Differences between wheat and rice in the enzymic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase and the relationship to photosynthetic gas exchange

The kinetic parameters of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) were determined by rapidly assaying the leaf extracts. The respective K _m and V _max values for carboxylase and oxygenase activities were significantly higher for wheat than for rice. In particular, the differences in the V _max values between the two species were greater. When the net activity of CO₂ exchange was calculated at the physiological CO₂-O₂ concentration from these kinetic parameters, it was 22% greater in wheat than in rice. This difference in the in-vitro RuBP-carboxylase/oxygenase activity between the two species reflected a difference in the CO₂-assimilation rate per unit of RuBP-carboxylase protein. However, there was no apparent difference in the CO₂-assimilation rate for a given leaf-nitrogen content between the two species. When the RuBP-carboxylase/oxygenase activity was estimated at the intercellular CO₂ pressure from the enzyme content and kinetic parameters, these estimated enzyme activities in wheat and rice were similar to each other for the same rate of CO₂ assimilation. These results indicate that the difference in the kinetic parameters of RuBP carboxylase between the two species was offset by the differences in RuBP-carboxylase content and conductance for a given leaf-nitrogen content.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic - PAR photosynthetically active radiation - RuBP ribulose-1,5-bisphosphate     

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.     

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     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

Purification and properties of ribulose 1,5-bisphosphate carboxylase from sunflower leaves

Extracts from sunflower leaves possess a high ribulose-1,5-bisphosphate (RuBP) carboxylase capacity but this enzyme activity is not stable. A purification procedure, developed with preservation of carboxylase activity by MgSO₄, yielded purified RuBP carboxylase with high specific activity (40 nkat mg^-1 protein). Measurement of kinetic parameters showed high Km values (RuBP, HCO ₃ ^- ) and high Vmax of the reaction catalyzed by this sunflower enzyme; the results are compared with those obtained for soybean carboxylase. Enzyme characteristics are discussed in relation to stabilization and activation procedures and to the high photosynthesis rates of this C₃ species.     

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

Abstract The evolutionary relationship of the RuBisCO large subunit gene(s) ( rbcL ) of several prokaryotes was examined using the technique of heterologous DNA hybridization. Restriction fragments of cloned rbcL from Anacystis nidulans 6301, Chlamydomonas reinhardtii, Rhodospirillum rubrum , and maize were nick-translated and used as probes. The C. reinhardtii and maize probes hybridized with restriction fragment(s) only from cyanobacteria: Agmenellum quadruplicatum, Fremyella diplosiphon , and Mastigocladus laminosus . In addition, the A. nidulans probe hybridized with restriction fragment(s) from Alcaligenes eutrophus, Chromatium vinosum, Nitrobacter hamburgensis, Paracoccus denitrificans, Pseudomonas oxalaticus, Rhodomicrobium vannielii, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides, Thiobacillus intermedius, Thiobacillus neapolitanus , and Thiothrix nivea . The elucidated fragment of Rhodopseudomonas species is presumably for the Form I RuBisCO LSU of these organisms. The R. rubrum probe hybridized only to a restriction fragment(s) from R. capsulata, R. palustris, R. sphaeroides, T. neapolitanus , and T. nivea . The fragment(s) of Rhodopseudomonas species is the Form II rbcL of these organisms. The restriction fragments of T. neapolitanus and T. nivea were also different from those elucidated by the A. nidulans probe, suggesting the presence of a second (different) rbcL in these organisms. Positive hybridization was not obtained using any of the probes with DNA from Beggiatoa alba, Chlorobium vibrioforme or Chloroflexus aurantiacus . It appears that all rbcL have evolved from a common ancestor. Our data are consistent with and supportive of the evolutionary scheme for RuBisCO proposed by Akazawa, Takabe, and Kobayashi [1].     

Light and metabolite regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the corresponding mRNAs in the unicellular alga Chlorogonium

In the unicellular green alga Chlorogonium elongatum, the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and its mRNAs is under the control of light and acetate. Acetate is the sole metabolizable organic carbon source for this organism. Light greatly promotes the synthesis of RuBPCase and the increase in the concentration of the mRNAs of both subunits of the enzyme while acetate has a strong inhibitory effect on this process. There is a good agreement between RuBPCase synthesis and the amount of translateable RuBPCase mRNA present in cells which are cultured under different conditions (autotrophic, heterotrophic, mixotrophic). During the transition period after transfer of the cells from heterotrophic to autotrophic growth conditions the amounts of the large and small subunits of the enzyme increase well coordinated. In contrast to the protein subunits the two subunit-mRNAs accumulate with different kinetics.Abbreviations LSU large subunit of RuBPCase - poly(A)^- RNA - poly(A)⁺RNA non-, poly-adenylated RNA - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase EC 4.1.1.39 - SSU small subunit of RuBPCase     

Long-term kinetics of the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in plants with and without 2-carboxyarabinitol 1-phosphate

The light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in two species: Phaseolus vulgaris L., which has high levels of the inhibitor of Rubisco activity, carboxyarabinitol 1-phosphate (CA1P), in the dark, and Chenopodium album L., which has little CA1P. In both species, the ratio of initial to fully-activated Rubisco activity declined by 40–50% within 60 min of a reduction in light from high a photosynthetic photon flux density (PPFD; >700 mol · m^–2 · s^–1) to a low PPFD (65 ± 15 mol · m^–2 · s^–1) or to darkness, indicating that decarbamylation of Rubisco is substantially involved in the initial regulatory response of Rubisco to a reduction in PPFD, even in species with potentially extensive CA1P inhibition. Total Rubisco activity was unaffected by PPFD in C. album, and prolonged exposure (2–6 h) to low light or darkness was accompanied by a slow decline in the activity ratio of this species. This indicates that the carbamylation state of Rubisco from C. album gradually declines for hours after the large initial drop in the first 60 min following light reduction. In P. vulgaris, the total activity of Rubisco declined by 10–30% within 1 h after a reduction in PPFD to below 100 mol · m^–2 · s^–1, indicating CA1P-binding contributes significantly to the reduction of Rubisco capacity during this period, but to a lesser extent than decarbamylation. With continued exposure of P. vulgaris leaves to very low PPFDs (< 30 mol · m^–2 · s^–1), the total activity of Rubisco declined steadily so that after 6–6.5 h of exposure to very low light or darkness, it was only 10–20% of the high-light value. These results indicate that while decarbamylation is more prominent in the initial regulatory response of Rubisco to a reduction in PPFD in P. vulgaris, binding of CA1P increases over time and after a few hours dominates the regulation of Rubisco activity in darkness and at very low PPFDs.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat substrate-saturated turnover rate of fully carbamylated enzyme - PPFD photosynthetically active photon flux density (400–700 nm) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate     

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 effect of divalent metal ions on the activity of Mg++ depleted ribulose-1,5-bisphosphate oxygenase

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg⁺⁺. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO₂. Incubation with Mg⁺⁺ and CO₂ restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn⁺⁺, Co⁺⁺, and Ni⁺⁺) and CO₂ fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO₂-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn⁺⁺ ions were present. Other divalent cations such as Ca⁺⁺ and Zn⁺⁺, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn⁺⁺ to the Mg⁺⁺ and CO₂ preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.Abbreviation RuBP ribulose-1,5-bisphosphate. The enyme unit for RuBP carboxylase is defined as mol CO₂ fixed·min^-1 and for the RuBP oxygenase as mol O₂ consumed · min^-1     

Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis, but O2 competes with CO2 for substrate ribulose 1,5-bisphosphate, leading to the loss of fixed carbon. Interest in genetically engineering improvements in carboxylation catalytic efficiency and CO2/O2 specificity has focused on the chloroplast-encoded large subunit because it contains the active site. However, there is another type of subunit in the holoenzyme of plants, which, like the large subunit, is present in eight copies. The role of these nuclear-encoded small subunits in Rubisco structure and function is poorly understood. Small subunits may have originated during evolution to concentrate large-subunit active sites, but the extensive divergence of structures among prokaryotes, algae, and land plants seems to indicate that small subunits have more-specialized functions. Furthermore, plants and green algae contain families of differentially expressed small subunits, raising the possibility that these subunits may regulate the structure or function of Rubisco. Studies of interspecific hybrid enzymes have indicated that small subunits are required for maximal catalysis and, in several cases, contribute to CO2/O2 specificity. Although small-subunit genetic engineering remains difficult in land plants, directed mutagenesis of cyanobacterial and green-algal genes has identified specific structural regions that influence catalytic efficiency and CO2/O2 specificity. It is thus apparent that small subunits will need to be taken into account as strategies are developed for creating better Rubisco enzymes.     

The specific activity of ribulose-1,5-bisphosphate carboxylase in relation to genotype in wheat

The specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) in crude extracts of leaves from euploid, amphiploid and alloplasmic lines of wheat fell into high or low categories (3.75 or 2.70 mol·mg^–1·min^–1, 30°C). For the alloplasmic lines, where the same hexaploid nuclear genome was substituted into different cytoplasms, the specific activity of RuBPCase was consistent with the type of cytoplasm (high for the B and S cytoplasms and low for the A and D cytoplasms). There was no evidence from the euploid and amphiploid lines that small subunits encoded in different nuclear genomes influenced the specific activity. High specific activity was conferred by possession of the chloroplast genome of the B-type cytoplasm which encodes the large subunit of RuBPCase. All lines with a cytoplasm derived from the Sitopsis section of wheat, with the exception of Aegilops longissima and A. speltoides 18940, had RuBPCase with high specific activity. In contrast with the euploid lines of A. longissima, the alloplasmic line containing A. longissima cytoplasm from a different source had RuBPCase with high specific activity. The difference in specific activity found here in-vitro was not apparent in-vivo when leaf gas exchange was measured.Abbreviation RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

The life of ribulose 1,5-bisphosphate carboxylase/oxygenase--posttranslational facts and mysteries

The life of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), from gene to protein to irreplaceable component of photosynthetic CO2 assimilation, has successfully served as a model for a number of essential cellular processes centered on protein chemistry and amino acid modifications. Once translated, the two subunits of Rubisco undergo a myriad of co- and posttranslational modifications accompanied by constant interactions with structurally modifying enzymes. Even after final assembly, the essential role played by Rubisco in photosynthetic CO2 assimilation is dependent on continuous conformation modifications by Rubisco activase. Rubisco is also continuously assaulted by various environmental factors, resulting in its turnover and degradation by processes that appear to be enhanced during plant senescence.     

Effect of temperature on the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light

Responses of the rate of net CO₂ assimilation (A) to the intercellular partial pressure of CO₂ (p _i ) were measured on intact spinach (Spinacia oleracea L.) leaves at different irradiances. These responses were analysed to find the value of p _i at which the rate of photosynthetic CO₂ uptake equalled that of photorespiratory CO₂ evolution. At this CO₂ partial pressure (denoted ), net rate of CO₂ assimilation was negative, indicating that there was non-photorespiratory CO₂ evolution in the light. Hence was lower than the CO₂ compensation point, . Estimates of were obtained at leaf temperatures from 15 to 30°C, and the CO₂/O₂ specificity of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (E.C. 4.1.1.39) was calculated from these data, taking into account changes in CO₂ and O₂ solubilities with temperature. The CO₂/O₂ specificity decreased with increasing temperature. Therefore we concluded that temperature effects on the ratio of photorespiration to photosynthesis were not solely the consequence of differential effects of temperature on the solubilities of CO₂ and O₂. Our estimates of the CO₂/O₂ specificity of RuBP carboxylase/oxygenase are compared with in-vitro measurements by other authors. The rate of nonphotorespiratory CO₂ evolution in the light (R _d ) was obtained from the value of A at . At this low CO₂ partial pressure, R _d was always less than the rate of CO₂ evolution in darkness and appeared to decrease with increasing irradiance. The decline was most marked up to about 100 mol quanta m^-2 s^-1 and less marked at higher irradiances. At one particular irradiance, however, R _d as a proportion of the rate of CO₂ evolution in darkness was similar in different leaves and this proportion was unaffected by leaf temperature or by [O₂] (ambient and greater). After conditions of high [CO₂] and high irradiance for several hours, the rate of CO₂ evolution in darkness increased and R _d also increased.Abbreviations and symbols A rate of net CO₂-assimilation - CO₂ compensation point - CO₂ compensation point in the absence of R _d - p _i intercellular partial pressure of CO₂ - R _d (day respiration) rate of non-photorespiratory CO₂ evolution in the light - R _n (night respiration) rate of CO₂ evolution in darkness - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase     

The CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase

The substrate specificity factor, V _cK_o/V_oK_c, of spinach (Spinacia oleracea L.) ribulose 1,5-bisphosphate carboxylase/oxygenase was determined at ribulosebisphosphate concentrations between 0.63 and 200 M, at pH values between 7.4 and 8.9, and at temperatures in the range of 5° C to 40° C. The CO₂/O₂ specificity was the same at all ribulosebisphosphate concentrations and largely independent of pH. With increasing temperature, the specificity decreased from values of about 160 at 5° C to about 50 at 40° C. The primary effects of temperature were on K _c [Km(CO₂)] and V _c [Vmax (CO₂)], which increased by factors of about 10 and 20, respectively, over the temperature range examined. In contrast, K _o [Ki (O₂)] was unchanged and V _o [Vmax (O₂)] increased by a factor of 5 over these temperatures. The CO₂ compensation concentrations () were calculated from specificity values obtained at temperatures between 5° C and 40° C, and were compared with literature values of . Quantitative agreement was found for the calculated and measured values. The observations reported here indicate that the temperature response of ribulose 1,5-bisphosphate carboxylase/oxygenase kinetic parameters accounts for two-thirds of the temperature dependence of the photorespiration/photosynthesis ratio in C₃ plants, with the remaining one-third the consequence of differential temperature effects on the solubilities of CO₂ and O₂.Abbreviations RuBPC/O(ase) ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - CO₂ compensation concentration     

The carboxylase activity of Rubisco and the photosynthetic performance in aquatic plants

Summary Activated carboxylase activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), as well as photosynthetic rates were measured for 42 species of freshwater and marine macrophytes. While the carboxylase activity varied greatly among the species investigated (0.2–12.5 mol CO₂ mg^–1 chlorophyll min^–1), the submersed freshwater plants showed significantly lower activities than emergent, floating leaved or secondary submersed forms. The variability in photosynthetic rates correlated with the carboxylase activity only for the marine macroalgae, and their photosynthesis to carboxylase activity ratios were close to 1. These plants also had a consistently high inorganic carbon transport capability, and it is suggested that ribulose-1,5-bisphosphate carboxylase/oxygenase activity is an important internal factor regulating the photosynthetic capacity within this plant group where, apparently, the internal CO₂ concentration is high and photorespiration is suppressed. Among the freshwater forms, it appears that their much lower inorganic carbon transport ability, rather than their carboxylase activity, limits the photosynthetic process.     

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9–13) 125 ng leaf^-1 day^-1; II (day 15–27) 11 ng leaf^-1 day^-1; III (day 29–49) 22 ng leaf^-1 day^-1. Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - TCA trichloroacetic acid Supported by the Wheat Industry Research Council of Australia and the Australian Research Grants Committee D2 74/15052     

Oxidative modification and breakdown of ribulose-1,5-bisphosphate carboxylase/oxygenase induced in Euglena gracitis by nitrogen starvation

When photoheterotrophic Euglena gracilis Z Pringsheim was subjected to nitrogen (N)-deprivation, the abundant photosynthetic enzyme ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was rapidly and selectively degraded. The breakdown began after a 4-h lag period and continued for a further 8 h at a steady rate. After 12 h of starvation, when the amount of Rubisco was reduced to 40%, the proteolysis of this enzyme slowed down while degradation of other proteins started at a similar pace. This resulted in a decline of culture growth, chloroplast disassembly — as witnessed by chlorophyll (Chl) loss — and cell bleaching. Experiments with spectinomycin, an inhibitor of chloroplastic translation, indicated that there was an absolute increase in the rate of Rubisco degradation in the N-deprived culture as compared with control conditions, where no significant carboxylase breakdown was detected. Oxidative aggregation of Rubisco (as detected by non-reductive electrophoresis) and association of the enzyme to membranes increased with time of N-starvation. Fluorescent labeling of oxidized cysteine (Cys) residues with monobromobimane indicated a progressive oxidation of Cys throughout the first hours of N-deprivation. It is concluded that Rubisco acts as an N store in Euglena, being first oxidized, and then degraded, during N-starvation. The mobilization of Rubisco allows sustained cell growth and division, at almost the same rate as the control (non-starved) culture, during 12 h of N-deprivation. Afterwards, breakdown is extended to other photosynthetic structures and the whole chloroplast is dismantled while cell growth is greatly reduced.Abbreviations Chl chlorophyll - Cys cysteine - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate We thank Drs. Pablo Vera and Ismael Rodrigo (Univ. Politécnica, Valencia, Spain) for advice and facilities in raising and collecting the anti-Rubisco serum. This work was supported by grants PB87-0353 and PB92-0821 of DGICYT and by a fellowship of the Spanish Ministerio de Educación y Ciencia (awarded to C.G.-F.).