Ribulose-1,5-bisphosphate Carboxylation Research Articles

Combined effects of elevated CO2 and warmer temperature on limitations to photosynthesis and carbon sequestration in yellow birch.

Elevated CO2 and warmer temperature occur simultaneously under the current climate change. However, their combined effects on the photosynthetic traits in boreal trees are not well understood. This study investigated the morphological and photosynthetic responses of yellow birch (Betula alleghaniensis Britt.) to a combined treatment of CO2 and temperature (ambient, ACT (400μmolmol-1 CO2 and current temperature) vs elevated, ECT (750μmolmol-1 CO2 and current +4 °C temperature)). It was found that ECT significantly reduced leaf-area based photosynthetic rate (An), maximum Rubisco carboxylation rate (Vcmax), photosynthetic electron transport rate (Jmax), leaf nitrogen concentration, respiration and mesophyll conductance. There were two interesting findings: first, the primary mechanism of photosynthetic limitation shifted from Ribulose-1,5-bisphosphate (RuBP) carboxylation (related to Vcmax) to RuBP regeneration (related to Jmax) in response to ECT, leading to decreased transition point (Ci-t and An-t) from RuBP carboxylation to regeneration; second, the increase in total leaf area in response to ECT more than compensated for the downregulation of leaf-area based photosynthesis, leading to greater biomass in ECT than in ACT. We proposed a new protocol for evaluating photosynthetic limitations by comparing the relative relationship between the transition point (Ci-t and An-t) and the photosynthetic rate at growth CO2 (Ci-g and An-g). Furthermore, we found that Jmax (RuBP regeneration) was the primary limitation to An under ECT.

Modelling the seasonal changes in the gas exchange response to CO2 in relation to short-term leaf temperature changes in Vitis vinifera cv. Shiraz grapevines grown in outdoor conditions

Effects of temperature on the photosynthetic response of Vitis vinifera cv. Shiraz leaves to CO2 were investigated across the growing season and modelling was used to determine relationships between photosynthesis and seasonal climate. Results indicated that photosynthetic rates declined from spring to summer, conforming to the deciduous habit of grapevines. Rates of ribulose 1,5-bisphosphate (RuBP) carboxylation and regeneration increased in a temperature dependent pattern throughout the season. However, the maximum rates decreased as the season progressed. There were also marked decreases in temperature sensitivity for each of these processes, consistent with the decreases occurring faster at high compared to low temperatures. There were no correlations between the seasonal climate and each of these photosynthetic processes but the effect of day was significant in all cases. CO2 saturated rates of photosynthesis (Amax) across the season were highly correlated with the maximum rates of RuBP carboxylation and regeneration. The transition temperature between RuBP regeneration and RuBP carboxylation-limited assimilation varied across the growing season, from 23 °C in spring, 35 °C in mid-summer and 30 °C at harvest and were highly correlated with mean day temperature. This suggested dynamic control of assimilation by carboxylation and regeneration processes occurred in these grapevines in tune with the seasonal climate.

Photosynthetic responses to CO2 at different leaf temperatures in leaves of apple trees (Malus domestica) grown in orchard conditions with different levels of soil nitrogen

The temperature-dependency of the photosynthetic response to CO2 was measured on apple (Malus domestica cv. Cox’s Orange) trees growing in orchard conditions with differing levels of nitrogen. Leaf nitrogen content varied from 1.7 to 2.2%. Nitrogen affected leaf attributes; larger areas but lower dry weights in high compared with low nitrogen trees. Across all leaf temperatures, higher rates of photosynthesis occurred in high nitrogen trees but rates differed mostly at 10 °C and progressively diminished to 35 °C. Rates of ribulose 1, 5-bisphosphate (RuBP) carboxylation, oxygenation and electron transport increased in a temperature-dependent pattern but nitrogen had no effect on rates at low (<25 °C) temperatures, whereas triose phosphate utilisation was higher and accounted for the increased photosynthesis. However, higher rates of RuBP carboxylation and regeneration in the high nitrogen trees accounted for the higher assimilation at temperatures above 30 °C. In addition, a stomatal limitation of assimilation at high temperatures was markedly increased by nitrogen but could not account for the increased photosynthesis, similarly, mesophyll conductance could not explain the effect. Basal (fruit subtending) leaves had lowest rates of assimilation and rates increased along the shoot. In addition, leaf nitrogen content also increased along the shoot and assimilation, carboxylation and regeneration all increased in concert, consistent with a causal relationship.

The short-term temperature-dependency of CO2 photosynthetic responses of two Vitis vinifera cultivars grown in a hot climate

A study of two grapevine cultivars, Vitis vinifera cv. Chardonnay and cv. Merlot revealed there were differences in photosynthesis. A more extensive comparative study of photosynthesis of these two cultivars in relation to internal CO2 (Ci) concentration at leaf temperatures from 15 to 45°C was, therefore, undertaken to explain these differences. Average rates of photosynthesis at ambient and saturating CO2 concentrations were confirmed to be higher in Merlot leaves across all leaf temperatures. The physiological explanation was partially attributable to much higher stomatal conductances at lower temperatures and by much higher rates of ribulose 1, 5-bisphosphate (RuBP) regeneration (Jmax) at all temperatures in Merlot leaves. By contrast, apparent rates of RuBP carboxylation did not vary between the cultivars except above 40°C where rates were highest in the Merlot leaves. There were cultivar differences in temperature optima for assimilation (25 and 30°C), but not for Vcmax (44, +45°C) but some differences in Jmax (37 and 39°C). The transition from RuBP regeneration limited to RuBP carboxylation-limited assimilation occurred at about 34°C in both cultivars, suggesting the high temperatures may have been detrimental to assimilation. Although the stomatal limitation to assimilation increased with increasing leaf temperature, there were no cultivar differences in the temperature dependency of the stomatal limitation to assimilation. Thus, the confirmed differences in assimilation in Merlot compared to Chardonnay leaves were concluded to be a result of stomatal conductance and an inherently greater capacity for RuBP regeneration.

Modelling seasonal changes in the temperature-dependency of CO2 photosynthetic responses in two Vitis vinifera cultivars.

A study of photosynthesis of two grapevine cultivars, Vitis vinifera L. cv. Chardonnay and cv. Merlot in relation to the seasonal climate and internal CO2 (Ci) concentration at leaf temperatures from 15 to 45°C was undertaken. Average rates of photosynthesis at saturating CO2 concentrations and all leaf temperatures were higher in Merlot compared with Chardonnay leaves. This was attributable to higher rates of ribulose 1,5-bisphosphate (RuBP) carboxylation (Vcmax) and regeneration (Jmax) in Merlot leaves. These differences in photosynthesis were extended as the season progressed, partly because rates of RuBP carboxylation and regeneration of Chardonnay leaves declined markedly whereas rates for Merlot leaves remained high. Although there was no cultivar difference in the seasonal average temperature optima for assimilation (34°C) and the underlying metabolism (40°C for Vcmax and 35°C for Jmax), for temperatures above 35°C, the Merlot leaves had 50% higher rates. Across the season, activation energies of the temperature sensitivity of Vcmax and Jmax declined in response to the seasonal climate but were consistently lower in Merlot than Chardonnay. This suggested some apparent differences in the biochemistry occurred between the two cultivars that limited assimilation in Chardonnay leaves, especially at higher temperatures, but did not limit assimilation in Merlot leaves.

Strong thermal acclimation of photosynthesis in tropical and temperate wet-forest tree species: the importance of altered Rubisco content.

Understanding of the extent of acclimation of light-saturated net photosynthesis (An ) to temperature (T), and associated underlying mechanisms, remains limited. This is a key knowledge gap given the importance of thermal acclimation for plant functioning, both under current and future higher temperatures, limiting the accuracy and realism of Earth system model (ESM) predictions. Given this, we analysed and modelled T-dependent changes in photosynthetic capacity in 10 wet-forest tree species: six from temperate forests and four from tropical forests. Temperate and tropical species were each acclimated to three daytime growth temperatures (Tgrowth ): temperate - 15, 20 and 25°C; tropical - 25, 30 and 35°C. CO2 response curves of An were used to model maximal rates of RuBP (ribulose-1,5-bisphosphate) carboxylation (Vcmax ) and electron transport (Jmax ) at each treatment's respective Tgrowth and at a common measurement T (25°C). SDS-PAGE gels were used to determine abundance of the CO2 -fixing enzyme, Rubisco. Leaf chlorophyll, nitrogen (N) and mass per unit leaf area (LMA) were also determined. For all species and Tgrowth , An at current atmospheric CO2 partial pressure was Rubisco-limited. Across all species, LMA decreased with increasing Tgrowth . Similarly, area-based rates of Vcmax at a measurement T of 25°C (Vcmax25 ) linearly declined with increasing Tgrowth , linked to a concomitant decline in total leaf protein per unit leaf area and Rubisco as a percentage of leaf N. The decline in Rubisco constrained Vcmax and An for leaves developed at higher Tgrowth and resulted in poor predictions of photosynthesis by currently widely used models that do not account for Tgrowth -mediated changes in Rubisco abundance that underpin the thermal acclimation response of photosynthesis in wet-forest tree species. A new model is proposed that accounts for the effect of Tgrowth -mediated declines in Vcmax25 on An , complementing current photosynthetic thermal acclimation models that do not account for T sensitivity of Vcmax25 .

Photosynthetic responses of apple leaves to CO2and temperature at pre- and postharvest growth stages

Field measurements of gas exchange in leaves of apple (Malus domestica) trees growing at the Universitat Hohenheim orchard were undertaken from mid stage of development until after harvest to enhance our understanding of seasonal limitations to photosynthesis. Photosynthetic responses to CO2 (A/ci) at different leaf temperature (10-35°C) were measured on leaves of selected bourse shoots each subtending a fruit and also when the fruit were harvested. A/ci responses were analysed to determine rates of carboxylation (V(cmax)) of Ribulose 1, 5 Bisphosphate (RuBP) and rates of RuBP regeneration (Jmax) at each temperature. Results showed that CO2 and light-saturated photosynthesis as well as Jmax were optimal at 25°C, but Vcmax was maximal at 30°C. It was also evident that at temperatures below about 25°C, photosynthesis was limited by regeneration of RuBP but above this, carboxylation of RuBP limited photosynthesis, in line with other species. From mid to late season, the maximum photosynthetic capacity remained about constant, at 31 µmol m-2 s-1 as did Jmax but Vcmax declined about 3-fold. After harvest, net photosynthesis was again reasonably constant but both Jmax and Vcmax increased. However, photosynthesis was primarily limited by RuBP regeneration because of the temperatures declining during this period. The conclusion of this study was that changes in sink capacity at harvest had no effect on the processes limiting photosynthesis. However, because of the temperature-dependent transition from RuBP regeneration to RuBP carboxylation, photosynthesis was most likely carboxylation-limited in the early season when temperatures were high whereas regeneration of RuBP limited photosynthesis at the cooler time of harvest.

Sun leaves up-regulate the photorespiratory pathway to maintain a high rate of CO2 assimilation in tobacco.

The greater rate of CO2 assimilation (An) in sun-grown tobacco leaves leads to lower intercellular and chloroplast CO2 concentrations and, thus, a higher rate of oxygenation of ribulose-1,5-bisphosphate (RuBP) than in shade-grown leaves. Impairment of the photorespiratory pathway suppresses photosynthetic CO2 assimilation. Here, we hypothesized that sun leaves can up-regulate photorespiratory pathway to enhance the An in tobacco. To test this hypothesis, we examined the responses of photosynthetic electron flow (JT) and CO2 assimilation to incident light intensity and intercellular CO2 concentration (Ci) in leaves of ‘k326’ tobacco plants grown at 95% sunlight (sun plants) or 28% sunlight (shade plants). The sun leaves had higher photosynthetic capacity and electron flow devoted to RuBP carboxylation (JC) than the shade leaves. When exposed to high light, the higher Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) content and lower Ci in the sun leaves led to greater electron flow devoted to RuBP oxygenation (JO). The JO/JC ratio was significantly higher in the sun leaves than in the shade leaves under strong illumination. As estimated from CO2-response curves, the maximum JO was linearly correlated with the estimated Rubisco content. Based on light-response curves, the light-saturated JO was linearly correlated with light-saturated JT and light-saturated photosynthesis. These findings indicate that enhancement of the photorespiratory pathway is an important strategy by which sun plants maintain a high An.

Seasonal changes in the photosynthetic response to CO2 and temperature in apple (Malus domestica cv. 'Red Gala') leaves during a growing season with a high temperature event.

Changes in the photosynthetic responses of Malus domestica cv. 'Red Gala' leaves to internal CO2 concentrations at leaf temperatures from 15°C to 40°C were followed across the growing season in field-grown trees exposed to a high temperature event in late summer. Light and CO2-saturated photosynthesis (Amax), maximum rates of ribulose 1,5-bisphosphate (RuBP) carboxylation (Vcmax) and maximum rates of electron transport (Jmax) were all highest in spring, although Amax was maximal at 30°C, and Vcmax and Jmax were maximal at 40°C. All attributes declined in late summer and reached minima that coincided with the occurrence of the high temperature event. Modelling suggested many of the changes were correlated with the seasonal climate, although the measurement or current temperature had an overriding effect, especially on stomatal conductance. Marked changes in the temperature-dependency of Vcmax and Jmax occurred across the season and appeared to relate to the seasonal changes in climate, especially temperature. The reduction in photosynthesis at high temperatures was, however, partly attributable to a stomatal limitation of up to 60% at the high temperatures. Non-stomatal reductions in photosynthesis during the heat event could be attributed to detrimental effect on RuBP carboxylation and on RuBP regeneration.

In vitro and in vivo analyses of the role of the carboxysomal β-type carbonic anhydrase of the cyanobacterium Synechococcus elongatus in carboxylation of ribulose-1,5-bisphosphate

The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50mM bicarbonate. With the initial concentrations of 50mM and 25mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22μmolmg(-1) protein min(-1)) was only 30% of that observed for the wild-type carboxysomes (0.71μmolmg(-1) protein min(-1)), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2% (v/v) CO2 in air, the mutant defective in ccaA as well as ccmLMNO required 5% (v/v) CO2 for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO2 by the cytoplasmically localized Rubisco by counteracting the action of the CO2 hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO2 fixation by the cyanobacterium at CO2 levels lower than 5%, unless Rubisco is properly organized into carboxysomes.

Rootstocks induce contrasting photosynthetic responses of orange plants to low night temperature without affecting the antioxidant metabolism

Low temperatures negatively impact the metabolism of orange trees, and the extent of damage can be influenced by the rootstock. We evaluated the effects of low nocturnal temperatures on Valencia orange scions grafted on Rangpur lime or Swingle citrumelo rootstocks. We exposed six-month-old plants to night temperatures of 20ºC and 8ºC under controlled conditions. After decreasing the temperature to 8ºC, there were decreases in leaf CO2 assimilation, stomatal conductance, mesophyll conductance and CO2 concentration in the chloroplasts, in plant hydraulic conductivity and in the maximum electron transport rate driven ribulose-1,5-bisphosphate (RuBP) regeneration in plants grafted on both rootstocks. However, the effects of low night temperature were more severe in plants grafted on Rangpur rootstock, which also presented reduction in the maximum rate of RuBP carboxylation and in the maximum quantum efficiency of the PSII. In general, irreversible damage due to night chilling was found in the photosynthetic apparatus of plants grafted on Rangpur lime. Low night temperatures induced similar changes in the antioxidant metabolism, preventing oxidative damage in citrus leaves on both rootstocks. As photosynthesis is linked to plant growth, our findings indicate that the rootstock may improve the performance of citrus trees in environments with low night temperatures, with Swingle rootstock improving the photosynthetic acclimation in leaves of orange plants.

Rubisco activity and regulation as targets for crop improvement

Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) enables net carbon fixation through the carboxylation of RuBP. However, some characteristics of Rubisco make it surprisingly inefficient and compromise photosynthetic productivity. For example, Rubisco catalyses a wasteful reaction with oxygen that leads to the release of previously fixed CO(2) and NH(3) and the consumption of energy during photorespiration. Furthermore, Rubisco is slow and large amounts are needed to support adequate photosynthetic rates. Consequently, Rubisco has been studied intensively as a prime target for manipulations to 'supercharge' photosynthesis and improve both productivity and resource use efficiency. The catalytic properties of Rubiscos from diverse sources vary considerably, suggesting that changes in turnover rate, affinity, or specificity for CO(2) can be introduced to improve Rubisco performance in specific crops and environments. While attempts to manipulate plant Rubisco by nuclear transformation have had limited success, modifying its catalysis by targeted changes to its catalytic large subunit via chloroplast transformation have been much more successful. However, this technique is still in need of development for most major food crops including maize, wheat, and rice. Other bioengineering approaches for improving Rubisco performance include improving the activity of its ancillary protein, Rubisco activase, in addition to modulating the synthesis and degradation of Rubisco's inhibitory sugar phosphate ligands. As the rate-limiting step in carbon assimilation, even modest improvements in the overall performance of Rubisco pose a viable pathway for obtaining significant gains in plant yield, particularly under stressful environmental conditions.

The coordination of leaf photosynthesis links C and N fluxes in C3 plant species.

Photosynthetic capacity is one of the most sensitive parameters in vegetation models and its relationship to leaf nitrogen content links the carbon and nitrogen cycles. Process understanding for reliably predicting photosynthetic capacity is still missing. To advance this understanding we have tested across C3 plant species the coordination hypothesis, which assumes nitrogen allocation to photosynthetic processes such that photosynthesis tends to be co-limited by ribulose-1,5-bisphosphate (RuBP) carboxylation and regeneration. The coordination hypothesis yields an analytical solution to predict photosynthetic capacity and calculate area-based leaf nitrogen content (N a). The resulting model linking leaf photosynthesis, stomata conductance and nitrogen investment provides testable hypotheses about the physiological regulation of these processes. Based on a dataset of 293 observations for 31 species grown under a range of environmental conditions, we confirm the coordination hypothesis: under mean environmental conditions experienced by leaves during the preceding month, RuBP carboxylation equals RuBP regeneration. We identify three key parameters for photosynthetic coordination: specific leaf area and two photosynthetic traits (k3, which modulates N investment and is the ratio of RuBP carboxylation/oxygenation capacity () to leaf photosynthetic N content (N pa); and J fac, which modulates photosynthesis for a given k 3 and is the ratio of RuBP regeneration capacity (J max) to). With species-specific parameter values of SLA, k 3 and J fac, our leaf photosynthesis coordination model accounts for 93% of the total variance in Na across species and environmental conditions. A calibration by plant functional type of k 3 and J fac still leads to accurate model prediction of N a, while SLA calibration is essentially required at species level. Observed variations in k3 and Jfac are partly explained by environmental and phylogenetic constraints, while SLA variation is partly explained by phylogeny. These results open a new avenue for predicting photosynthetic capacity and leaf nitrogen content in vegetation models.

Rubisco activity is associated with photosynthetic thermotolerance in a wild rice (Oryza meridionalis)

Oryza meridionalis is a wild species of rice, endemic to tropical Australia. It shares a significant genome homology with the common domesticated rice Oryza sativa. Exploiting the fact that the two species are highly related but O. meridionalis has superior heat tolerance, experiments were undertaken to identify the impact of temperature on key events in photosynthesis. At an ambient CO(2) partial pressure of 38 Pa and irradiance of 1500 µmol quanta m(-2) s(-1), the temperature optimum of photosynthesis was 33.7 ± 0.8°C for O. meridionalis, significantly higher than the 30.6 ± 0.7°C temperature optimum of O. sativa. To understand the basis for this difference, we measured gas exchange and rubisco activation state between 20 and 42°C and modeled the response to determine the rate-limiting steps of photosynthesis. The temperature response of light respiration (R(light)) and the CO(2) compensation point in the absence of respiration (Γ(*)) were determined and found to be similar for the two species. C3 photosynthesis modeling showed that despite the difference in susceptibility to high temperature, both species had a similar temperature-dependent limitation to photosynthesis. Both rice species were limited by ribulose-1,5-bisphosphate (RuBP) regeneration at temperatures of 25 and 30°C but became RuBP carboxylation limited at 35 and 40°C. The activation state of rubisco in O. meridionalis was more stable at higher temperatures, explaining its greater heat tolerance compared with O. sativa.

Modelling photosynthetic responses to temperature of grapevine (Vitis vinifera cv. Semillon) leaves on vines grown in a hot climate

Field measurements of photosynthesis of Vitis vinifera cv. Semillon leaves in relation to a hot climate, and responses to photon flux densities (PFDs) and internal CO(2) concentrations (c(i) ) at leaf temperatures from 20 to 40 °C were undertaken. Average rates of photosynthesis measured in situ decreased with increasing temperature and were 60% inhibited at 45 °C compared with 25 °C. This reduction in photosynthesis was attributed to 15-30% stomatal closure. Light response curves at different temperatures revealed light-saturated photosynthesis optimal at 30 °C but also PFDs saturating photosynthesis increased from 550 to 1200 µmol (photons) m(-2)s(-1) as temperatures increased. Photosynthesis under saturating CO(2) concentrations was optimal at 36 °C while maximum rates of ribulose 1,5-bisphosphate (RuBP) carboxylation (V(cmax)) and potential maximum electron transport rates (J(max)) were also optimal at 39 and 36 °C, respectively. Furthermore, the high temperature-induced reduction in photosynthesis at ambient CO(2) was largely eliminated. The chloroplast CO(2) concentration at the transition from RuBP regeneration to RuBP carboxylation-limited assimilation increased steeply with an increase in leaf temperature. Semillon assimilation in situ was limited by RuBP regeneration below 30 °C and above limited by RuBP carboxylation, suggesting high temperatures are detrimental to carbon fixation in this species.

Subunit Interface Dynamics in Hexadecameric Rubisco

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L 2 dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance.

Substrate-induced Assembly of Methanococcoides burtoniid-Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Dimers into Decamers

Like many enzymes, the biogenesis of the multi-subunit CO(2)-fixing enzyme ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) in different organisms requires molecular chaperones. When expressed in Escherichia coli, the large (L) subunits of the Rubisco from the archaeabacterium Methanococcoides burtonii assemble into functional dimers (L(2)). However, further assembly into pentamers of L(2) (L(10)) occurs when expressed in tobacco chloroplasts or E. coli producing RuBP. In vitro analyses indicate that the sequential assembly of L(2) into L(10) (via detectable L(4) and L(6) intermediates) occurs without chaperone involvement and is stimulated by protein rearrangements associated with either the binding of substrate RuBP, the tight binding transition state analog carboxyarabinitol-1,5-bisphosphate, or inhibitory divalent metal ions within the active site. The catalytic properties of L(2) and L(10) M. burtonii Rubisco (MbR) were indistinguishable. At 25 degrees C they both shared a low specificity for CO(2) over O(2) (1.1 mol x mol(-1)) and RuBP carboxylation rates that were distinctively enhanced at low pH (approximately 4 s(-1) at pH 6, relative to 0.8 s(-1) at pH 8) with a temperature optimum of 55 degrees C. Like other archaeal Rubiscos, MbR also has a high O(2) affinity (K(m)(O(2)) = approximately 2.5 microM). The catalytic and structural similarities of MbR to other archaeal Rubiscos contrast with its closer sequence homology to bacterial L(2) Rubisco, complicating its classification within the Rubisco superfamily.

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO(2) fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg(2+) for catalysis and, like RuBisCO, was stimulated by CO(2). Four residues that are essential for the enolization reaction of RuBisCO, Lys(175), Lys(201), Asp(203), and Glu(204), were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys(123), the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a K(i) value of 103 mum. RuBP and d-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.

Photosynthetic acclimation to CO&lt;sub&gt;2&lt;/sub&gt; enrichment related to ribulose-1,5-bisphosphate carboxylation limitation in wheat

Net photosynthetic rate (P N) measured at the same CO2 concentration, the maximum in vivo carboxylation rate, and contents of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPCO) and RuBPCO activase were significantly decreased, but the maximum in vivo electron transport rate and RuBP content had no significant change in CO2-enriched [EC, about 200 µmol mol−1 above the ambient CO2 concentration (AC)] wheat leaves compared with those in AC grown wheat leaves. Hence photosynthetic acclimation in wheat leaves to EC is largely due to RuBP carboxylation limitation.

Phylogenetic and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms.

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) catalyses the key reaction by which inorganic carbon may be assimilated into organic carbon. Phylogenetic analyses indicate that there are three classes of bona fide RubisCO proteins, forms I, II and III, which all catalyse the same reactions. In addition, there exists another form of RubisCO, form IV, which does not catalyse RuBP carboxylation or oxygenation. Form IV is actually a homologue of RubisCO and is called the RubisCO-like protein (RLP). Both RubisCO and RLP appear to have evolved from an ancestor protein in a methanogenic archaeon, and comprehensive analyses indicate that the different forms (I, II, III and IV) contain various subgroups, with individual sequences derived from representatives of all three kingdoms of life. The diversity of RubisCO molecules, many of which function in distinct milieus, has provided convenient model systems to study the ways in which the active site of this protein has evolved to accommodate necessary molecular adaptations. Such studies have proven useful to help provide a framework for understanding the molecular basis for many important aspects of RubisCO catalysis, including the elucidation of factors or functional groups that impinge on RubisCO carbon dioxide/oxygen substrate discrimination.