Affixing the O to Rubisco: discovering the source of photorespiratory glycolate and its regulation

The source of glycolate in photorespiration and its control, a particularly active and controversial research topic in the 1970s, was resolved in large part by several discoveries and observations described here. George Bowes discovered that the key carboxylation enzyme Rubisco (ribulosebisphosphate carboxylase/oxygenase) is competitively inhibited by O₂ and that O₂ substitutes for CO₂ in the initial `dark' reaction of photosynthesis to yield glycolate-P, the substrate for photorespiration. William Laing derived an equation from basic enzyme kinetics that describes the CO₂, O₂, and temperature dependence of photosynthesis, photorespiration, and the CO₂ compensation point in C₃ plants. Jerome Servaites established that photosynthesis cannot be increased by inhibiting the photorespiratory pathway prior to the release of photorespiratory CO₂, and Douglas Jordan discovered substantial natural variation in the Rubisco oxygenase/carboxylase ratio. A mutant Arabidopsis plant with defective glycolate-P phosphatase, isolated by Chris Somerville, definitively established the role of O₂ and Rubisco in providing photorespiratory glycolate. Selection techniques to isolate photorespiration-deficient plants were devised by Jack Widholm and by Somerville, but no plants with reduced photorespiration were found. Somerville's approach, directed mutagenesis of Arabidopsis plants, was subsequently successful in the isolation of numerous other classes of mutants and revolutionized the science of plant biology. This revised version was published online in August 2006 with corrections to the Cover Date.     

Oxygen Requirement and Inhibition of C4 Photosynthesis : An Analysis of C4 Plants Deficient in the C3 and C4 Cycles

The basis for O₂ sensitivity of C₄ photosynthesis was evaluated using a C₄-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C₃-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C₄-cycle-limited mutant showed that atmospheric levels of O₂ (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O₂ partial pressure for photosynthesis was reduced from approximately 5 kPa O₂ to 1 to 2 kPa O₂, becoming similar to that of C₃ plants. Therefore, the higher O₂ requirement for optimal C₄ photosynthesis is specifically associated with the C₄ function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O₂ than in the wild type. When CO₂ fixation by Rubisco is limited, an increase in the CO₂ concentration in bundle-sheath cells via the C₄ cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O₂ while increasing CO₂ leakage and overcycling of the C₄ pathway. These results indicate that in C₄ plants the investment in the C₃ and C₄ cycles must be balanced for maximum efficiency.     

Photosynthetic Plasticity in Flaveria brownii: Growth Irradiance and the Expression of C(4) Photosynthesis

Photosynthesis was examined in leaves of Flaveria brownii A. M. Powell, grown under either 14% or 100% full sunlight. In leaves of high light grown plants, the CO₂ compensation point and the inhibition of photosynthesis by 21% O₂ were significantly lower, while activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and various C₄ cycle enzymes were considerably higher than those in leaves grown in low light. Both the CO₂ compensation point and the degree of O₂ inhibition of apparent photosynthesis were relatively insensitive to the light intensity used during measurements with plants from either growth conditions. Partitioning of atmospheric CO₂ between Rubisco of the C₃ pathway and phosphoenolpyruvate carboxylase of the C₄ cycle was determined by exposing leaves to ¹⁴CO₂ for 3 to 16 seconds, and extrapolating the labeling curves of initial products to zero time. Results indicated that ~94% of the CO₂ was fixed by the C₄ cycle in high light grown plants, versus ~78% in low light grown plants. Thus, growth of F. brownii in high light increased the expressed level of C₄ photosynthesis. Consistent with the carbon partitioning patterns, photosynthetic enzyme activities (on a chlorophyll basis) in protoplasts from leaves of high light grown plants showed a more C₄-like pattern of compartmentation. Pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase were more enriched in the mesophyll cells, while NADP-malic enzyme and ribulose 1,5-bisphosphate carboxylase/oxygenase were relatively more abundant in the bundle sheath cells of high light than of low light grown plants. Thus, these results indicate that F. brownii has plasticity in its utilization of different pathways of carbon assimilation, depending on the light conditions during growth.     

High CO(2) Requiring Mutant of Anacystis nidulans R(2)

Some physiological characteristics of a mutant (E₁) of Anacystis nidulans R₂, incapable of growing at air level of CO₂, are described. E₁ is capable of accumulating inorganic carbon (C_i) internally as efficiently as the wild type (R₂). The apparent photosynthetic affinity for C_i in E₁, however, is some 1000 times lower than that of R₂. The kinetic parameters of ribulose 1,5-bisphosphate carboxylase/oxygenase from E₁ are similar to those observed in R₂. The mutant appears to be defective in its ability to utilize the intracellular C_i pool for photosynthesis and depends on extracellular supply of Ci in the form of CO₂. The very high apparent photosynthetic K_m (CO₂) of the mutant indicate a large diffusion resistance for CO₂. Data obtained here are used to calculate the permeability coefficient for CO₂ between the bulk medium and the carboxylation site of cyanobacteria.     

Temperature-conditional nuclear mutation of Chlamydomonas reinhardtii decreases the CO2/O2 specificity of chloroplast ribulosebisphosphate carboxylase/oxygenase

The Chlamydomonas reinhardtii (Dangeard) temperature-conditional mutant 68-11AR is phenotypically indistinguishable from the wild type at the permissive temperature (25°C), but has greatly reduced photosynthetic ability and requires acetate for growth at the restrictive temperature (35°C). The mutant strain is deficient in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) holoenzyme when grown at 35°C. This decrease in the level of enzyme appears to be due to degradation of assembled holoenzyme rather than to a reduction in the synthesis of enzyme subunits. When grown at 25°C, the mutant has a substantial amount of Rubisco. Enzyme purified from 25°C-grown mutant cells was found to have a 16% decrease in the CO₂/O₂ specificity factor when compared to the wild-type enzyme. This alteration was accompanied by changes in the kinetic constants for both carboxylation and oxygenation. Although the Rubisco active site is located on the chloroplast-encoded large subunit, genetic analysis showed that the 68-11AR strain arose from a nucleargene mutation. The two nuclear genes that encode the Rubisco small subunits (rbcS1 and rbcS2) were cloned from mutant 68-11AR and completely sequenced, but no mutation was found. Analysis of restriction-fragment length polymorphisms also failed to detect linkage between mutant and rbcS gene loci. These results indicate that nuclear genes can influence Rubisco catalysis without necessarily encoding polypeptides that reside within the holoenzyme.Abbreviations and Symbols K _c Michaelis constant for CO₂ - K _o Michaelis constant for O₂ - mt mating type - pf paralyzed flagella - RFLP restriction-fragment length polymorphism - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V _c V _max for carboxylation - V _o V _max for oxygenation - CO₂/O₂ specificity factor C. G. gratefully acknowledges fellowship support from the Consejo Superior de Investigaciones Cientificas (Spain). This work was supported by National Science Foundation grant MCB-9005547, and is published as Paper No. 10481, Journal Series, Nebraska Agricultural Research Division.     

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO₂ requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO₂ concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O₂. In contrast to a CO₂ compensation concentration of near zero in wild type at all O₂ concentrations examined, the mutant exhibited a high, O₂-stimulated CO₂ compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of ¹⁴CO₂. At air levels of CO₂ and O₂, the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO₂ concentration. At ambient CO₂, wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO₂ limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO₂ concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO₂, creating a high internal CO₂ concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO₂ concentration.     

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits: ENGINEERED rbcS cDNA FOR EXPRESSION IN CHLAMYDOMONAS*?

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO₂ fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO₂/O₂ specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO₂/O₂ specificity but a lower carboxylation V_max than Chlamydomonas Rubisco, the hybrid enzymes have 3–11% increases in CO₂/O₂ specificity and retain near normal V_max values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO₂ is concentrated for optimal photosynthesis.     

Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase

Kinetic properties of soybean net photosynthetic CO₂ fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO₂ concentration, and O₂ concentration. With leaves, O₂ inhibition of net photosynthetic CO₂ fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO₂ fixation at higher temperatures was caused by a reduced affinity of the leaf for CO₂ and an increased affinity of the leaf for O₂. With purified ribulose 1,5-diphosphate carboxylase, O₂ inhibition of CO₂ incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O₂ sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO₂ and a slightly increased affinity of the enzyme for O₂. The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO₂ and O₂ provides further evidence that the carboxylase regulates the O₂ response of photosynthetic CO₂ fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO₂ and O₂ fixation in vivo is proposed.     

Peroxisomal malate dehydrogenase is not essential for photorespiration in Arabidopsis but its absence causes an increase in the stoichiometry of photorespiratory CO2 release

Peroxisomes are important for recycling carbon and nitrogen that would otherwise be lost during photorespiration. The reduction of hydroxypyruvate to glycerate catalyzed by hydroxypyruvate reductase (HPR) in the peroxisomes is thought to be facilitated by the production of NADH by peroxisomal malate dehydrogenase (PMDH). PMDH, which is encoded by two genes in Arabidopsis (Arabidopsis thaliana), reduces NAD⁺ to NADH via the oxidation of malate supplied from the cytoplasm to oxaloacetate. A double mutant lacking the expression of both PMDH genes was viable in air and had rates of photosynthesis only slightly lower than in the wild type. This is in contrast to other photorespiratory mutants, which have severely reduced rates of photosynthesis and require high CO₂ to grow. The pmdh mutant had a higher O₂-dependent CO₂ compensation point than the wild type, implying that either Rubisco specificity had changed or that the rate of CO₂ released per Rubisco oxygenation was increased in the pmdh plants. Rates of gross O₂ evolution and uptake were similar in the pmdh and wild-type plants, indicating that chloroplast linear electron transport and photorespiratory O₂ uptake were similar between genotypes. The CO₂ postillumination burst and the rate of CO₂ released during photorespiration were both greater in the pmdh mutant compared with the wild type, suggesting that the ratio of photorespiratory CO₂ release to Rubisco oxygenation was altered in the pmdh mutant. Without PMDH in the peroxisome, the CO₂ released per Rubisco oxygenation reaction can be increased by over 50%. In summary, PMDH is essential for maintaining optimal rates of photorespiration in air; however, in its absence, significant rates of photorespiration are still possible, indicating that there are additional mechanisms for supplying reductant to the peroxisomal HPR reaction or that the HPR reaction is altogether circumvented.     

Inhibition of inorganic carbon transport by oxygen in a high CO2-requiring mutant (E1) of Anacystis nidulans R2

The effect of O₂ on inorganic carbon (C_i) transport was studied with a high CO₂-requiring mutant (E₁) of Anacystis nidulans R₂. Oxygen (above 2%) inhibited C_i transport by 15–35|X% at CO₂ concentrations above 200 μl/l, but had no apparent effect at low, limiting CO₂ concentration. The action spectra for C_i transport measured in the presence or absence of 20% O₂ showed two peaks around 684 and 625 nm, corresponding to chlorophyll a and phycocyanin absorption, respectively. The difference between these two spectra (anaerobic minus aerobic) showed one peak around 625 nm, which indicates that a linear electron transport from water to O₂ is involved in the O₂ inhibition of C_i transport. Dithiothreitol could overcome the inhibition by O₂. The results suggested that the O₂ inhibition is a result of inactivation of the C_i-transporting system.     

A Functional Calvin Cycle Is Not Indispensable for the Light Activation of C4 Phosphoenolpyruvate Carboxylase Kinase and Its Target Enzyme in the Maize Mutant bundle sheath defective2-mutable1

We used a pale-green maize (Zea mays L.) mutant that fails to accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to test the working hypothesis that the regulatory phosphorylation of C₄ phosphoenolpyruvate carboxylase (PEPC) by its Ca²⁺-insensitive protein-serine/threonine kinase (PEPC kinase) in the C₄ mesophyll cytosol depends on cross-talk with a functional Calvin cycle in the bundle sheath. Wild-type (W22) and bundle sheath defective2-mutable1 (bsd2-m1) seeds were grown in a controlled environment chamber at 100 to 130 μmol m⁻² s⁻¹ photosynthetic photon flux density, and leaf tissue was harvested 11 d after sowing, following exposure to various light intensities. Immunoblot analysis showed no major difference in the amount of polypeptide present for several mesophyll- and bundle-sheath-specific photosynthetic enzymes apart from Rubisco, which was either completely absent or very much reduced in the mutant. Similarly, leaf net CO₂-exchange analysis and in vitro radiometric Rubisco assays showed that no appreciable carbon fixation was occurring in the mutant. In contrast, the sensitivity of PEPC to malate inhibition in bsd2-m1 leaves decreased significantly with an increase in light intensity, and there was a concomitant increase in PEPC kinase activity, similar to that seen in wild-type leaf tissue. Thus, although bsd2-m1 mutant plants lack an operative Calvin cycle, light activation of PEPC kinase and its target enzyme are not grossly perturbed.     

A Mutant of Arabidopsis thaliana Which Lacks Activation of RuBP Carboxylase In Vivo

A mutant of Arabidopsis thaliana has been isolated in which ribulose-1,5-bisphosphate carboxylase is present in a nonactivatable form in vivo. The mutation appears to affect carboxylase activation specifically, and not any other enzyme of the photosynthesis or photorespiratory cycles. The effect of the mutation on carboxylase activation is indirect, inasmuch as the properties of ribulose-1,5-bisphosphate carboxylase purified from the mutant are not distinguishable from those of the wild type enzyme. The mutant requires high levels of atmospheric CO₂ for growth because photosynthesis is severely impaired in atmospheres containing normal levels of CO₂, irrespective of the atmospheric O₂ concentration. In this respect, the mutant is distinguished from previously described high-CO₂ requiring mutants of Arabidopsis which have defects in photorespiratory carbon or nitrogen metabolism.     

Rubisco Evolution in C4 Eudicots: An Analysis of Amaranthaceae Sensu Lato

Background

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses the key reaction in the photosynthetic assimilation of CO₂. In C₄ plants CO₂ is supplied to Rubisco by an auxiliary CO₂-concentrating pathway that helps to maximize the carboxylase activity of the enzyme while suppressing its oxygenase activity. As a consequence, C₄ Rubisco exhibits a higher maximum velocity but lower substrate specificity compared with the C₃ enzyme. Specific amino-acids in Rubisco are associated with C₄ photosynthesis in monocots, but it is not known whether selection has acted on Rubisco in a similar way in eudicots.

Methodology/Principal Findings

We investigated Rubisco evolution in Amaranthaceae sensu lato (including Chenopodiaceae), the third-largest family of C₄ plants, using phylogeny-based maximum likelihood and Bayesian methods to detect Darwinian selection on the chloroplast rbcL gene in a sample of 179 species. Two Rubisco residues, 281 and 309, were found to be under positive selection in C₄ Amaranthaceae with multiple parallel replacements of alanine by serine at position 281 and methionine by isoleucine at position 309. Remarkably, both amino-acids have been detected in other C₄ plant groups, such as C₄ monocots, illustrating a striking parallelism in molecular evolution.

Conclusions/Significance

Our findings illustrate how simple genetic changes can contribute to the evolution of photosynthesis and strengthen the hypothesis that parallel amino-acid replacements are associated with adaptive changes in 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     

Acclimation of photosynthesis to increasing atmospheric CO2: The gas exchange perspective

The nature of photosynthetic acclimation to elevated CO₂ is evaluated from the results of over 40 studies focusing on the effect of long-term CO₂ enrichment on the short-term response of photosynthesis to intercellular CO₂ (the A/C_i response). The effect of CO₂ enrichment on the A/C_i response was dependent on growth conditions, with plants grown in small pots (< 5 L) or low nutrients usually exhibiting a reduction of A at a given C_i, while plants grown without nutrient deficiency in large pots or in the field tended to exhibit either little reduction or an enhancement of A at a given C_i following a doubling or tripling of atmospheric CO₂ during growth. Using theoretical interpretations of A/C_i curves to assess acclimation, it was found that when pot size or nutrient deficiency was not a factor, changes in the shape of A/C_i curves which are indicative of a reallocation of resources within the photosynthetic apparatus typically were not observed. Long-term CO₂ enrichment usually had little effect or increased the value of A at all C_i. However, a minority of species grown at elevated CO₂ exhibited gas exchange responses indicative of a reduced amount of Rubisco and an enhanced capacity to metabolize photosynthetic products. This type of response was considered beneficial because it enhanced both photosynthetic capacity at high CO₂ and reduced resource investment in excessive Rubisco capacity. The ratio of intercellular to ambient CO₂ (the C_i/C_a ratio) was used to evaluate stomatal acclimation. Except under water and humidity stress, C_i/C_a exhibited no consistent change in a variety of C₃ species, indicating no stomatal acclimation. Under drought or humidity stress, C_i/C_a declined in high-CO₂ grown plants, indicating stomata will become more conservative during stress episodes in future high CO₂ environments.Abbreviations A net CO₂ assimilation rate - C_i (C_a) intercellular (ambient) partial pressure of CO₂ - operational C_i intercellular partial pressure of CO₂ at a given ambient partial pressure of CO₂ - g_s stomatal conductance - normal CO₂ current atmospheric mole fraction of CO₂ (330 to 355 mol mol^–1) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Effect of inorganic carbon supply on the photosynthetic physiology of Gracilaria tenuistipitata

Gracilaria tenuistipitata Zhang et Xia was cultured for 15 d at low, normal and high inorganic carbon concentrations under constant light, temperature and nutrient conditons. Carbonic anhydrase (CA; EC 4.2.1.1.) activity, ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco; EC 4.1.1.39) content, pigment content and C/N ratio were measured, and also the photosynthesis and growth rates. Both Rubisco content and CA activity increased under conditions of low inorganic carbon (C_i) but decreased at high C_i with respect to the control. The amount of pigments declined considerably at high C_i and was slightly higher at low C_i. The maximum rate of photosynthesis and the photosynthetic efficiency increased in low C_i and the opposite was found at high C_i concentration. The effects of C_i concentration on maximum rate of photosynthesis and photosynthetic efficiency are discussed in relation to the variation in pigment and Rubisco contents and CA activity. The data indicate that C_i may be an important factor controlling the photosynthetic physiology of G. tenuistipitata with regard, not only to the enzymes of C_i metabolism, but also to the pigment content.Abbreviations APS_max maximum apparent photosynthetic rate - CA carbonic anhydrase - Chl chlorophyll - C_i inorganic carbon - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work has been supported by grants No. PB91-0962 and No. MAR90-0365 from Spanish Direction for Science and Technology (DIGICYT). M.J. G-S holds a fellowship from the DIGICYT.     

Is There a Role for the 42 Kilodalton Polypeptide in Inorganic Carbon Uptake by Cyanobacteria?

Cyanobacterial cells accumulate substantial amounts of a membrane-associated 42 kilodalton polypeptide during adaptation to low CO₂ conditions. The role of this polypeptide in the process of adaptation and in particular in the large increase in the ability to accumulate inorganic carbon (C_i), which accompanies this process, is not yet understood. We have isolated a mutant Synechococcus PCC7942 that does not accumulate the 42 kilodalton polypeptide. The mutant requires a high-CO₂ concentration for growth and exhibits a very low apparent photosynthetic affinity for extracellular C_i. The latter might be attributable to the observed defective ability of the mutant to utilize the intracellular C_i pool for photosynthesis. The 42 kilodalton polypeptide does not appear to participate directly in the active transport of C_i, since the difference between the observed capabilities for CO₂ and HCO₃⁻ uptake of the mutant and the wild type is not sufficient to account for their different growth and photosynthetic performance. Furthermore, high CO₂-grown wild-type cells, where we could not detect the 42 kilodalton polypeptide, transported CO₂ faster than the mutant. An analysis of the curves relating the rate of accumulation of C_i to the concentration of CO₂ or HCO₃⁻ supplied, in the presence or absence of carbonic anhydrase, indicated that under the experimental conditions used here, CO₂ was the preferred C_i species taken up by Synechococcus.     

An Excel tool for deriving key photosynthetic parameters from combined gas exchange and chlorophyll fluorescence: theory and practice

Combined photosynthetic gas exchange and modulated fluorometres are widely used to evaluate physiological characteristics associated with phenotypic and genotypic variation, whether in response to genetic manipulation or resource limitation in natural vegetation or crops. After describing relatively simple experimental procedures, we present the theoretical background to the derivation of photosynthetic parameters, and provide a freely available Excel‐based fitting tool (EFT) that will be of use to specialists and non‐specialists alike. We use data acquired in concurrent variable fluorescence–gas exchange experiments, where A/C_i and light–response curves have been measured under ambient and low oxygen. From these data, the EFT derives light respiration, initial PSII (photosystem II) photochemical yield, initial quantum yield for CO₂ fixation, fraction of incident light harvested by PSII, initial quantum yield for electron transport, electron transport rate, rate of photorespiration, stomatal limitation, Rubisco (ribulose 1·5‐bisphosphate carboxylase/oxygenase) rate of carboxylation and oxygenation, Rubisco specificity factor, mesophyll conductance to CO₂ diffusion, light and CO₂ compensation point, Rubisco apparent Michaelis–Menten constant, and Rubisco CO₂‐saturated carboxylation rate. As an example, a complete analysis of gas exchange data on tobacco plants is provided. We also discuss potential measurement problems and pitfalls, and suggest how such empirical data could subsequently be used to parameterize predictive photosynthetic models.     

Effects of O(2) and CO(2) Concentration on the Steady-State Fluorescence Yield of Single Guard Cell Pairs in Intact Leaf Discs of Tradescantia albiflora: Evidence for Rubisco-Mediated CO(2) Fixation and Photorespiration in Guard Cells

A procedure for following changes in the steady-state yield of chlorophyll a fluorescence (F_s) from single guard cell pairs in variegated leaves of Tradescantia albiflora is described. As an indicator of photosynthetic electron transport, F_s is a very sensitive indirect measure of the balance of adenosine 5′-triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), producing reactions with the sink reactions that utilize those light-generated products. We found that F_s under constant light is sensitive to manipulation of ambient CO₂ concentrations, as would be expected if either phosphoenolpyruvate carboxylase or ribulose-1, 5 bisphosphate carboxylase/oxygenase (Rubisco)-dependent CO₂ fixation is the sink for photosynthetic ATP and NADPH in guard cells. However, we also found that changing O₂ concentration had a strong effect on fluorescence yield, and that O₂ sensitivity was only evident when the concentration of CO₂ was low. This finding provides evidence that both O₂ and CO₂ can serve as sinks for ATP and NADPH produced by photosynthetic electron transport in guard cell chloroplasts. Identical responses were observed with mesophyll cell chloroplasts in intact leaves. This finding is difficult to reconcile with the view that guard cell chloroplasts have fundamentally different pathways of photosynthetic metabolism from other chloroplasts in C₃ plants. Indeed, Rubisco has been detected at low levels in guard cell chloroplasts, and our studies indicate that it is active in the pathways for photosynthetic carbon reduction and photorespiration in guard cells.     

Simple Determination of the CO(2)/O(2) Specificity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by the Specific Radioactivity of [C]Glycerate 3-Phosphate

A new method is presented for measurement of the CO₂/O₂ specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [¹⁴C]3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. ¹⁴CO₂ fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO₂ in O₂-saturated water and carboxylase only with 160 micromolar CO₂ under N₂. Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the [¹⁴C]PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 ± 4), from the green alga Chlamydomonas reinhardtii (66 ± 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods.