Rubisco activities, properties, and regulation in three different C4 grasses under drought.
ABSTRACT: In C4 plants, water deficit may decrease photosynthetic CO2 assimilation independently of changes in stomatal conductance, suggesting decreased turnover by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The activity and biochemistry of Rubisco was studied in three different C4 grasses: Paspalum dilatatum, Cynodon dactylon, and Zoysia japonica. The objectives were to characterize the C4 Rubisco in these species and to identify factors associated with decreased photosynthetic rates caused by drought. Rubisco isolated from each of the three C4 grasses was characterized by smaller specificity factors (SC/O), larger Michaelis-Menten constants for CO2 (Kc) and O2 (Ko), and larger maximum carboxylation velocities (Vc) than Rubisco from wheat, which can be rationalized in terms of the CO2-rich environment of C4 Rubisco in the bundle sheath. During leaf dehydration the quantity and maximum activity of Rubisco remained unchanged but the initial and total activities declined slightly, possibly due to increased inhibition. Tight-binding inhibitors were present in the light but were more abundant in the dark, especially in Z. japonica, and increased in quantity with drought stress. The inhibitor from darkened leaves of Z. japonica was identified as 2-carboxyarabinitol-1-phosphate (CA1P). Consistent with the presence of CA1P, the total activity of Rubisco was decreased after 12 h darkness in Z. japonica. Ribulose-1,5-bisphosphate (RuBP) in the leaves decreased with drought stress, to quantities approximating those of Rubisco catalytic sites. The magnitude of the decrease in RuBP suggested that, at least in C. dactylon and Z. japonica, it could contribute to the drought-induced decrease in photosynthesis.
Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity is modulated in vivo either by reaction with CO2 and Mg2+ to carbamylate a lysine residue in the catalytic site, or by the binding of inhibitors within the catalytic site. Binding of inhibitors blocks either activity or the carbamylation of the lysine residue that is essential for activity. At night, in many species, 2-carboxyarabinitol-1-phosphate (CA1P) is formed which binds tightly to Rubisco, inhibiting catalytic activity. Recent work has shown that tight-binding inhibitors can also decrease Rubisco activity in the light and contribute to the regulation of Rubisco activity. Here we determine the influence that such inhibitors of Rubisco exert on catalytic activity during drought stress. In tobacco plants, 'total Rubisco activity', i.e. the activity following pre-incubation with CO2 and Mg2+, was positively correlated with leaf relative water content. However, 'total Rubisco activity' in extracts from leaves with low water potential increased markedly when tightly bound inhibitors were removed, thus increasing the number of catalytic sites available. This suggests that in tobacco the decrease of Rubisco activity under drought stress is not primarily the result of changes in activation by CO2 and Mg2+ but due rather to the presence of tight-binding inhibitors. The amounts of inhibitor present in leaves of droughted tobacco based on the decrease in Rubisco activity per mg soluble protein were usually much greater than the amounts of the known inhibitors (CA1P and 'daytime inhibitor') that can be recovered in acid extracts. Alternative explanations for the difference between maximal and total activities are discussed.
Project description:Most physiology comparisons of C3 and C4 plants are made under current or elevated concentrations of atmospheric CO2 which do not reflect the low CO2 environment under which C4 photosynthesis has evolved. Accordingly, photosynthetic nitrogen (PNUE) and water (PWUE) use efficiency, and the activity of the photosynthetic carboxylases [Rubisco and phosphoenolpyruvate carboxylase (PEPC)] and decarboxylases [NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PEP-CK)] were compared in eight C4 grasses with NAD-ME, PCK, and NADP-ME subtypes, one C3 grass, and one C3-C4 grass grown under ambient (400 ?l l(-1)) and glacial (180 ?l l(-1)) CO2. Glacial CO2 caused a smaller reduction of photosynthesis and a greater increase of stomatal conductance in C4 relative to C3 and C3-C4 species. Panicum bisulcatum (C3) acclimated to glacial [CO2] by doubling Rubisco activity, while Rubisco was unchanged in Panicum milioides (C3-C4), possibly due to its high leaf N and Rubisco contents. Glacial CO2 up-regulated Rubisco and PEPC activities in concert for several C4 grasses, while NADP-ME and PEP-CK activities were unchanged, reflecting the high control exerted by the carboxylases relative to the decarboxylases on the efficiency of C4 metabolism. Despite having larger stomatal conductance at glacial CO2, C4 species maintained greater PWUE and PNUE relative to C3-C4 and C3 species due to higher photosynthetic rates. Relative to other C4 subtypes, NAD-ME and PEP-CK grasses had the highest PWUE and PNUE, respectively; relative to C3, the C3-C4 grass had higher PWUE and similar PNUE at glacial CO2. Biomass accumulation was reduced by glacial CO2 in the C3 grass relative to the C3-C4 grass, while biomass was less reduced in NAD-ME grasses compared with NADP-ME and PCK grasses. Under glacial CO2, high resource use efficiency offers a key evolutionary advantage for the transition from C3 to C4 photosynthesis in water- and nutrient-limited environments.
Project description:There is a long-standing controversy as to whether drought limits photosynthetic CO2 assimilation through stomatal closure or by metabolic impairment in C3 plants. Comparing results from different studies is difficult due to interspecific differences in the response of photosynthesis to leaf water potential and/or relative water content (RWC), the most commonly used parameters to assess the severity of drought. Therefore, we have used stomatal conductance (g) as a basis for comparison of metabolic processes in different studies. The logic is that, as there is a strong link between g and photosynthesis (perhaps co-regulation between them), so different relationships between RWC or water potential and photosynthetic rate and changes in metabolism in different species and studies may be 'normalized' by relating them to g. Re-analysing data from the literature using light-saturated g as a parameter indicative of water deficits in plants shows that there is good correspondence between the onset of drought-induced inhibition of different photosynthetic sub-processes and g. Contents of ribulose bisphosphate (RuBP) and adenosine triphosphate (ATP) decrease early in drought development, at still relatively high g (higher than 150 mmol H20 m(-2) s(-1)). This suggests that RuBP regeneration and ATP synthesis are impaired. Decreased photochemistry and Rubisco activity typically occur at lower g (<100 mmol H20 m(-2) s(-1)), whereas permanent photoinhibition is only occasional, occurring at very low g (<50 mmol H20 m(-2) s(-1)). Sub-stomatal CO2 concentration decreases as g becomes smaller, but increases again at small g. The analysis suggests that stomatal closure is the earliest response to drought and the dominant limitation to photosynthesis at mild to moderate drought. However, in parallel, progressive down-regulation or inhibition of metabolic processes leads to decreased RuBP content, which becomes the dominant limitation at severe drought, and thereby inhibits photosynthetic CO2 assimilation.
Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of the Calvin-Benson-Bassham cycle of photosynthesis, requires conformational repair by Rubisco activase for efficient function. Rubisco mediates the fixation of atmospheric CO2 by catalyzing the carboxylation of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP). It is a remarkably inefficient enzyme, and efforts to increase crop yields by bioengineering Rubisco remain unsuccessful. This is due in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. To function, Rubisco must undergo an activation process that involves carboxylation of an active site lysine by a non-substrate CO2 molecule and binding of a Mg2+ ion. Premature binding of the substrate RuBP results in an inactive enzyme. Moreover, Rubisco can also be inhibited by a range of sugar phosphates, some of which are "misfire" products of its multistep catalytic reaction. The release of the inhibitory sugar molecule is mediated by the AAA+ protein Rubisco activase (Rca), which couples hydrolysis of ATP to the structural remodeling of Rubisco. Rca enzymes are found in the vast majority of photosynthetic organisms, from bacteria to higher plants. They share a canonical AAA+ domain architecture and form six-membered ring complexes but are diverse in sequence and mechanism, suggesting their convergent evolution. In this review, we discuss recent advances in understanding the structure and function of this important group of client-specific AAA+ proteins.
Project description:Plants operating C3 and C4 photosynthetic pathways exhibit differences in leaf anatomy and photosynthetic carbon fixation biochemistry. Fully understanding this underpinning biochemical variation is requisite to identifying solutions for improving photosynthetic efficiency and growth. Here we refine assay methods for accurately measuring the carboxylase and decarboxylase activities in C3 and C4 plant soluble protein. We show that differences in plant extract preparation and assay conditions are required to measure NADP-malic enzyme and phosphoenolpyruvate carboxylase (pH 8, Mg(2+), 22 °C) and phosphoenolpyruvate carboxykinase (pH 7, >2mM Mn(2+), no Mg(2+)) maximal activities accurately. We validate how the omission of MgCl2 during leaf protein extraction, lengthy (>1min) centrifugation times, and the use of non-pure ribulose-1,5-bisphosphate (RuBP) significantly underestimate Rubisco activation status. We show how Rubisco activation status varies with leaf ontogeny and is generally lower in mature C4 monocot leaves (45-60% activation) relative to C3 monocots (55-90% activation). Consistent with their >3-fold lower Rubisco contents, full Rubisco activation in soluble protein from C4 leaves (<5min) was faster than in C3 plant samples (<10min), with addition of Rubisco activase not required for full activation. We conclude that Rubisco inactivation in illuminated leaves primarily stems from RuBP binding to non-carbamylated enzyme, a state readily reversible by dilution during cellular protein extraction.
Project description:Representatives of the genus Beijerinckia are known as heterotrophic, dinitrogen-fixing bacteria which utilize a wide range of multicarbon compounds. Here we show that at least one of the currently known species of this genus, i.e., Beijerinckia mobilis, is also capable of methylotrophic metabolism coupled with the ribulose bisphosphate (RuBP) pathway of C1 assimilation. A complete suite of dehydrogenases commonly involved in the sequential oxidation of methanol via formaldehyde and formate to CO2 was detected in cell extracts of B. mobilis grown on CH3OH. Carbon dioxide produced by oxidation of methanol was further assimilated via the RuBP pathway as evidenced by reasonably high activities of phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Detection and partial sequence analysis of genes encoding the large subunits of methanol dehydrogenase (mxaF) and form I RubisCO (cbbL) provided genotypic evidence for methylotrophic autotrophy in B. mobilis.
Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is responsible for almost all biological CO2 assimilation, but forms inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. The distantly related AAA+ proteins rubisco activase and CbbX remodel inhibited rubisco complexes to effect inhibitor release in plants and ?-proteobacteria, respectively. Here we characterize a third class of rubisco activase in the chemolithoautotroph Acidithiobacillus ferrooxidans. Two sets of isoforms of CbbQ and CbbO form hetero-oligomers that function as specific activases for two structurally diverse rubisco forms. Mutational analysis supports a model wherein the AAA+ protein CbbQ functions as motor and CbbO is a substrate adaptor that binds rubisco via a von Willebrand factor A domain. Understanding the mechanisms employed by nature to overcome rubisco's shortcomings will increase our toolbox for engineering photosynthetic carbon dioxide fixation.
Project description:Green oceanic microalgae are efficient converters of solar energy into the biomass via the photosynthesis process, with the first step of carbon fixation in the photosynthesis being controlled by the enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), which is a large proteinaceous machine composed of large (L, 52 kDa) and small (S, 12 kDa) subunits arranged as a L8S8 hexadecamer that catalyzes the formation of 2 phosphoglyceric acid molecules from one ribulose 1,5-bisphosphate (RuBP) molecule and one of carbon dioxide (CO2) and that is considered as the most abundant protein on Earth. The catalytic efficiency of this protein is controlled by the RuBisCO activase (RCA) that interacts with RuBisCO and promotes the CO2 entrance to the active site of RuBisCO by removing RuBP. One of the peculiar features of RCA is the presence of functional disordered tails that might play a role in RCA-RuBisCO interaction. Based on their ability to move, microalgae are grouped into 2 major class, motile and non-motile. Motile microalgae have an obvious advantage over their non-motile counterparts because of their ability to actively migrate within the water column to find the most optimal environmental conditions. We hypothesizes that the RCA could be functionally different in the non-motile and motile microalgae. To check this hypothesis, we conducted a comparative computational analysis of the RCAs from the representatives of the non-motile (Ostreococcus tauri) and motile (Tetraselmis sp. GSL018) green oceanic microalgae.
Project description: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.
Project description:Salinity and drought are two often simultaneously occurring abiotic stresses that limit the production of food crops worldwide. This study aimed to distinguish between the separate and combined impacts of drought and salinity on the plant response. Panicum antidotale was cultivated in a greenhouse under the following growth conditions: control, 100 mM NaCl (100) and 300 mM NaCl (300) salinity, drought (D; 30% irrigation), and two combinations of salinity and drought (100 + D and 300 + D). The growth response was as follows: 0 ? 100 > 100 + D > > D ? 300 ? 300 + D. Growth correlated directly with photosynthesis. The net photosynthesis, stomatal conductance, intercellular CO2, transpiration, ribulose 1,5-bisphosphate carboxylase (Rubisco), ribulose 1,5-bisphosphate (RuBP) regeneration, and triose phosphate utilization protein (e.g., phosphoenolpyruvate carboxylase) were highest in the control and declined most at 300 + D, while 100 + D performed significantly better as compared to drought. Maximum and actual photosystem II (PSII) efficiencies, along with photochemical quenching during light harvesting, resemble the plant growth and contemporary CO2/H2O gas exchange parameters in the given treatments. Plant improves water use efficiency under salt and drought treatments, which reflects the high water conservation ability of Panicum. Our findings indicate that the combination of low salinity with drought was able to minimize the deleterious effects of drought alone on growth, chlorophyll content, cell integrity, photosynthesis, leaf water potential, and water deficit. This synergetic effect demonstrates the positive role of Na+ and Cl- in carbon assimilation and osmotic adjustment. In contrast, the combination of high salinity and drought enforced the negative response of plants in comparison to single stress, demonstrating the antagonistic impact of water availability and ion toxicity.