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     

Relationship of photosynthetic acclimation to changes of Rubisco activity in field-grown winter wheat and barley during growth in elevated carbon dioxide

The responses of photosynthesis, Rubisco activity, Rubisco protein, leaf carbohydrates and total soluble protein to three carbon dioxide treatments were studied in winter wheat [Triticum aestivum (L.)] and barley [Hordeum vulgare (L.)]. Barley and wheat plants were grown in small field plots during 1995 and 1996 in clear, acrylic chambers (1.2–2.4 m²) and were provided with continuous carbon dioxide fertilization at concentrations of 350, 525 and 700 mol mol^–1. Photosynthetic rates of barley penultimate leaves and wheat flag leaves measured at growth carbon dioxide concentrations decreased with leaf age in all three CO₂ treatments during 1995 and 1996. Photosynthetic acclimation to elevated CO₂ was observed on seven of eight measurement dates for barley and ten of eleven measurement dates for wheat over both years. Initial Rubisco activity, total soluble protein and Rubisco protein in barley penultimate leaves and wheat flag leaves also decreased with leaf age. Total Rubisco activity was not used because of enzyme degradation. There was a significant CO₂ treatment effect on initial Rubisco activity, total soluble protein and Rubisco protein for wheat in 1995 and 1996 and for barley in 1995. Responses of barley penultimate leaf Rubisco activity and leaf protein concentrations to elevated carbon dioxide were nonsignificant in 1996. A significant CO₂ treatment effect also was detected when means of Rubisco activity, soluble protein and Rubisco protein for wheat flag leaves were combined over harvests and years. These three flag leaf parameters were not significantly different in the 350 and 525 mol mol^–1 CO₂ treatments but were decreased during growth in 700 mol mol^–1 CO₂ relative to the other two CO₂ treatments. Ratios of photosynthesis at 700 and 350 mol mol^–1 were compared to ratios of Rubisco activity at 700 and 350 mol mol^–1 using wheat flag leaf data from 1995 and 1996. Regression analysis of these data were linear [y = 0.586 + 1.103t x (r² = 0.432)] and were significant at P 0.05. This result indicated that photosynthetic acclimation was positively correlated with changes of initial Rubisco activity in wheat flag leaves in response to CO₂ enrichment. Effects of elevated CO₂ on wheat leaf proteins during 1995 and 1996 and on barley during 1995 were consistent with an acceleration of senescence.     

Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803rbc, were detected within 8 days when grown under 5% CO₂ in air. These transformants were unable to grow in air (0.03% CO₂). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO₂ in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.Abbreviations Cm^r chloramphenicol resistance - Km^r kanamycin resistance - HCR high CO₂ requirer - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SSC sodium chloride and sodium citrate - wt wild-type     

On the functional significance of the polypeptide PsbY for photosynthetic water oxidation in the cyanobacterium<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. strain PCC 6803

Recent investigations have revealed that the cyanobacterial photosystem II complex contains more than 26 polypeptides. The functions of most of the low-molecular-mass polypeptides, including PsbY, have remained elusive. Here we present a comparative characterization of the wild-type Synechocystis sp. strain PCC 6803 and a PsbY-free mutant derived from it. The results show that growth of the PsbY-free mutant was comparable to that of the wild-type when cells were cultivated in complete BG11 medium or under initial manganese or chloride limitation, and when illuminated at 20 or 200 E m^–2 s^–1. However, while growth rates of both the wild-type and the PsbY-free mutant were reduced when cells were cultivated in BG11 medium in the absence of calcium, the reduction was significantly greater in the case of the PsbY-free mutant. This differential effect on growth of the mutant relative to the wild-type in CaCl₂ deficient medium was detected when the cells were illuminated with high-intensity light (200 E m^–2 s^–1) but not when light levels were lower (20 E m^–2 s^–1). The differential effect on growth was associated with lower O₂ evolving activity in the mutant compared to wild-type cells. The mutant was also found to be more sensitive to photoinhibition, and showed an altered pattern of fluorescence emission at 77 K. In addition, mass spectrometric analysis revealed that PsbY-free cells cultivated in CaCl₂ sufficient medium (in which no growth reduction was observed) had a significantly higher O₂ evolution from hydrogen peroxide and a lower O₂ evolution from water under flash light illumination than wild-type cells. These results imply that photosystem II is slightly impaired in the PsbY-free mutant, and that the mutant is less capable of coping with low levels of Ca²⁺ than the wild-type.Communicated by R. G. Herrmann     

Characteristics of a photorespiratory mutant of barley (Hordeum vulgare L.) deficient in phosphogly collate phosphatase

A barley mutant RPr84/90 has been isolated by selecting for plants which grow poorly in natural air, but normally in air enriched to 0.8% CO₂. After 5 minutes of photosynthesis in air containing¹⁴CO₂ this mutant incorporated 26% of the¹⁴C carbon into phosphoglycollate, a compound not normally labelled in wild type (cv. Maris Mink) leaves.The activity of phosphoglycollate phosphatase (EC 3.1.1.18) was 1.2 nkat mg^–1 protein at 30°C in RPr 84/90 compared to 19.2 nkat mg^–1 protein in the wild-type leaves. Phosphoglycollate phosphatase activity was not detected after protein separation by electrophoresis of leaf extracts from the mutant on polyacrylamide gels; on linear 5% acrylamide gels three bands with enzyme activity were separated from extracts of wild type plants. Gradient gel electrophoresis followed by activity staining showed two bands in Maris Mink tracks of MW 86,000 and 96,000, but no bands in 84/90. This is the first report of isozymes of phosphoglycollate phosphatase in barley which were absent in the mutant extracts. Our results confirm an earlier report of isozymes of this phosphatase in Phaseolus vulgaris [18].The photosynthetic rate of RPr 84/90 in 1% O₂, 350 l CO₂ l^–1 was 9–12 mg CO₂ dm^–2 h^–1 at 20°C, whereas the wild-type rate was 27–29 mg CO₂ dm^–2 h^–1 at 20°C. In 21% O₂, 350 l CO₂ l^–1 the rate was 2–3 mg CO₂ dm^–2 h^–1 in the mutant and 20 mg CO₂ dm^–2 h^–1 in the wild type.Genetic analysis has shown that the mutation segregates as a single recessive nuclear gene.     

Photosynthetic gas exchange response of poplars to steady-state and dynamic light environments

The steady-state and dynamic photosynthetic response of two poplar species (Populus tremuloides and P. fremontii) to variations in photon flux density (PFD) were observed with a field portable gas exchange system. These poplars were shown to be very shade intolerant with high light saturation (800 to 1300 mol photons m^–2 s^–1) and light compensation (70 to 100 mol m^–2 s^–1) points. Understory poplar leaves showed no physiological acclimation to understory light environments. These plants become photosynthetically induced quickly (10 min). Activation of Rubisco was the primary limitation for induction, with stomatal opening playing only a minor role. Leaves maintained high stomatal conductances and stomata were unresponsive to variations in PFD. Leaves were very efficient at utilizing rapidly fluctuating light environments similar to those naturally occurring in canopies. Post-illumination CO₂ fixation contributed proportionally more to the carbon gain of leaves during short frequent lightflecks than longer less frequent ones. The benefits of a more dynamic understory light environment for the carbon economy of these species are discussed.     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with ‘antisense’ rbcS

Transgenic tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to produce a series of plants with a progressive decrease in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been used to investigate the contribution of Rubsico to the control of photosynthesis at different irradiance, CO₂ concentrations and vapour-pressure deficits. Assimilation rates, transpiration, the internal CO₂ concentration and chlorophyll fluorescence were measured in each plant. (i) The flux-control coefficient of Rubisco was estimated from the slope of the plot of Rubisco content versus assimilation rate. The flux-control coefficient had a value of 0.8 or more in high irradiance, (1050 mol·m^–2·s^–1), low-vapour pressure deficit (4 mbar) and ambient CO₂ (350 bar). Control was marginal in enhanced CO₂ (450 bar) or low light (310 mol·m^–2·s^–1) and was also decreased at high vapour-pressure deficit (17 mbar). No control was exerted in 5% CO₂. (ii) The flux-control coefficients of Rubisco were compared with the fractional demand placed on the calculated available Rubisco capacity. Only a marginal control on photosynthetic flux is exerted by Rubisco until over 50% of the available capacity is being used. Control increases as utilisation rises to 80%, and approaches unity (i.e. strict limitation) when more than 80% of the available capacity is being used. (iii) In low light, plants with reduced Rubisco have very high energy-dependent quenching of chlorophyll fluorescence (qE) and a decreased apparent quantum yield. It is argued that Rubisco still exerts marginal control in these conditions because decreased Rubisco leads to increased thylakoid energisation and high-energy dependent dissipation of light energy, and lower light-harvesting efficiency. (iv) The flux-control coefficient of stomata for photosynthesis was calculated from the flux-control coefficient of Rubisco and the internal CO₂ concentration, by applying the connectivity theorem. Control by the stomata varies between zero and about 0.25. It is increased by increased irradiance, decreased CO₂ or decreased vapour-pressure deficit. (v) Photosynthetic oscillations in saturating irradiance and CO₂ are suppressed in decreased-activity transformants before the steady-state rate of photosynthesis is affected. This provides direct evidence that these oscillations reveal the presence of excess Rubisco. (vi) Comparison of the flux-control coefficients of Rubisco with mechanistic models of photosynthesis provides direct support for the reliability of these models in conditions where Rubisco has a flux-control coefficient approach unity (i.e. limits photosynthesis), but also indicates that these models are less useful in conditions where control is shared between Rubisco and other components of the photosynthetic apparatus.Abbreviations A assimilation rate - C_i intercellular CO₂ concentration in the leaf - C_R flux-control coefficient of Rubisco for photosynthesis - qE high-energy-state-dependent quenching of chlorophyll fluorescence - Q_A primary acceptor of PSII - rbc S gene for the nuclear-encoded small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - Ru1,5bisP ribulose-1,5-bisphosphate - VPD vapour-pressure deficit     

Rubisco Activase mRNA Expression in Spinach: Modulation by Nanoanatase Treatment

Characterized by a photocatalysis property, nanoanatase is closely related to the photosynthesis of spinach. It could not only improve light absorbance, transformation from light energy to electron energy, and active chemical energy, but also promote carbon dioxide (CO₂) assimilation of spinach. However, the molecular mechanism of carbon reaction promoted by nanoanatase remains largely unclear. In this study, we report that the amounts of Rubisco activase (rca) mRNA in the nanoanatase-treated spinach were increased by about 51%, whereas bulk-TiO₂ treatment produced an increase of only 5%. Accordingly, the protein level of Rubisco activase from the nanoanatase-treated spinach was increased by 42% compared with the control; however, bulk-TiO₂ treatment resulted in a 5% improvement. Further analysis indicated that the activity of Rubisco activase in the nanoanatase-treated spinach was significantly higher than the control by up to 2.75 times, and bulk-TiO₂ treatment had no such significant effects. Together, one of the molecular mechanisms of carbon reaction promoted by nanoanatase is that the nanoanatase treatment results in the enhancement of rca mRNA expressions, protein levels, and activities of Rubisco activase, thereby leading to the improvement of Rubisco carboxylation and the high rate of photosynthetic carbon reaction.     

Leaf cavity CO2 concentrations and CO2 exchange in onion,Allium cepa L.

Onion (Allium cepa L.) plants were examined to determine the photosynthetic role of CO₂ that accumulates within their leaf cavities. Leaf cavity CO₂ concentrations ranged from 2250 L L^–1 near the leaf base to below atmospheric (<350 L L^–1) near the leaf tip at midday. There was a daily fluctuation in the leaf cavity CO₂ concentrations with minimum values near midday and maximum values at night. Conductance to CO₂ from the leaf cavity ranged from 24 to 202 mol m^–2 s^–1 and was even lower for membranes of bulb scales. The capacity for onion leaves to recycle leaf cavity CO₂ was poor, only 0.2 to 2.2% of leaf photosynthesis based either on measured CO₂ concentrations and conductance values or as measured directly by ¹⁴CO₂ labeling experiments. The photosynthetic responses to CO₂ and O₂ were measured to determine whether onion leaves exhibited a typical C₃-type response. A linear increase in CO₂ uptake was observed in intact leaves up to 315 L L^–1 of external CO₂ and, at this external CO₂ concentration, uptake was inhibited 35.4±0.9% by 210 mL L^–1 O₂ compared to 20 mL L^–1 O₂. Scanning electron micrographs of the leaf cavity wall revealed degenerated tissue covered by a membrane. Onion leaf cavity membranes apparently are highly impermeable to CO₂ and greatly restrict the refixation of leaf cavity CO₂ by photosynthetic tissue.Abbreviations C_a external CO₂ concentration - C_i intercellular CO₂ concentration - CO₂ compensation concentration - PPFR photosynthetic photon fluence rate     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS

Experiments were carried out to determine how decreased expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) affects photosynthetic metabolism in ambient growth conditions. In a series of tobacco (Nicotiana tabacum L.) plants containing progressively smaller amounts of Rubisco the rate of photosynthesis was measured under conditions similar to those in which the plants had been grown (310 mol photons · m^–2 · s^–1, 350 bar CO₂, 22° C). (i) There was only a marginal inhibition (6%) of photosynthesis when Rubisco was decreased to about 60% of the amount in the wildtype. The reduced amount of Rubisco was compensated for by an increase in Rubisco activation (rising from 60 to 100%), with minor contributions from an increase of its substrates (ribulose-1,5-bisphosphate and the internal CO₂ concentration) and a decrease of its product (glycerate-3-phosphate). (ii) The decreased amount of Rubisco was accompanied by an increased ATP/ADP ratio that may be causally linked to the increased activation of Rubisco. An increase of highenergy-state chlorophyll fluorescence shows that thylakoid membrane energisation and high-energy-state-dependent energy dissipation at photosystem two had also increased. (iii) A further decrease of Rubisco (in the range of 50–20% of the wildtype level) resulted in a strong and proportional inhibition of CO₂ assimilation. This was accompanied by a decrease of fructose-1,6-bisphosphatase activity, coupling-factor 1 (CF₁)-ATP-synthase protein, NADP-malate dehydrogenase protein, and chlorophyll. The chlorophyll a/b ratio did not change, and enolase and sucrose-phosphate synthase activity did not decrease. It is argued that other photosynthetic enzymes are also decreased once Rubisco decreases to the point at which it becomes strongly limiting for photosynthesis. (iv) It is proposed that the amount of Rubisco in the wildtype represents a balance between the demands of light, water and nitrogen utilisation. The wildtype overinvests about 15% more protein in Rubisco than is needed to avoid a strict Rubisco limitation of photosynthesis. However, this excess Rubisco allows the wildtype to operate with lower thylakoid energisation, and decreased high-energy-state-dependent energy dissipation, hence increasing light-use efficiency by about 6%. It also allows the wildtype to operate with a lower internal CO₂ concentration in the leaf and a lower stomatal conductance at a given rate of photosynthesis, so that instantaneous water-use efficiency is marginally (8%) increased.Abbreviations C_i CO₂ concentration in the air spaces within the leaf - CF₁ coupling factor 1 - Chl chlorophyll Fru1 - 6bisP fructose-1,6-bisphosphate - F_m fluorescence yield with a saturating pulse in dark-adapted material - F_o ground-level of fluorescence obtained using a weak non-actinic modulated beam in the dark - PGA glycerate-3-phosphate - rbcS gene for the nuclear-encoded small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - Ru1, 5bisP ribulose-1,5-bisphosphate     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS

Tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to decrease the expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) have been used to investigate the contribution of Rubisco to the control of photosynthesis in plants growing at different irradiances. Tobacco plants were grown in controlled-climate chambers under ambient CO₂ at 20°C at 100, 300 and 750 mol·m^–2·s^–1 irradiance, and at 28°C at 100, 300 and 1000 mol·m^–2·s^–1 irradiance. (i) Measurement of photosynthesis under ambient conditions showed that the flux control coefficient of Rubisco (C _infRubisco ^supA ) was very low (0.01–0.03) at low growth irradiance, and still fairly low (0.24–0.27) at higher irradiance. (ii) Short-term changes in the irradiance used to measure photosynthesis showed that C _infRubisco ^supA increases as incident irradiance rises, (iii) When low-light (100 mol·m^–2·s^–1)-grown plants are exposed to high (750–1000 mol·m^–2·s^–1) irradiance, Rubisco is almost totally limiting for photosynthesis in wild types. However, when high-light-grown leaves (750–1000 mol·m^–2·s^–1) are suddenly exposed to high and saturating irradiance (1500–2000 mol·m^–2·s^–1), C _infRubisco ^supA remained relatively low (0.23–0.33), showing that in saturating light Rubisco only exerts partial control over the light-saturated rate of photosynthesis in sun leaves; apparently additional factors are co-limiting photosynthetic performance, (iv) Growth of plants at high irradiance led to a small decrease in the percentage of total protein found in the insoluble (thylakoid fraction), and a decrease of chlorophyll, relative to protein or structural leaf dry weight. As a consequence of this change, high-irradiance-grown leaves illuminated at growth irradiance avoided an inbalance between the light reactions and Rubisco; this was shown by the low value of C _infRubisco ^supA (see above) and by measurements showing that non-photochemical quenching was low, photochemical quenching high, and NADP-malate dehydrogenase activation was low at the growth irradiance. In contrast, when a leaf adapted to low irradiance was illuminated at a higher irradiance, Rubisco exerted more control, non-photochemical quenching was higher, photochemical quenching was lower, and NADP-malate dehydrogenase activation was higher than in a leaf which had grown at that irradiance. We conclude that changes in leaf composition allow the leaf to avoid a one-sided limitation by Rubisco and, hence, overexcitation and overreduction of the thylakoids in high-irradiance growth conditions, (v) Antisense plants with less Rubisco contained a higher content of insoluble (thylakoid) protein and chlorophyll, compared to total protein or structural leaf dry weight. They also showed a higher rate of photosynthesis than the wild type, when measured at an irradiance below that at which the plant had grown. We propose that N-allocation in low light is not optimal in tobacco and that genetic manipulation to decrease Rubisco may, in some circumstances, increase photosynthetic performance in low light.Abbreviations A rate of photosynthesis - C _infRubisco ^supA flux control coefficient of Rubisco for photosynthesis - c_i internal CO₂ concentration - qE energy-dependent quenching of chlorophyll fluorescense - qQ photochemical quenching of chlorophyll fluorescence - NADP-MDH NADP-dependent malate dehydrogenase - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - RuBP ribulose-1,5-bisphosphate This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137).     

Photosynthetic Metabolism and Activity of Carboxylating Enzymes in Emergent,Floating, and Submersed Leaves of Hydrophytes

Radioisotope techniques were used to compare photosynthetic CO₂ fixation, activities of carboxylating enzymes, and the composition of photosynthates in 42 species of aquatic plants (emergent, floating, and submersed hydrophytes) collected from rivers Sysert' and Iset' in Sverdlovsk oblast (Russia). The submersed leaves, in comparison with the emergent and floating leaves, featured lower rates of potential photosynthesis (by 2.2 mg CO²/(dm² h) on average), low content of the fraction I protein, and low activity of Rubisco and phosphoenolpyruvate carboxylase (PEPC). The averaged activities of Rubisco and PEPC were diminished in submersed leaves by 10 and 1 mg/(dm² h), respectively. Different hydrophyte groups showed similar composition of assimilates accumulated after 5-min photosynthesis and did not differ in this respect from terrestrial plants. However, the incorporation of ¹⁴C into sucrose and starch in submersed leaves (30 and 9% of total labeling, respectively) was lower than in emergent and floating leaves (45 and 15%, respectively). At the same time, the incorporation of ¹⁴C into C₄ acids (malate and aspartate) was 1.5 times higher in submersed leaves than in other leaf types. Analysis of leaf differentiation, the Rubisco/PEPC activity ratio, the PEPC activity, and the composition of primary photosynthates in the pulse–chase experiments revealed no evidence of the C₄ effect in the submersed hydrophytes examined. The adaptation of hydatophytes to specific conditions of an aquatic environment was structurally manifested in the reduction (by a factor of 3–5) in the number of chloroplasts per 1 cm² leaf area. This small number of chloroplasts was responsible for low photosynthetic rates in submersed leaves, although metabolic activities of individual chloroplasts were similar for all three hydrophyte groups.     

Can the Cyanobacterial Carbon-Concentrating Mechanism Increase Photosynthesis in Crop Species? A Theoretical Analysis

Experimental elevation of [CO₂] around C₃ crops in the field has been shown to increase yields by suppressing the Rubisco oxygenase reaction and, in turn, photorespiration. Bioengineering a cyanobacterial carbon-concentrating mechanism (CCM) into C₃ crop species provides a potential means of elevating [CO₂] at Rubisco, thereby decreasing photorespiration and increasing photosynthetic efficiency and yield. The cyanobacterial CCM is an attractive alternative relative to other CCMs, because its features do not require anatomical changes to leaf tissue. However, the potential benefits of engineering the entire CCM into a C₃ leaf are unexamined. Here, a CO₂ and HCO₃⁻ diffusion-reaction model is developed to examine how components of the cyanobacterial CCM affect leaf light-saturated CO₂ uptake (A_sat) and to determine whether a different Rubisco isoform would perform better in a leaf with a cyanobacterial CCM. The results show that the addition of carboxysomes without other CCM components substantially decreases A_sat and that the best first step is the addition of HCO₃⁻ transporters, as a single HCO₃⁻ transporter increased modeled A_sat by 9%. Addition of all major CCM components increased A_sat from 24 to 38 µmol m⁻² s⁻¹. Several Rubisco isoforms were compared in the model, and increasing ribulose bisphosphate regeneration rate will allow for further improvements by using a Rubisco isoform adapted to high [CO₂]. Results from field studies that artificially raise [CO₂] suggest that this 60% increase in A_sat could result in a 36% to 60% increase in yield.C₃ species include the major grain crops rice (Oryza sativa) and wheat (Triticum aestivum) and overall accounted for approximately 75% of all primary foodstuff production in 2012 (FAOSTAT, 2013). The yield of many crop species has been substantially improved through breeding and agronomy, but advancement in yield has substantially slowed in many of the major C₃ crops in the last decade, suggesting that limits on yield improvement using these techniques are being reached and that other approaches are needed (Long and Ort, 2010; Ray et al., 2012).One largely unexploited approach would be to improve the efficiency of photosynthesis in these species (Zhu et al., 2010). The photosynthetic enzyme Rubisco catalyzes the reaction of CO₂ with ribulose bisphosphate (RuBP), which eventually forms carbohydrates. However, Rubisco will also react with oxygen as the first step in photorespiration. This reaction is considered wasteful, since energy is consumed to recover RuBP and CO₂ is lost in the process. In C₃ plants at 25°C and current atmospheric [CO₂], photorespiration results in an approximately 30% decrease in net carbon assimilation (Zhu et al., 2010). Thus, it is a large inefficiency in carbon uptake and a target for improvement.Since CO₂ and oxygen act competitively at Rubisco, photorespiration can be decreased by increasing [CO₂] around Rubisco. That this will increase yield is demonstrated by the many studies that have artificially increased atmospheric [CO₂] around C₃ crops growing in the field (Kimball et al., 2002; Long et al., 2006b). Other photosynthetic organisms have evolved mechanisms to internally elevate [CO₂] at Rubisco to decrease or eliminate photorespiration. Such carbon-concentrating mechanisms (CCMs) include C₄ photosynthesis, as in maize (Zea mays), and the carboxysome and pyrenoid CCMs of single-celled cyanobacteria and algae. The C₄ pathway requires the addition of the photosynthetic C₄ dicarboxylate cycle and inner photosynthetic cells (i.e. bundle sheath), where Rubisco is localized. Converting a C₃ crop to a C₄ crop will require a coordination of changes in photosynthetic tissue differentiation and enzyme and transporter localization. In contrast, cyanobacteria achieve the same effect in a single cell by localizing Rubisco to specialized subcellular compartments called carboxysomes, so in theory they would require fewer changes. Carboxysomes are polyhedral bodies with a protein shell that encloses carbonic anhydrase (CA) and Rubisco packed in an ordered or semiordered array (Long et al., 2007; Yeates et al., 2011). Bicarbonate is actively transported from the environment into the cytosol of the cyanobacteria, and CO₂ in the cytosol is actively hydrated to HCO₃⁻ using NADH. CO₂ is also hydrated to HCO₃⁻, which serves to increase the [CO₂] gradient between the medium and cytosol, increasing CO₂ flux, and also serves to refix CO₂ that leaks from the carboxysome. Since the cytosol lacks CA and the plasma membrane has low permeability to HCO₃⁻, a high cytosolic [HCO₃⁻] far from equilibrium with [CO₂] is achieved. Bicarbonate diffuses through the protein shell of the carboxysome, and since CA is localized to the inner side of the shell, it is rapidly converted to CO₂, given the disequilibrium. The resulting high [CO₂] around Rubisco inside the carboxysome accelerates carboxylation and competitively inhibits the oxygenation reaction (for review, see Price et al., 2008, 2011, 2013; Espie and Kimber, 2011).The C₄ pathway has been well characterized, and several projects are attempting to engineer it into C₃ plants with some success (Slewinski, 2013). However, a primary obstacle has been achieving the necessary two tissue types with the correct localization of the key enzymes (Covshoff and Hibberd, 2012). In this regard, cyanobacterial CCM may provide an attractive parallel approach to eliminating photorespiration in C₃ plants for two reasons. First, the biochemical, structural, and genetic components of the CCM are well understood, in that all of the necessary proteins and their genes have been identified. Second, chloroplasts of higher plants evolved from a common ancestor with modern cyanobacteria (Raven and Allen, 2003) and, therefore, are structurally similar. Thus, engineering carboxysomes into the chloroplast could involve introducing an operon containing the genes associated with the cyanobacterial CCM into the chloroplast genome.There are, however, multiple proteins in the cyanobacterial CCM, and simultaneously transforming all of the required genes into an organism may be particularly challenging. Furthermore, some of the proteins appear to have a similar function, and others may be deleterious if introduced without the full apparatus. A random sequence of gene stacking would likely be inefficient, while the addition of some genes may simply be unnecessary. However, the biophysical reactions involved are well understood, and there are some measurements of the kinetics of the enzymatic reactions and transporters. With this information, a mathematical model can be created to simulate the CCM. Such a model will allow deduction of the minimal component set necessary for an improvement of leaf photosynthetic efficiency and identify a logical sequence of gene additions to deliver progressive improvements and avoid any lethal effects. This model will also be used to determine which aspects of the CCM exert the most control over the overall process of photosynthetic CO₂ assimilation, information that can then be used to optimize the CCM, help identify the ideal set of components, or identify parameters that need to be more accurately measured in order to improve the effectiveness of the CCM model. Similar kinetic models have been applied to propose systems optimization of the Calvin cycle when plants are grown in elevated [CO₂] (Zhu et al., 2007), of the whole C₃ photosynthetic system (Zhu et al., 2013), C₄ photosynthesis (Yu et al., 2014), and to determine the mechanistic basis of mesophyll conductance (Tholen and Zhu, 2011).This paper develops a kinetic model to determine the potential of the cyanobacterial CCM engineered into C₃ crops for improving photosynthesis by determining the necessary components and estimating the potential improvement of CO₂ assimilation rate and efficiency. There are four distinct features to the cyanobacterial CCM: (1) a carboxysome with internally localized Rubisco and CA; (2) active transport of HCO₃⁻ from the medium into the cyanobacterial cytosol (the equivalent compartment in higher plants is the stroma of the chloroplast); (3) active hydration of CO₂ to HCO₃⁻ within the cytosol; and (4) the absence of CA in the cyanobacterial cytosol, in contrast to the higher plant stroma, which contains high activities of CA. Cyanobacteria have three HCO₃⁻ transporters and two CO₂ hydration enzymes that have different kinetics and are induced under different conditions (Price et al., 2011). All of these enzymes were examined, giving a total of seven individual components (listed in Fig. 1A) to the full cyanobacterial CCM model. Because of the common ancestry between cyanobacteria and higher plant chloroplasts, the two have homologous membranes and compartments. Proteins were modeled within a C₃ leaf using locations equivalent to those in cyanobacteria. That is, bicarbonate transporters were localized to the inner chloroplast membrane, and carboxysomes were localized to the stroma. The CO₂ hydration enzymes are bound to the thylakoid and plasma membranes in cyanobacteria, but the reaction for both occurs within the cyanobacterial cytosol; therefore, in this model, hydration of CO₂ by these enzymes was localized to the stroma. This model was used to determine the necessity of each of the features noted above, potential improvements in light-saturated CO₂ uptake (A_sat; irradiance of 1,800 µmol photons m⁻² s⁻¹) that would be achieved on incorporation of each feature in turn, a possible sequence of gene additions that would give an incremental improvement, and the key components of the CCM. The model was also used to test the value of using higher plant isoforms of Rubisco versus prokaryotic isoforms adapted to incorporation within the carboxysome. Sources of uncertainty in the model are also defined.Open in a separate windowFigure 1.A_sat of leaf models with sequential addition of components of the cyanobacterial CCM. Each point represents a leaf that contains the component listed for that column and all of the components in the columns to the left of it. The columns represent the sequence of transformations that produce the fastest increase in A_sat assuming that it is not possible to simultaneously add carboxysomes and remove stromal CA and that it is not desirable to add CO₂ hydration enzymes before removal of stromal CA (A) and assuming that it is possible to simultaneously add carboxysomes and remove stromal CA (B).     

Reduction in chlorophyll content without a corresponding reduction in photosynthesis and carbon assimilation enzymes in yellow-green oil yellow mutants of maize

The photosynthetic properties of a yellow lethal mutant, Oy/oy, and two yellow-green mutants of maize which are allelic (a homozygous recessive oy/oy and a heterozygous dominant Oy/+) were examined. Although Oy/oy had little or no chlorophyll or capacity for CO₂ fixation compared to normal siblings, it had 28% as much ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activity, and from 40% to near normal activities of C₄ cycle enzymes.Both yellow-green mutants had only half as much chlorophyll per leaf area as normal green seedlings in greenhouse-grown plants in winter and spring. However, the absorbance of light by the mutants was relatively high, as their transmittance was only 5 to 8% greater than normal leaves. In winter-grown greenhouse plants, the activities of Rubisco and several C₄ cycle enzymes in the mutants were unaffected and similar to those of normal seedlings on a leaf area basis. After allowing for small differences in leaf absorbance, the light response curves for photosynthesis in the mutants were similar on a leaf area basis but much higher on a chlorophyll basis than those of the normal seedlings. In spring-grown greenhouse plants the enzyme activities and photosynthesis rates were about 30% lower per leaf area in the yellow-green mutant leaves compared to the wild type. The maximum carboxylation efficiency (measured under low CO₂ and 1000 mol quanta m^-2 s^-1) in the mutants and normal leaves was similar on a Rubisco protein basis. The results indicate that maize can undergo a 50% reduction in chlorophyll content without a corresponding reduction in enzymes of carbon assimilation, and still maintain a high capacity for photosynthesis.Abbreviations Chl chlorophyll - PEP phosphoenolypruvate - Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase This research was supported by CSIRO and by USDA Competitive Grant 86-CRCR-1-2036.     

A model of photosynthetic CO2 assimilation and carbon-isotope discrimination in leaves of certain C3−C4 intermediates

A model of leaf, photosynthesis has been developed for C₃–C₄ intermediate species found in the generaPanicum, Moricandia, Parthenium andMollugo where no functional C₄ pathway has been identified. Model assumptions are a functional C₃ cycle in both mesophyll and bundle-sheath cells and that glycine formed in the mesophyll, as a consequence of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco, EC 4.1.1.39), diffuses to the bundle sheath, where most of the photorespiratory CO₂ is released. The model describes the observed gas-exchange characteristics of these C₃–C₄ intermediates, such as low CO₂-compensation points () at an O₂ pressure of 200 mbar, a curvilinear response of to changing O₂ pressures, and typical responses of CO₂-assimilation rate to intercellular CO₂ pressure. The model predicts that bundle-sheath CO₂ concentration is highest at low mesophyll CO₂ pressures and decreases as mesophyll CO₂ pressure increases. A partitioning of 5–15% of the total leaf Rubisco into the bundle-sheath cells and a bundlesheath conductance similar to that proposed for C₄ species best mimics the gas-exchange results. The model predicts C₃-like carbon-isotope discrimination for photosynthesis at atmospheric levels of CO₂, but at low CO₂ pressures it predicts a higher discrimination than is typically found during C₃ photosynthesis at lower CO₂ pressures.Abbreviations and symbols PEP phosphoenolpyruvate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) - RuBP ribulose-1,5-bisphosphate - p(CO₂) partial pressure of CO₂ - p(O₂) partial pressure of O₂. See also p. 471     

Characterisation of carbon dioxide and bicarbonate transport during steady-state photosynthesis in the marine cyanobacterium Synechococcus strain PCC7002

Net O₂ evolution, gross CO₂ uptake and net HCO _inf3 ^su– uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO₂ concentrations. Regardless of the CO₂ concentration during growth, all cells had the capacity to transport both CO₂ and HCO _inf3 ^su– ; however, the activity of HCO _inf3 ^su– transport was more than twofold higher than CO₂ transport even in cyanobacteria grown at high concentration of inorganic carbon (C_i = CO₂ + HCO _inf3 ^su– ). In low-C_i cells, the affinities of CO₂ and HCO _inf3 ^su– transport for their substrates were about 5 (CO₂ uptake) and 10 (HCO _inf3 ^su– uptake) times higher than in high-C_i cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated C_i pool reached 18, 32 and 55 mM in high-C_i, air-grown and low-C_i cells, respectively, when measured at 1 mM external C_i. Photosynthetic O₂ evolution, maximal CO₂ and HCO _inf3 ^su– transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO₂ provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO₂ concentrations. Transport studies with high-C_i cells revealed that CO₂ and HCO _inf3 ^su– uptake were equally inhibited when CO₂ fixation was reduced by the addition of glycolaldehyde. In contrast, in low-C_i cells steady-state CO₂ transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO₂ and HCO _inf3 ^su– uptake as well as O₂ evolution in both cell types. In high-C_i cells, the degree of inhibition was similar for HCO _inf3 ^su– transport and O₂ evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO₂ was much more sensitive to the inhibitor than HCO _inf3 ^su– transport, with an apparent I₅₀ value of around 250 M ethoxyzolamide for CO₂ uptake. The implications of our results are discussed with respect to C_i utilisation in the marine Synechococcus strain.Abbreviations Chl chlorophyll - C_i inorganic carbon (CO₂ + HCO _inf3 ^su– ) - CA carbonic anhydrase - CCM CO₂-concentrating mechanism - EZA ethoxyzolamide - GA glycolaldehyde - K_1/2 concentration required for half-maximal response - Rubisco ribulose-1,5,-bisphosphate carboxylase-oxygenase D.S. is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (D.F.G.). In addition, we are grateful to Donald A. Bryant, Department of Molecular and Cell Biology and Center of Biomolecular Structure Function, Pennsylvania State University, USA, for sending us the wild-type strain of Synechococcus PCC7002.     

Simultaneous oxygen production and nitrogenase activity of an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis in a co-culture with wheat

Summary A mutant strain of Anabaena variabilis, strain SA-1 that supported growth of wheat plants in a hydroponic co-culture in nitrogen (N) free medium also produced enough oxygen (O₂) to support root respiration. The steady-state concentration of net O₂ in the co-culture was dependent on incident light intensity. At an incident photosynthetic photoflux (PPF) of 1000 mol photons·m^–2·s^–1, net O₂ evolution by the co-culture in the root zone reached a maximum value of about 220 mol O₂ evolved·h^–1·mg chlorophyll (Chl)^–1. The O₂ concentration in the rhizosphere of the co-culture stayed above the ambient air level. O₂ uptake in the dark by strain SA-1-supplemented wheat roots washed free of cyanobacterium was higher than the root respiration of nitrate-grown plants. Nitrate-grown plants required aeration for maximum growth while the wheat-cyanobacterial co-culture can be cultured without aeration. These results show that O₂ produced by strain SA-1 can be used to supply the O₂ needs for root respiration of wheat. Respiration reduced net O₂ evolution by the mutant SA-1, decreasing the partial pressure of O₂ at the sites of cyanobacterial attachment to the roots. This led to an increase in the specific activity of nitrogenase of the co-culture at the high light intensities used to support wheat growth. This activity of about 30 mol ethylene produced·mg Chl^–1·h^–1 was three-fold higher than the activities obtained with the free-living strain SA-1 assayed at the same light intensity. In the co-culture, ammonia produced by the mutant strain SA-1 was not detectable. The NH _inf4 ^sup+ produced by strain SA-1 was used by the wheat plants and, under these conditions, the total N content of the plants reached as high as 85% of the total N content of nitrate-grown plants. In the co-culture system the metabolism of wheat and the cyanobacterium complemented each other, leading to higher plant growth in N-free medium. Offprint requests to: M. Gunasekaran     

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     

Effects of genetic manipulation of the activity of photorespiration on the redox state of photosystem I and its robustness against excess light stress under CO<Subscript>2</Subscript>-limited conditions in rice

Under CO₂-limited conditions such as during stomatal closure, photorespiration is suggested to act as a sink for excess light energy and protect photosystem I (PSI) by oxidizing its reaction center chlorophyll P700. In this study, this issue was directly examined with rice (Oryza sativa L.) plants via genetic manipulation of the amount of Rubisco, which can be a limiting factor for photorespiration. At low [CO₂] of 5 Pa that mimicked stomatal closure condition, the activity of photorespiration in transgenic plants with decreased Rubisco content (RBCS-antisense plants) markedly decreased, whereas the activity in transgenic plants with overproduction of Rubisco (RBCS-sense plants) was similar to that in wild-type plants. Oxidation of P700 was enhanced at [CO₂] of 5 Pa in wild-type and RBCS-sense plants. PSI was not damaged by excess light stress induced by repetitive saturated pulse-light (rSP) in the presence of strong steady-state light. On the other hand, P700 was strongly reduced in RBCS-antisense plants at [CO₂] of 5 Pa. PSI was also damaged by rSP illumination. These results indicate that oxidation of P700 and the robustness of PSI against excess light stress are hampered by the decreased activity of photorespiration as a result of genetic manipulation of Rubisco content. It is also suggested that overproduction of Rubisco does not enhance photorespiration as well as CO₂ assimilation probably due to partial deactivation of Rubisco.     

Effects of phosphorus nutrition on the response of photosynthesis to CO2 and O2, activation of ribulose bisphosphate carboxylase and amounts of ribulose bisphosphate and 3-phosphoglycerate in spinach leaves

Phosphorus-deficient spinach plants were grown by transferring them to nutrient solutions without PO₄. Photosynthetic rates were measured at a range of intercellular CO₂ partial pressures from 50–500 bar and then the leaves were freeze-clamped in situ to measure ribulose bisphosphate carboxylase (Rubisco) activity and metabolite concentrations. Compared with control leaves, deficient leaves had significantly lower photosynthetic rates, percentage activation of Rubisco, and amounts of ribulose bisphosphate and 3-phosphoglycerate at all CO₂ partial pressures. After feeding 10 mM PO₄ to the petioles of detached deficient leaves, all these measurements increased within 2 hours. At atmospheric CO₂ partial pressure the photosynthetic rate was stimulated in 19 mbar O₂ compared with 200 mbar. At higher CO₂ partial pressures this stimulation was less but the percentage stimulation in deficient leaves was no different from controls in either CO₂ partial pressure. It was concluded that phosphorus deficiency affects both Rubisco activity and the capacity for ribulose bisphosphate regeneration, and possible causes are discussed.Abbreviations A CO₂ assimilation rate - C_i intercellular CO₂ partial pressure - PGA 3-phosphoglycerate - RuP₂ ribulose 1,5-bisphosphate - Rubisco RuP₂ carboxylase/oxygenase