Reversible light-activation of ribulose bisphosphate carboxylase/oxygenase in isolated barley protoplasts and chloroplasts

The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase displayed near-maximal activity in isolated, intact barley (Hordeum vulgare L. cv. Pennrad) mesophyll protoplasts. The carboxylase deactivated 40 to 50% in situ when protoplasts were dark-incubated 20 minutes in air-equilibrated solutions. Enzyme activity was fully restored after 1 to 2 minutes of light. Addition of 5 millimolar NaHCO₃ to the incubation medium prevented dark-inactivation of the carboxylase. There was no permanent CO₂-dependent activation of the protoplast carboxylase either in light or dark. Activation of the carboxylase from ruptured protoplasts was not increased significantly by in vitro preincubation with CO₂ and Mg²⁺. In contrast to the enzyme in protoplasts, the carboxylase in intact barley chloroplasts was not fully reactivated by light at atmospheric CO₂ levels. The lag phase in carbon assimilation was not lengthened by dark-adapting protoplasts to low CO₂ demonstrating that light-activation of the carboxylase was not involved in photosynthetic induction. Irradiance response curves for reactivation of the the carboxylase and for CO₂ fixation by isolated barley protoplasts were similar. The above results show that there was a fully reversible light-activation of the carboxylase in isolated barley protoplasts at physiologically significant CO₂ levels.     

Glyoxylate and glutamate effects on photosynthetic carbon metabolism in isolated chloroplasts and mesophyll cells of spinach

Addition of millimolar sodium glyoxylate to spinach (Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of ¹⁴CO₂ under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO₂ concentrations. Incorporation of ¹⁴C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [¹⁴C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO₂ uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased ¹⁴C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased ¹⁴CO₂ labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net ¹⁴CO₂ fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase ¹⁴CO₂ incorporation by 15% in isolated mesophyll cells under air levels of CO₂.     

Steady-state photosynthesis in alfalfa leaflets: effects of carbon dioxide concentration

When the CO₂ concentration to which Medicago sativa L. var. El Unico leaflets were exposed was increased from half-saturation to saturation (doubled rate of photosynthesis), glycolate and glycine production apparently decreased due to inhibition of a portion of the glycolate pathway. Serine and glycerate production was not inhibited. We conclude that serine and glycerate were made from 3-phosphoglycerate and not from glycolate and that the conversion of glycine to serine may not be the major source of photorespiratory CO₂ in alfalfa. In investigations of glycolate and photorespiratory metabolism, separate labeling data should be obtained for glycine and serine as those two amino acids may be produced from different precursors and respond differently to environmental perturbations. The increased photosynthetic rate (at saturating CO₂) resulted in greater labeling of both soluble and insoluble products. Sucrose labeling increased sharply, but there was no major shift of tracer carbon flow into sucrose relative to other metabolites. The flow of carbon from the reductive pentose phosphate cycle into the production of tricarboxylic acid cycle intermediates and amino acids increased. Only small absolute increases occurred in steady-state pool sizes of metabolites of the reductive pentose phosphate cycle at elevated CO₂, providing further evidence that the cycle is well regulated.     

Inactivation of the photosynthetic carbon reduction cycle in isolated mesophyll protoplasts subjected to freezing stress

Isolated mesophyll protoplasts from Valerianella locusta L. were subjected to freeze-thaw cycles. Subsequently, steady-state pool sizes of ¹⁴C-labeled intermediates of the photosynthetic carbon reduction cycle were determined by high performance liquid chromatography. Protoplasts in which CO₂ fixation was inhibited by preceding freezing stress, showed a strong increase in the proportion of fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate and triose phosphates. These results indicate an inhibition of the activities of stromal fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase. Furthermore, freezing stress caused a slight increase in the proportion of labeled ribulose-1,5-bisphosphate, which may be based on an inhibition or ribulose bisphosphate carboxylase activity. It was shown earlier (Rumich-Bayer and Krause 1986) that freezing-thawing readily affects photosynthetic CO₂ assimilation independently of thylakoid inactivation. The present results are interpreted in terms of an inhibition of the light-activation system of the photosynthetic carbon reduction cycle, caused by freezing stress.Abbreviations FBP Fructose-1,6-bisphosphate - HMP Hexose Monophosphates - PGA 3-phosphoglycerate - PMP Pentose Monophosphates - RBP Ribulose-1,5-bisphosphate - SBP Sedoheptulose-1,7-bisphosphate - TP Triose Phosphates     

Mechanism of C4 photosynthesis in Chloris gayana: pool sizes and kinetics of 14CO2 incorporation into 4-carbon and 3-carbon intermediates.

In one group of C₄ species, including Chloris gayana, C₄ acids are decarboxylated via phosphoenolpyruvate carboxykinase to give phosphoenolpyruvate as the initial C₃ product. This paper presents an analysis of the kinetics of labeling of various photosynthetic intermediates in Chloris gayana leaves exposed to ¹⁴CO₂, and the pool sizes of these intermediates, primarily to provide information about the subsequent metabolism of phosphoenolpyruvate. Saturation labeling of the C-4 of aspartate and malate, and the C-1 of 3-phosphoglycerate, indicated photosynthetically active pools of 0.45, 0.22, and 0.95 μol/mg chlorophyll, respectively. For aspartate and 3-phosphoglycerate, the total leaf pools and the photosynthetic pools were of similar size, but the total pool of malate was about 100 times larger than the photosynthetically active pool. From the relative rates of labeling of phosphoenolpyruvate, pyruvate, alanine, and C-1, C-2 plus C-3 of aspartate, during steady-state ¹⁴CO₂ assimilation, relative pool sizes were calculated to be about 10:11:78:100, respectively. Pulse/chase labeling of leaves provided estimates of relative photosynthetic pool sizes in the ratio of about 6:15:90:100, respectively, where aspartate is arbitrarily assigned a value of 100 in both cases. Notably, labeling of alanine was consistent with its derivation from the C-1, C-2 plus C-3 carbons of aspartate, and the alanine pool was at least eight times larger than the phosphoenolpyruvate pool that showed similar labeling kinetics. Results were consistent with the view that at least most of the phosphoenolpyruvate produced by C₄ acid decarboxylation is metabolized via alanine.     

Phosphoserine and phosphohydroxypyruvic Acid: evidence for their role as early intermediates in photosynthesis

Photosynthetic fixation of ¹⁴CO₂ in the bean Phaseolus vulgaris, cv. Pencil Pod Black Wax, resulted in the appearance of labeled compounds that were characterized as phosphoserine and phosphohydroxypyruvate by chromatographic separation and by the synthesis of chemical derivatives. In ¹⁴CO₂/¹²CO₂ pulse-chase experiments these metabolites demonstrated the rapid pool saturation and depletion of ¹⁴C characteristic of early intermediates in photosynthetic carbon fixation. They were present in sufficient amounts to account for about 35% of total carbon fixed in 1 minute.     

Compartmental modelling of photorespiration and carbon metabolism of water stressed leaves

Abstract Carbon fluxes in photosynthesis and photorespiration of water stressed leaves have been analysed in a steady state model based on the ribulose diphosphate carboxylase (RuDP carboxylase) and RuDP oxygenase enzyme activities and the CO₂ and O₂ concentrations in the leaf. Agreement between predicted and observed photorespiration (Lawlor & Fock, 1975) and C flux in the glycollate pathway is good over much of the range of water stress, but not at severe stress. An alternative source of respiratory CO₂ is suggested to explain the discrepancy. The model suggests that resistance to CO₂ fixation is mainly in the carboxylation reactions, not in CO₂ transport. Using the steady state model, the kinetics of ¹⁴C incorporation into photosynthetic and photorespiratory intermediates are simulated. The predicted rate of ¹⁴C incorporation is faster than observed and delay terms in the model are used to simulate the slow rates of mixing and metabolic reactions. Inactive pools of glycine and serine are suggested to explain the observed specific activities of glycine and serine. Three models of carbon flux between the glycollate pathway, the photosynthetic carbon reduction cycle and sucrose synthesis are considered. The most satisfactory simulation is for glycollate pathway carbon feeding into the PCR cycle pool of 3-phosphoglyceric acid which provides the carbon for sucrose synthesis. Simulation of the specific activity of CO₂ released in photorespiration suggests that a source of unlabelled carbon may contribute to photorespiration.     

13CO2 labeling kinetics in maize reveal impaired efficiency of C4 photosynthesis under low irradiance

C₄ photosynthesis allows faster photosynthetic rates and higher water and nitrogen use efficiency than C₃ photosynthesis, but at the cost of lower quantum yield due to the energy requirement of its biochemical carbon concentration mechanism. It has also been suspected that its operation may be impaired in low irradiance. To investigate fluxes under moderate and low irradiance, maize (Zea mays) was grown at 550 µmol photons m⁻² s^−l and ¹³CO₂ pulse-labeling was performed at growth irradiance or several hours after transfer to 160 µmol photons m⁻² s⁻¹. Analysis by liquid chromatography/tandem mass spectrometry or gas chromatography/mass spectrometry provided information about pool size and labeling kinetics for 32 metabolites and allowed estimation of flux at many steps in C₄ photosynthesis. The results highlighted several sources of inefficiency in low light. These included excess flux at phosphoenolpyruvate carboxylase, restriction of decarboxylation by NADP-malic enzyme, and a shift to increased CO₂ incorporation into aspartate, less effective use of metabolite pools to drive intercellular shuttles, and higher relative and absolute rates of photorespiration. The latter provides evidence for a lower bundle sheath CO₂ concentration in low irradiance, implying that operation of the CO₂ concentration mechanism is impaired in this condition. The analyses also revealed rapid exchange of carbon between the Calvin–Benson cycle and the CO₂-concentration shuttle, which allows rapid adjustment of the balance between CO₂ concentration and assimilation, and accumulation of large amounts of photorespiratory intermediates in low light that provides a major carbon reservoir to build up C₄ metabolite pools when irradiance increases.

Analysis of metabolite pools, sizes, and fluxes reveals that multiple interlocking factors decrease the efficiency of photosynthesis in low irradiance in maize.     

Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells

The effects of added glycine hydroxamate on the photosynthetic incorporation of ¹⁴CO₂ into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO₂ and 20% O₂) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO₂ and 2.7% O₂). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO₂ and NH₄⁺. During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the “reverse” flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.     

Mechanism of c(4) photosynthesis: the size and composition of the inorganic carbon pool in bundle sheath cells

We sought to characterize the inorganic carbon pool (CO₂ plus HCO₃⁻) formed in the leaves of C₄ plants when C₄ acids derived from CO₂ assimilation in mesophyll cells are decarboxylated in bundle sheath cells. The size and kinetics of labeling of this pool was determined in six species representative of the three metabolic subgroups of C₄ plants. The kinetics of labeling of the inorganic carbon pool of leaves photosynthesizing under steady state conditions in ¹⁴CO₂ closely paralleled those for the C-4 carboxyl of C₄ acids for all species tested. The inorganic carbon pool size, determined from its ¹⁴C content at radioactivity saturation, ranged between 15 and 97 nanomoles per milligram of leaf chlorophyll, giving estimated concentrations in bundle sheath cells of between 160 and 990 micromolar. The size of the pool decreased, together with photosynthesis, as light was reduced from 900 to 95 microeinsteins per square meter per second or as external CO₂ was reduced from 400 to 98 microliters per liter. A model is developed which suggests that the inorganic carbon pool existing in the bundle sheath cells of C₄ plants during steady state photosynthesis will comprise largely of CO₂; that is, CO₂ will only partially equlibrate with bicarbonate. This predominance of CO₂ is believed to be vital for the proper functioning of the C₄ pathway.     

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO₂ assimilation was determined by CO₂ exchange studies at three, limiting CO₂ concentrations with a ca-1 mutant of Chlamydomonas reinhardiii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO₂ assimilation did not respond to the varying photon flux densities because of CO₂ limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100 mol photons m^-2 s^-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO₂ assimilation responded to external CO₂ concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO₂ into the cells supplies most of the CO₂ for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO₂ concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO₂ exchange method to determine whether inorganic carbon accumulation and photosynthetic CO₂ assimilation compete for energy at low photon flux densities proved inconclusive.     

Mapping the carbon reduction cycle: a personal retrospective

The photosynthetic carbon reduction cycle was elucidated through the use of ¹⁴CO₂ during photosynthesis to label metabolic intermediates. Mapping and proof of the cycle required identification of labeled metabolites, observation of changes in levels of labeled metabolites during transitions from light to dark and from high to low CO₂ levels, determination of intramolecular distribution of ¹⁴C within the metabolites after a few seconds of photosynthesis with ¹⁴CO₂, and estimation of metabolite concentrations, used to calculate true free energy changes at each step in the cycle. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biochemical components of the photosynthetic CO2 compensation point of higher plants.

Barley, Panicum milioides and Panicum maximum were exposed to ¹⁴CO₂ near their photosynthetic CO₂ compensation points and their respective ¹⁴C-products were determined. In short exposure times Panicum maximum had 100% of its ¹⁴C in malate and aspartate whereas Panicum milioides and barley had 16 and 3% of their respective ¹⁴C in C₄ organic acids. Near the respective CO₂ compensation points a linear relationship occurs in plotting the ratio of glycine, serine, and glycerate to C₄ organic acids. The ratio of ribulose 1,5-bisphosphate oxygenase to phosphoenolpyruvate carboxylase is linear with their CO₂ compensation points. The photosynthetic CO₂ compensation point apparently is controlled by the activity of enzymes producing photorespiration metabolites and the activity of phospheonolpyruvate carboxylase.     

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L

A mass spectrometric method combining ¹⁶O/¹⁸O and ¹²C/¹³C isotopes was used to quantify the unidirectional fluxes of O₂ and CO₂ during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O₂ uptake and CO₂ evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O₂ (61 micromoles of O₂ per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O₂ per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO₂ in darkness at a rate of 27 micromoles of CO₂ per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O₂ evolution and CO₂ fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO₂ evolution by guard cell protoplasts was sharply decreased (37%), while O₂ uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO₂ assimilation and may be dissipated for other purposes such as ion uptake.     

Diurnal changes in allocation and partitioning of recently assimilated carbon in loblolly pine seedlings

We examined diurnal fluctuations in acquisition and partitioning of recently assimilated ¹⁴CO₂, and in subsequent allocation and partitioning to roots of loblolly pine (Pinus taeda L.) seedlings. Nonmycorrhizal seedlings were grown under optimal nutrient conditions in continuously flowin solution culture. Shoots of 15-week-old loblolly pine seedlings were labeled with ¹⁴CO₂ for 30 min at four separate labeling times: 1000, 1200, 1400 and 1600 h. Six whole plant harvests were conducted during a 48 h chase period, i.e. 0, 4, 8 12, 24 and 48 h after the end of the labeling and evacuation periods. Although assimilation of ¹⁴CO₂ was constant between 1000 and 1400 h, there were significant differences in partitioning of ¹⁴C-labeled assimilate in needles of all age classes. The highest percentage of recently assimilated ¹⁴CO₂ in the ethanol-soluble fraction of photosynthesizing tissue was observed near the beginning and end of the photoperiod. Partitioning of ¹⁴C in the ethanol-soluble fraction declined between the 1000 and 1400 h labeling eriods, and was accompanied by an increase in partitioning of recently assimilated ¹⁴CO₂ toward starch and a decrease in respiratory losses. These data suggest that most of the ¹⁴CO₂ assimilated at 1000 h was used to support shoot metabolic activities and possibly restore soluble sugar reserves. Peak starch accumulation in needles during the 1400 h labeling period, concomitant with minimal respiratory loss, indicated that photosynthate production exceeded demand and export out of source leaves. A possible feedback regulation of photosynthesis by starch and/or sugar accumulation may be responsible for the observed decline in assimilation of ¹⁴CO₂ during the 1600 h labeling period. Net accumulation of recently assimilated ¹⁴CO₂ in roots was correlated with assimilation rate of ¹⁴CO₂, but independent of partitioning of recently assimilated carbon in photosynthetic tissue. However, the percentage of total seedling ¹⁴C allocated to roots was essentially the same throughout the 48 h chase, regardless of time of labeling and assimilation rate. The data suggest a strong diurnal regulation of starch and soluble sugars synthesized from recently assimilated carbon in needles of loblolly pine seedlings that was independent of assimilation rate. Allocation and transport of recently assimilated carbon to roots of loblolly pine seedlings were not subject to short-term fluctuations in supply and demand.     

Detection of a variable intracellular acid‐labile carbon pool in Thalassiosira weissflogii (Heterokontophyta) and Emiliania huxleyi (Haptophyta) in response to changes in the seawater carbon system

Accumulation of an intracellular pool of carbon (C_i pool) is one strategy by which marine algae overcome the low abundance of dissolved CO₂ (CO_2(aq)) in modern seawater. To identify the environmental conditions under which algae accumulate an acid‐labile C_i pool, we applied a ¹⁴C pulse‐chase method, used originally in dinoflagellates, to two new classes of algae, coccolithophorids and diatoms. This method measures the carbon accumulation inside the cells without altering the medium carbon chemistry or culture cell density. We found that the diatom Thalassiosira weissflogii [(Grunow) G. Fryxell & Hasle] and a calcifying strain of the coccolithophorid Emiliania huxleyi [(Lohmann) W. W. Hay & H. P. Mohler] develop significant acid‐labile C_i pools. Ci pools are measureable in cells cultured in media with 2–30 µmol l^?1 CO_2(aq), corresponding to a medium pH of 8.6–7.9. The absolute C_i pool was greater for the larger celled diatoms. For both algal classes, the C_i pool became a negligible contributor to photosynthesis once CO_2(aq) exceeded 30 µmol l^?1. Combining the ¹⁴C pulse‐chase method and ¹⁴C disequilibrium method enabled us to assess whether E. huxleyi and T. weissflogii exhibited thresholds for foregoing accumulation of DIC or reduced the reliance on bicarbonate uptake with increasing CO_2(aq). We showed that the C_i pool decreases with higher CO₂:HCO₃^? uptake rates.     

Photosynthesis and inorganic carbon transport in isolated asparagus mesophyll cells

The possibility of HCO₃⁻ transport into isolated leaf mesophyll cells of Asparagus sprengeri Regel has been investigated. Measurement of the inorganic carbon pool in these cells over an external pH range 6.2 to 8.0, using the silicone-fluid filtration technique, indicated that the pool was larger than predicted by passive ¹⁴CO₂ distribution, suggesting that HCO₃⁻ as well as CO₂ crosses the plasmalemma. Intracellular pH values, calculated from the distribution of ¹⁴CO₂ between the cells and the medium, were found to be higher (except at pH 8.0) than those previously determined by 5,5-dimethyl[2-¹⁴C]oxazolidine-2,4-dione distribution. It is suggested that the inorganic carbon accumulated above predicted concentrations may be bound to proteins and membranes and thus may not represent inorganic carbon actively accumulated by the cells, inasmuch as in a closed system at constant CO₂ concentration, the photosynthetic rates at pH 7.0 and 8.0 were 5 to 8 times lower than the maximum rate which could be supported by CO₂ arising from the spontaneous dehydration of HCO₃⁻. Furthermore, CO₂ compensation points of the cells in liquid media at 21% O₂ at pH 7.0 and 8.0, and the K_½ CO₂ (CO₂ concentration supporting the half maximal rate of O₂ evolution) at 2% O₂ at pH 7.0 and 8.0 are not consistent with HCO₃⁻ transport. These results indicate that the principal inorganic carbon species crossing the plasmalemma in these cells is CO₂.     

Effect of oxygen on photosynthesis by spinach leaf protoplasts

The photosynthetic CO₂ fixation by spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts was inhibited by substituting an atmosphere of N₂ with one of either air (21% O₂) or 100% O₂. The inhibitory effect of 100% O₂ was greater than that of air. The mode of inhibition by 100% O₂ and air was competitive with respect to CO₂; Ki(O₂) value was 0.32 mM at pH 7 and 0.28 mM at pH 8.5 The labeling patterns of compounds in protoplasts exposed to ¹⁴CO₂ in light after transferring them from N₂ to O₂ atmospheres were examined. There was no detectable ¹⁴CO₂ incorporation into glycolate under anaerobic and O₂ atmospheres; a more marked labeling of glycine occurred under an oxidative environment compared to that under the anaerobic condition, presumably because of a rapid transformation of glycolate to glycine in the protoplasts.     

Photosynthetic and Photorespiratory Carbon Metabolism in Mesophyll Protoplasts and Chloroplasts Isolated from Isogenic Diploid and Tetraploid Cultivars of Ryegrass (Lolium perenne L.)

Photosynthetic ¹⁴CO₂ fixation, [¹⁴C]glycolate formation, and the decarboxylation of [1-¹⁴C]glycolate and [1-¹⁴C]glycine by leaf mesophyll protoplasts isolated from isogenic diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) were examined. The per cent O₂ inhibition of photosynthesis in protoplasts from the tetraploid cultivar was less than that of the diploid line at both 21 and 49% O₂. Kinetic studies revealed that the K_m (CO₂) for photosynthesis by the diploid protoplasts was about twice that of the tetraploid line. In contrast, the K_i (O₂) for protoplast photosynthesis was similar in both cultivars, as was the potential for oxidizing glycolate and glycine to CO₂ via the photorespiratory carbon oxidation cycle. Although the maximal rates of glycolate accumulation by the isolated protoplasts in the presence of 21% O₂ and a glycolate oxidase inhibitor were similar in the two cultivars, the percentage of total fixed ¹⁴C entering the [¹⁴C]glycolate pool and the ratio of the rate of [¹⁴C]glycolate formation to ¹⁴CO₂ fixation at 21% O₂ and low pCO₂ were about two times greater in protoplasts and intact chloroplasts isolated from the diploid line compared to the tetraploid. These results fully support the recent observation that a doubling of ploidy in various ryegrass cultivars reduced the K_m (CO₂) of purified ribulose bisphosphate carboxylase-oxygenase by about one-half without affecting the K_i (O₂) (Garrett 1978 Nature 274: 913-915).