of CO₂ assimilation rate to chloroplast CO₂ concentration (C_c) at different light intensities and temperatures were used to estimate the maximum carboxylation rate of Rubisco (V_cmax) and the chloroplast electron transport rate (J). Maximum electron transport rates were linearly related to cyt f content at any given temperature (e.g. 115 and 179 µmol electrons mol^?1 cyt f s^?1 at 25 and 40 °C, respectively). The chloroplast CO₂ concentration (C_trans) at which the transition from RuBP carboxylation to RuBP regeneration limitation occurred increased with leaf temperature and was independent of growth light intensity, consistent with the constant ratio of cyt f/Rubisco. In tobacco, CO₂ assimilation rate at 380 µmol mol^?1 CO₂ concentration and high light was limited by RuBP carboxylation above 32 °C and by RuBP regeneration below 32 °C.     

Stability and activity of rubisco in chestnut plantlets transferred to <Emphasis Type="Italic">ex vitro</Emphasis> conditions under elevated CO<Subscript>2</Subscript>

Summary Heterotrophic plantlets obtained by in vitro propagation are biochemically different compared to autotrophic plantlets. When heterotrophic plantlets are transferred to ex vitro conditions, higher irradiance levels are generally applied. Irradiance levels higher than those used in vitro lead to oxidative stress symptoms, that can be counteracted by CO₂ concentrations above normal. We analyzed the stability and activity of Rubisco and leaf-soluble sugars and starch contents in chestnut plantlets transferred from in vitro to ex vitro conditions under four treatments obtained by associating two irradiances of 150 (low light, LL) and 300 (high light, HL) μmolm⁻²s⁻¹, respectively three and six times in vitro irradiance, with two CO₂ levels of 350 (low CO₂, LCO₂) and 700 (high CO₂, HCO₂) μll⁻¹. In in vitro plantlets it was possible to immunodetect apparent products of degradation of Rubisco large subunit (LSU). In ex vitro plantlets, these degradation products were no longer dtected except under LL associated with LCO₂. The decrease in soluble sugars and starch in plantlets under HL HCO₂ gave an indication of a faster acquisition of autotrophic characteristics. However, under the same treatment, a down-regulation of Rubisco activity was observed. From the results taken as a whole, two aspects seem to be confirmed: HL HCO₂ is more efficient in inducing an autotrophic behavior in chestnut ex vitro plantlets; actively growing systems as ex vitro plantlets reflect the down-regulation of Rubisco by HCO₂ without accumulation of carbohydrates.     

A facile method to determine the CO2/O2 specificity factor for ribulose bisphosphate carboxylase/oxygenase

A rapid method to determine the CO₂/O₂ specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase is presented. The assay measures the amount of CO₂ and O₂ fixation at varying CO₂/O₂ ratios to determine the relative rates of each reaction. CO₂ fixation is measured by the incorporation of the moles of¹⁴CO₂ into 3-phosphoglycerate, while O₂ fixation is determined by subtraction of the moles of CO₂ fixed from the moles of RuBP consumed in each reaction. By analyzing the inorganic phosphate specifically hydrolyzed from RuBP under alkaline conditions, the amount of RuBP present before and after catalysis by rubisco can be determined.     

Dissociation of ribulose-1,5-bisphosphate bound to ribulose-1,5-bisphosphate carboxylase/oxygenase and its enhancement by ribulose-1,5-bisphosphate carboxylase/oxygenase activase-mediated hydrolysis of ATP

Ribulose bisphosphate (RuBP), a substrate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is an inhibitor of Rubisco activation by carbamylation if bound to the inactive, noncarbamylated form of the enzyme. The effect of Rubisco activase on the dissociation kinetics of RuBP bound to this form of the enzyme was examined and characterized with the use of ³H-labeled RuBP and proteins purified from spinach (Spinacia oleracea L.) In the absence of Rubisco activase and in the presence of a large excess of unlabeled RuBP, the dissociation rate of bound [1-³H]RuBP was much faster after a short (30 second) incubation than after an extended incubation (1 hour). After 1 hour of incubation, the dissociation rate constant (K_off) of the bound RuBP was 4.8 × 10⁻⁴ per second, equal to a half-time of about 35 minutes, whereas the rate after only 30 seconds was too fast to be accurately measured. This time-dependent change in the dissociation rate was reflected in the subsequent activation kinetics of Rubisco in the presence of RuBP, CO₂, and Mg²⁺, and in both the absence or presence of Rubisco activase. However, the activation of Rubisco also proceeded relatively rapidly without Rubisco activase if the RuBP level decreased below the estimated catalytic site concentration. High pH (pH 8.5) and the presence of Mg²⁺ in the medium also enhanced the dissociation of the bound RuBP from Rubisco in the presence of RuBP. In the presence of Rubisco activase, Mg²⁺, ATP (but not the nonhydrolyzable analog, adenosine-5′-O-[3-thiotriphosphate]), excess RuBP, and an ATP-regenerating system, the dissociation of [1-³H]RuBP from Rubisco was increased in proportion to the amount of Rubisco activase added. This result indicates that Rubisco activase-mediated hydrolysis of ATP is required for promotion of the enhanced dissociation of the bound RuBP from Rubisco. Furthermore, product analysis by ion-exchange chromatography demonstrated that the release of the bound RuBP, in an unchanged form, was considerably faster than the observed increase in Rubisco activity. Thus, RuBP dissociation was experimentally separated from activation and precedes the subsequent formation of active, carbamylated Rubisco during activation of Rubisco by Rubisco activase.     

The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco

Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO₂ assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO₂ partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO₂ assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO₂, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, k_cat, of 3.5+0.2 mol CO₂·(mol sites)^–1·s^–1 at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO₂·(mol sites)^–1·^–1 in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO₂ and O₂, the rate of CO₂ assimilation was measured at 25° C at different intercellular partial pressures of CO₂ and O₂. These measurements were combined with carbon-isotope analysis (¹³C/¹²C) of CO₂ in the air passing over the leaf to estimate the conductance for transfer of CO₂ from the substomatal cavities to the sites of carboxylation (0.3 mol·m^–2·s^–1·bar^–1) and thus the partial pressure of CO₂ at the sites of carboxylation. The calculated Michaelis-Menten constants for CO₂ and O₂ were 259 ±57 bar (8.6±1.9M) and 179 mbar (226 M), respectively, and the effective Michaelis-Menten constant for CO₂ in 200 mbar O₂ was 549 bar (18.3 M). From measurements of the photocompensation point (_* = 38.6 ubar) we estimated Rubisco's relative specificity for CO₂, as opposed to O₂ to be 97.5 in vivo. These values were dependent on the size of the estimated CO₂-transfer conductance.Abbreviations and Symbols A CO₂-assimilation rate - g_w conductance for CO₂ transfer from the substomatal cavities to the sites of carboxylation - K_c, K_o Michaelis-Menten constants for carboxylation, oxygenation of Rubisco - k_cat V_cmax/[active site] - O partial pressure of O₂ at the site of carboxylation - p_c partial pressure of CO₂ at the site of carboxylation - p_i intercellular CO₂ partial pressure - R_d day respiration (non-photorespiratory CO₂ evolution) - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - S_c/o relative specificity factor for Rubisco - SSu small subunit of Rubisco - V_cmax, V_omax maximum rates of Rubisco carboxylation, oxygenation - _* partial pressure of CO₂ in the chloroplast at which photorespiratory CO₂ evolution equals the rate of carboxylation     

Measurement of the Concentration of the Active Centers of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo

Methods for in vivo measurement of the concentration of the reactive centers of ribulose-1,5,-bisphosphate carboxylase/oxygenase (Rubisco) are suggested that are based on saturation of the active centers with RuBP and determination of the concentration of the Rubisco–RuBP complex. The total concentration of potentially reactive centers is calculated from the dependence of the concentration of this complex on CO₂ concentration at a steady-state photosynthetic rate with further extrapolation of the carbon dioxide dependence curve to a zero CO₂ concentration. The concentration of centers that possessed a catalytic activity under given environmental conditions was measured after transferring leaves having a steady-state photosynthetic rate into a medium devoid of CO₂ and O₂. This procedure ensured the saturation of the carboxylation centers with RuBP. The carboxylation rates were measured during a short-term exposure to ¹⁴CO₂, and the concentration of the complex was calculated using the values of CO₂ concentration during the exposure time, as well as the carboxylation rate and constant. Rubisco activity was found to decrease at elevated CO₂ concentrations due to a lower concentration of catalytically active enzyme centers.     

Rubisco activity of scenedesmus ecornis: What is wrong with initial activity determination?

A procedure was devised to measure the initial and total Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities for the green microalga, Scenedesmus ecornis. Total Rubisco activities corresponded well with photosynthetic carbon assimilation rates. Initial activities ranged from 10 to 40% of the total activities and did not correlate with photosynthetic rates. Investigations into potential causes of the reduced initial activities yielded modest increases in percentage of the total activity. Values of K_m for ribulose-1,5-bisphosphate (RuBP) were similar for both initial and CO₂-Mg²⁺ activated enzyme. Total activities increased with increasing concentrations of RuBP to 400 μm, the assay concentration. However, concentrations above the K_m, 25 μm RuBP, were inhibitory for the initial Rubisco form. Inhibition increased with increasing RuBP concentration. The addition of Mg²⁺ in the extraction solution did not prevent RuBP inhibition. The results suggest that the low initial Rubisco activities are principally due to decarbamylation of the active sites of the enzyme during extraction.     

Inadequacy of Rubisco initial and total activities to account for observed rates of photosynthetic carbon dioxide assimilation by Scenedesmus ecornis

Both initial and total activity of ribulose-1,5, bisphosphate carboxylase/oxygenase (Rubisco) measured for the green alga Scenedesmus ecornis are affected by the experimental procedure and they are not sufficiently high to account for the rates of ¹⁴C fixation by photosynthesis. The very low β-carboxylase activities detected (less than 3% of the Rubisco total activity) cannot explain the difference in CO₂ fixation. Attempts to obtain possible optimal conditions (pH, duration of activation with Mg²⁺ and HCO^- ₃, absence of proteases, linearity of ¹⁴C fixation with time) did not lead to increased activity yields. The substrate ribulose-1,5-bisphosphate was found to decrease the initial activity at concentrations higher than 25 μM for algae harvested by centrifugation and having thus experienced several minutes of darkness. Deactivation seems to be primarily responsible for this loss of activity. Furthermore, initial and total activities decrease when the delay before freezing increases, suggesting accumulation of an inhibitor from the light-dark transition metabolism during the first minutes of harvesting.     

From chaperonins to Rubisco assembly and metabolic repair

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO₂ in photosynthesis by catalyzing the carboxylation of the 5‐carbon sugar ribulose‐1,5‐bisphosphate (RuBP). Despite its pivotal role, Rubisco is an inefficient enzyme and thus has been a key target for bioengineering. However, efforts to increase crop yields by Rubisco engineering remain unsuccessful, due in part to the complex machinery of molecular chaperones required for Rubisco biogenesis and metabolic repair. While the large subunit of Rubisco generally requires the chaperonin system for folding, the evolution of the hexadecameric Rubisco from its dimeric precursor resulted in the dependence on an array of additional factors required for assembly. Moreover, Rubisco function can be inhibited by a range of sugar‐phosphate ligands. Metabolic repair of Rubisco depends on remodeling by the ATP‐dependent Rubisco activase and hydrolysis of inhibitors by specific phosphatases. This review highlights our work toward understanding the structure and mechanism of these auxiliary machineries.     

Effects of periodic fluctuations of photon flux density on anatomical and photosynthetic characteristics of soybean leaves

The development of soybean leaves grown at fluctuating photon flux density between 100 and 1500M m^-2s^-1 with a period of 160 sec were compared to leaves developed under continuous light with the same mean photon flux density. Number of epidermal cells and stomata, leaf area and specific leaf weight were not affected by the periodic fluctuation of photon flux density. Chloroplastic pigment concentration and chlorophyll fluorescence reveal some photoinhibitory effects of the high photon flux density phase. Stomatal and internal CO₂ conductance and the quantum yield were not affected by the light regime. In contrast ribulose 1.5 bisphosphate carboxylase/oxygenase activity before in vitro activation by CO₂ and Mg⁺⁺ was stimulated by the periodic illumination whereas the total amount of the enzyme and the internal leaf CO₂ conductance remained steady. In conclusion, there was no major difference between leaves of plant grown either under a steady or under a periodic fluctuation of the photon flux density except some photoinhibitory symptoms under fluctuating illumination, and a higher in vivo level of activation of the Rubisco.     

In vitro enzyme activities and products of 14CO2 assimilation in flag leaf and ear parts of wheat (Triticum aestivum L.)

Activities of key enzymes of Calvin cycle and C₄ metabolism, rate of ¹⁴CO₂ fixation in light and dark and the initial products of photosynthetic ¹⁴CO₂ fixation were determined in flag leaf and different ear parts of wheat viz. pericarp, awn and glumes. Compared to the activities of RuBP carboxylase and other Calvin cycle enzymes viz. NADP-glyceraldehyde-3-phosphate dehydrogenase, NAD-glyceraldehyde-3-phosphate dehydrogenase and ribulose-5-phosphate kinase, the levels of PEP carboxylase and other enzymes of C₄ metabolism viz. NADP-malate dehydrogenase, NAD-malate dehydrogenase, NADP-malic enzyme, NAD-malic enzyme, glutamate oxaloacetate transaminase genase, NADP-malic enzyme, NAD-malic enzyme, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase, were generally greater in ear parts than in the flag leaf. In contrast to CO₂ fixation in light, the various ear parts incorporated CO₂ in darkness at much higher rates than flag leaf. In short term assimilation of ¹⁴CO₂ by illuminated ear parts, most of the ¹⁴C was in malate with less in 3-phosphoglyceric acid, whereas flag leaves incorporated most into 3-phosphoglyceric acid. It seems likely that ear parts have the capability of assimilating CO₂ by the C₄ pathway of photosynthesis and utilise PEP carboxylase for recapturing the respired CO₂.