A study of photorespiratory ammonia production in the C4 plant Amaranthus edulis, using mutants with altered photosynthetic capacities

Previous studies have indicated that the rate of photorespiration in C₄ plants is low or negligible. In this study, wild-type and mutant leaves of the C₄ plant Amaranthus edulis were treated with the glutamine synthetase inhibitor, phosphinothricin and the glycine decarboxylase inhibitor, aminoacetonitrile, at different concentrations of CO₂. The time course of ammonia accumulation in leaves of the wild type was compared with a mutant lacking phosphoenolpyruvate carboxylase activity (EC 4.1.1.31), and with three different mutants that accumulated glycine. The increase in the concentration of ammonia in the leaves, stimulated by the treatments was used as a measurement of the rate of photorespiration in C₄ plants. The application of glutamine and glycine maintained the rate of photorespiratory ammonia production for a longer period in the wild type, and increased the rate in a mutant lacking phosphoenolpyruvate carboxylase suggesting that there was a lack of amino donors in these plants. The calculated rate of photorespiration in Amaranthus edulis wild-type leaves when the supply of amino donors was enough to maintain the photorespiratory nitrogen flow, accounted for approximately 6% of the total net photosynthetic CO₂ assimilation rate. In a mutant lacking phosphoenolpyruvate carboxylase, however, this rate increased to 48%, when glutamine was fed to the leaf, a value higher than that found in some C₃ plants. In mutants of Amaranthus edulis that accumulated glycine, the rate of photorespiration was reduced to 3% of the total net CO₂ assimilation rate. The rate of ammonia produced during photorespiration was 60% of the total produced by all metabolic reactions in the leaves. The data suggests that photorespiration is an active process in C₄ plants, which can play an important role in photosynthetic metabolism in these plants.     

A phosphopantetheinyl transferase that is essential for mitochondrial fatty acid biosynthesis

In this study we report the molecular genetic characterization of the Arabidopsis mitochondrial phosphopantetheinyl transferase (mtPPT), which catalyzes the phosphopantetheinylation and thus activation of mitochondrial acyl carrier protein (mtACP) of mitochondrial fatty acid synthase (mtFAS). This catalytic capability of the purified mtPPT protein (encoded by AT3G11470) was directly demonstrated in an in vitro assay that phosphopantetheinylated mature Arabidopsis apo‐mtACP isoforms. The mitochondrial localization of the AT3G11470‐encoded proteins was validated by the ability of their N‐terminal 80‐residue leader sequence to guide a chimeric GFP protein to this organelle. A T‐DNA‐tagged null mutant mtppt‐1 allele shows an embryo‐lethal phenotype, illustrating a crucial role of mtPPT for embryogenesis. Arabidopsis RNAi transgenic lines with reduced mtPPT expression display typical phenotypes associated with a deficiency in the mtFAS system, namely miniaturized plant morphology, slow growth, reduced lipoylation of mitochondrial proteins, and the hyperaccumulation of photorespiratory intermediates, glycine and glycolate. These morphological and metabolic alterations are reversed when these plants are grown in a non‐photorespiratory condition (i.e. 1% CO₂ atmosphere), demonstrating that they are a consequence of a deficiency in photorespiration due to the reduced lipoylation of the photorespiratory glycine decarboxylase.     

Glycine decarboxylase controls photosynthesis and plant growth

Photorespiration makes oxygenic photosynthesis possible by scavenging 2-phosphoglycolate. Hence, compromising photorespiration impairs photosynthesis. We examined whether facilitating photorespiratory carbon flow in turn accelerates photosynthesis and found that overexpression of the H-protein of glycine decarboxylase indeed considerably enhanced net-photosynthesis and growth of Arabidopsis thaliana. At the molecular level, lower glycine levels confirmed elevated GDC activity in vivo, and lower levels of the CO₂ acceptor ribulose 1,5-bisphosphate indicated higher drain from CO₂ fixation. Thus, the photorespiratory enzyme glycine decarboxylase appears as an important feed-back signaller that contributes to the control of the Calvin-Benson cycle and hence carbon flow through both photosynthesis and photorespiration.     

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.     

Deletion of glycine decarboxylase in Arabidopsis is lethal under nonphotorespiratory conditions

The mitochondrial multienzyme glycine decarboxylase (GDC) catalyzes the tetrahydrofolate-dependent catabolism of glycine to 5,10-methylene-tetrahydrofolate and the side products NADH, CO(2), and NH(3). This reaction forms part of the photorespiratory cycle and contributes to one-carbon metabolism. While the important role of GDC for these two metabolic pathways is well established, the existence of bypassing reactions has also been suggested. Therefore, it is not clear to what extent GDC is obligatory for these processes. Here, we report on features of individual and combined T-DNA insertion mutants for one of the GDC subunits, P protein, which is encoded by two genes in Arabidopsis (Arabidopsis thaliana). The individual knockout of either of these two genes does not significantly alter metabolism and photosynthetic performance indicating functional redundancy. In contrast, the double mutant does not develop beyond the cotyledon stage in air enriched with 0.9% CO(2). Rosette leaves do not appear and the seedlings do not survive for longer than about 3 to 4 weeks under these nonphotorespiratory conditions. This feature distinguishes the GDC-lacking double mutant from all other known photorespiratory mutants and provides evidence for the nonreplaceable function of GDC in vital metabolic processes other than photorespiration.     

Photorespiratory metabolism and immunogold localization of photorespiratory enzymes in leaves of C3 and C3-C4 intermediate species of Moricandia

Photorespiratory metabolism of the C₃-C₄ intermediate species Moricandia arvensis (L.) DC has been compared with that of the C₃ species, Moricandia moricandioides (Boiss.) Heywood. Assays of glycollate oxidase (EC 1.1.3.1), glyoxylate aminotransferases (EC 2.6.1.4, EC 2.6.1.45) and hydroxypyruvate reductase (EC 1.1.1.29) indicate that the capacity for flux through the photorespiratory cycle is similar in both species. Immunogold labelling with monospecific antibodies was used to investigate the cellular locations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), glycollate oxidase, and glycine decarboxylase (EC 2.1.2.10) in leaves of the two species. Ribulose 1,5-bisphosphate carboxylase/oxygenase was confined to the stroma of chloroplasts and glycollate oxidase to the peroxisomes of all photosynthetic cells in leaves of both species. However, whereas glycine decarboxylase was present in the mitochondria of all photosynthetic cells in M. moricandioides, it was only found in the mitochondria of bundle-sheath cells in M. arvensis. We suggest that localized decarboxylation of glycine in the leaves of M. arvensis will lead to improved recapture of photorespired CO₂ and hence a lower rate of photorespiration.Abbreviations kDa kilodalton - RuBP ribulose-1,5-bisphosphate     

The afterglow thermoluminescence band as an indicator of changes in the photorespiratory metabolism of the model legume Lotus japonicus

The afterglow (AG) luminescence is a delayed chlorophyll fluorescence emitted by the photosystem II that seems to reflect the level of assimilatory potential (NADPH+ATP) in chloroplast. In this work, the thermoluminescence (TL) emissions corresponding to the AG band were investigated in plants of the WT and the Ljgln2‐2 photorespiratory mutant from Lotus japonicus grown under either photorespiratory (air) or non‐photorespiratory (high concentration of CO₂) conditions. TL glow curves obtained after two flashes induced the strongest overall TL emissions, which could be decomposed in two components: B band (t_max = 27–29°C) and AG band (t_max = 44–45°C). Under photorespiratory conditions, WT plants showed a ratio of 1.17 between the intensity of the AG and B bands (I_AG/I_B). This ratio increased considerably under non‐photorespiratory conditions (2.12). In contrast, mutant Ljgln2‐2 plants grown under both conditions showed a high I_AG/I_B ratio, similar to that of WT plants grown under non‐photorespiratory conditions. In addition, high temperature thermoluminescence (HTL) emissions associated to lipid peroxidation were also recorded. WT and Ljgln2‐2 mutant plants grown under photorespiratory conditions showed both a significant HTL band, which increased significantly under non‐photorespiratory conditions. The results of this work indicate that changes in the amplitude of I_AG/I_B ratio could be used as an in vivo indicator of alteration in the level of photorespiratory metabolism in L. japonicus chloroplasts. Moreover, the HTL results suggest that photorespiration plays some role in the protection of the chloroplast against lipid peroxidation.     

High-to-Low CO(2) Acclimation Reveals Plasticity of the Photorespiratory Pathway and Indicates Regulatory Links to Cellular Metabolism of Arabidopsis

Background

Photorespiratory carbon metabolism was long considered as an essentially closed and nonregulated pathway with little interaction to other metabolic routes except nitrogen metabolism and respiration. Most mutants of this pathway cannot survive in ambient air and require CO₂-enriched air for normal growth. Several studies indicate that this CO₂ requirement is very different for individual mutants, suggesting a higher plasticity and more interaction of photorespiratory metabolism as generally thought. To understand this better, we examined a variety of high- and low-level parameters at 1% CO₂ and their alteration during acclimation of wild-type plants and selected photorespiratory mutants to ambient air.

Methodology and Principal Findings

The wild type and four photorespiratory mutants of Arabidopsis thaliana (Arabidopsis) were grown to a defined stadium at 1% CO₂ and then transferred to normal air (0.038% CO₂). All other conditions remained unchanged. This approach allowed unbiased side-by-side monitoring of acclimation processes on several levels. For all lines, diel (24 h) leaf growth, photosynthetic gas exchange, and PSII fluorescence were monitored. Metabolite profiling was performed for the wild type and two mutants. During acclimation, considerable variation between the individual genotypes was detected in many of the examined parameters, which correlated with the position of the impaired reaction in the photorespiratory pathway.

Conclusions

Photorespiratory carbon metabolism does not operate as a fully closed pathway. Acclimation from high to low CO₂ was typically steady and consistent for a number of features over several days, but we also found unexpected short-term events, such as an intermittent very massive rise of glycine levels after transition of one particular mutant to ambient air. We conclude that photorespiration is possibly exposed to redox regulation beyond known substrate-level effects. Additionally, our data support the view that 2-phosphoglycolate could be a key regulator of photosynthetic-photorespiratory metabolism as a whole.     

Control of photosynthesis in barley plants with reduced activities of glycine decarboxylase

A mutant (LaPr 87/30) of barley (Hordeum vulgare L.) deficient in glycine decarboxylase (GDC; EC 2.1.2.10) was crossed with wild-type plants to generate heterozygous plants with reduced GDC activities. Plants of the F₂ generation were grown in air and analysed for reductions in GDC proteins and GDC activity. The leaves of heterozygous plants contained reduced amounts of H-protein, and when the content of H-protein was lower than 60% of the wild-type, the P-protein was also reduced. The contents of the other two proteins of the GDC complex, T-protein and L-protein were not affected. Glycine decarboxylase activities, measured as the decarboxylation of [1-¹⁴C]glycine by intact mitochondria released from protoplasts, were between 47% and 63% of the wild-type activity in heterozygous plants and between 86% and 100% in plants with normal contents of H-protein. The enzyme activity was linearly correlated with the relative content of H-protein. Plants with reduced GDC activities developed normally and did not show major pleiotropic effects. In air, the reduction in GDC activity had no effect on the leaf metabolite content or photosynthesis, but under conditions of enhanced photorespiration (low CO₂ and high light), glycine accumulated and the rates of photosynthesis decreased compared to the wild-type. The accumulation of glycine did not lead to a depletion of amino donors or to the accumulation of glyoxylate. The lower rates of photosynthesis were probably caused by an impaired recycling of carbon in the photorespiratory pathway. It is concluded that GDC has no control over CO₂ assimilation under normal growth conditions, but appreciable control by GDC becomes apparent under conditions leading to higher rates of photorespiration. Received: 24 November 1996 / Accepted: 23 January 1997     

Photorespiration-deficient Mutants of Arabidopsis thaliana Lacking Mitochondrial Serine Transhydroxymethylase Activity

Three allelic mutants of Arabidopsis thaliana which lack mitochondrial serine transhydroxymethylase activity due to a recessive nuclear mutation have been characterized. The mutants were shown to be deficient both in glycine decarboxylation and in the conversion of glycine to serine. Glycine accumulated as an end product of photosynthesis in the mutants, largely at the expense of serine, starch, and sucrose formation. The mutants photorespired CO₂ at low rates in the light, but this evolution of photorespiratory CO₂ was abolished by provision of exogenous NH₃. Exogenous NH₃ was required by the mutants for continued synthesis of glycine under photorespiratory conditions. These and related results with wild-type Arabidopsis suggested that glycine decarboxylation is the sole site of photorespiratory CO₂ release in wild-type plants but that depletion of the amino donors required for glyoxylate amination may lead to CO₂ release from direct decarboxylation of glyoxylate. Photosynthetic CO₂ fixation was inhibited in the mutants under atmospheric conditions which promote photorespiration but could be partially restored by exogenous NH₃. The magnitude of the NH₃ stimulation of photosynthesis indicated that the increase was due to the suppression of glyoxylate decarboxylation. The normal growth of the mutants under nonphotorespiratory atmospheric conditions indicates that mitochondrial serine transhydroxymethylase is not required in C₃ plants for any function unrelated to photorespiration.     

Predominant localization of mitochondria enriched with glycine-decarboxylating enzymes in bundle sheath cells of Alternanthera tenella, a C3–C4 intermediate species

Mesophyll protoplasts and bundle sheath cells were prepared by enzymatic digestion of leaves of Alternanthera tenella, a C₃-C₄ intermediate species. The intercellular distribution of selected photosynthetic, photorespiratory and respiratory (mitochondrial) enzymes in these meso-phyll and bundle sheath cells was studied. The activity levels of photosynthetic enzymes such as PEP carboxylase (EC 4.1.1.31) or NAD-malic enzyme (EC 1.1.1.39) and photorespiratory enzymes such as glycolate oxidase (EC 1.1.3.1) or NADH-hydroxypyruvate reductase (EC 1.1.1.29) were similar in the two cell types. The activity levels of mitochondrial TCA cycle enzymes such as citrate synthase (EC 4.1.3.7) or fumarase (EC 4.2.1.2) were 2- to 3-fold higher in bundle sheath cells. On the other hand, the activity levels of mitochondrial photorespiratory enzymes, namely glycine decarboxylase (EC 2.1.2.10) and serine hydroxymethyltransferase (EC 2.1.2.1), were 6-9-fold higher in bundle sheath cells than in mesophyll protoplasts. Such preferential localization of mitochondria enriched with the glycine-decarboxylating system in the inner bundle sheath cells would result in efficient refixa-tion of CO₂ from not only photorespiration but also dark respiration before its exit from the leaf. We propose that predominant localization of mitochondria specialized in glycine decarboxylation in bundle sheath cells may form the basis of reduced photorespiration in this C₃-C₄ intermediate species.     

Two photosynthetic mechanisms mediating the low photorespiratory state in submersed aquatic angiosperms

The submersed angiosperms Myriophyllum spicatum L. and Hydrilla verticillata (L.f.) Royal exhibited different photosynthetic pulse-chase labeling patterns. In Hydrilla, over 50% of the ¹⁴C was initially in malate and aspartate, but the fate of the malate depended upon the photorespiratory state of the plant. In low photorespiration Hydrilla, malate label decreased rapidly during an unlabeled chase, whereas labeling of sucrose and starch increased. In contrast, for high photorespiration Hydrilla, malate labeling continued to increase during a 2-hour chase. Thus, malate formation occurs in both photorespiratory states, but reduced photorespiration results when this malate is utilized in the light. Unlike Hydrilla, in low photorespiration Myriophyllum, ¹⁴C incorporation was via the Calvin cycle, and less than 10% was in C₄ acids.

Ethoxyzolamide, a carbonic anhydrase inhibitor and a repressor of the low photorespiratory state, increased the label in glycolate, glycine, and serine of Myriophyllum. Isonicotinic acid hydrazide increased glycine labeling of low photorespiration Myriophyllum from 14 to 25%, and from 12 to 48% with high photorespiration plants. Similar trends were observed with Hydrilla. Increasing O₂ increased the per cent [¹⁴C]glycine and the O₂ inhibition of photosynthesis in Myriophyllum. In low photorespiration Myriophyllum, glycine labeling and O₂ inhibition of photosynthesis were independent of the CO₂ level, but in high photorespiration plants the O₂ inhibition was competitively decreased by CO₂. Thus, in low but not high photorespiration plants, glycine labeling and O₂ inhibition appeared to be uncoupled from the external [O₂]/[CO₂] ratio.

These data indicate that the low photorespiratory states of Hydrilla and Myriophyllum are mediated by different mechanisms, the former being C₄-like, while the latter resembles that of low CO₂-grown algae. Both may require carbonic anhydrase to enhance the use of inorganic carbon for reducing photorespiration.

     

Markedly low requirement of added CO2 for photosynthesis by mesophyll protoplasts of pea (Pisum sativum): possible roles of photorespiratory CO2 and carbonic anhydrase

Mesophyll protoplasts of pea required only 74.1 μM CO₂ for maximal photosynthesis, unlike chloroplasts, which required up to 588 μM CO₂. Such a markedly low requirement for CO₂ could be because of an internal carbon source and/or a CO₂ concentrating mechanism in mesophyll protoplasts. Ethoxyzolamide (EZA), an inhibitor of internal carbonic anhydrase (CA) suppressed photosynthesis by mesophyll protoplasts at low CO₂ (7.41 μM) but had no significant effect at high CO₂ (741 μM). However, acetazolamide, another inhibitor of CA, did not exert as much dramatic effect as EZA. Three photorespiratory inhibitors, aminoacetonitrile or glycine hydroxamate (GHA) or aminooxyacetate inhibited markedly photosynthesis at low CO₂ but not at high CO₂. Inhibitors of glycolysis or tricarboxylic acid cycle (NaF, sodium malonate) or phosphoenolpyruvate carboxylase (3,3‐dichloro‐2‐dihydroxy phosphinoyl‐methyl‐2‐propenoate) had no significant effect on photosynthesis. The CO₂ requirement of protoplast photosynthesis and the sensitivity of photosynthesis to EZA were much higher at low oxygen (65 nmol ml^?1) than that at normal oxygen (212 nmol ml^?1). In contrast, the inhibitory effect of photorespiratory inhibitors on protoplast photosynthesis was similar in both normal and low oxygen medium. The marked elevation of glycine/serine ratio at low O₂ or in presence of GHA confirmed the suppression of photorespiratory decarboxylation by GHA. While demonstrating interesting difference between the response of protoplasts and chloroplasts to CO₂, we suggest that photorespiration could be a significant source of CO₂ for photosynthesis in mesophyll protoplasts at limiting CO₂ and at atmospheric levels of oxygen. Obviously, carbonic anhydrase is essential to concentrate or retain CO₂ in mesophyll cells.     

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.     

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.     

The role of photorespiration during drought stress: an analysis utilizing barley mutants with reduced activities of photorespiratory enzymes

C _i, intercellular CO₂ concentration
F_v/F_m, quantum efficiency of excitation capture by open photosystem II centres
FBPase, fructose-1,6-bisphosphatase
GAPDH, glyceraldehyde-3-phosphate dehydrogenase
GDC, glycine decarboxylase
GS-2, chloroplastic glutamine synthetase
HPR, hydroxypyruvate reductase
PFD, photon flux density
ΦCO₂, quantum efficiency of CO₂ assimilation
ΦPSII, quantum efficiency of photosystem II electron transport
ψ, water potential
q_N, non-photochemical chlorophyll a fluorescence quenching
q_P, photochemical chlorophyll a fluorescence quenching
RuBP, ribulose-1,5-bisphosphate
Rubisco, ribulose-1,5-bisphosphate carboxylase-oxygenase
SBPase, sedoheptulose-1,7-bisphosphatase
SGAT, serine : glyoxylate aminotransferase

The significance of photorespiration in drought-stressed plants was studied by withholding water from wild-type barley (Hordeum vulgare L.) and from heterozygous mutants with reduced activities of chloroplastic glutamine synthetase (GS-2), glycine decarboxylase (GDC) or serine : glyoxylate aminotransferase (SGAT). Well-watered plants of all four genotypes had identical rates of photosynthesis. Under moderate drought stress (leaf water potentials between –1 and –2 MPa), photosynthesis was lower in the mutants than in the wild type, indicating that photorespiration was increased under these conditions. Analysis of chlorophyll a fluorescence revealed that, in the GDC and SGAT mutants, the lower rates of photosynthesis coincided with a decreased quantum efficiency of photosystem II and increased non-photochemical dissipation of excitation energy. Correspondingly, the de-epoxidation state of xanthophyll-cycle carotenoids was increased several-fold in the drought-stressed GDC and SGAT mutants compared with the wild type. Accumulation of glycine in the GDC mutant was further evidence for increased photorespiration in drought-stressed barley. The effect of drought on the photorespiratory enzymes was determined by immunological detection of protein abundance. While the contents of GS-2 and P- and H-protein of the GDC complex remained unchanged as drought stress developed, the content of NADH-dependent hydroxypyruvate reductase increased. Enzymes of the Benson–Calvin cycle, on the other hand, were either not affected (ribulose-1,5-bisphosphate carboxylase-oxygenase and plastidic fructose-1,6-bisphosphatase) or declined (sedoheptulose- 1,7-bisphosphatase and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase). These data demonstrate that photorespiration was enhanced during drought stress in barley and that the control exerted by photorespiratory enzymes on the rate of photosynthetic electron transport and CO₂ fixation was increased.     

Influence of Photorespiration on ATP/ADP Ratios in the Chloroplasts, Mitochondria, and Cytosol, Studied by Rapid Fractionation of Barley (Hordeum vulgare) Protoplasts

Using the principle described by R McC Lilley, M Stitt, G Mader, HW Heldt (1982 Plant Physiol 70: 965-970), an apparatus for rapid fractionation of barley leaf (Hordeum vulgare) protoplasts by membrane filtration was built. From studies of ATP/ADP ratios, it is concluded that the quenching of metabolic reactions is very fast, making it possible to use the method for studies on metabolic interactions between different compartments in plant cells. The fractionation method was used to study the influence of photorespiration on ATP/ADP ratios in the chloroplasts, mitochondria, and cytosol of barley leaf protoplasts. The cytosolic ATP/ADP ratio was higher under photorespiratory conditions than under nonphotorespiratory conditions. Aminoacetonitrile, an inhibitor of the photorespiratory conversion of glycine to serine, had a very small effect on the ATP/ADP ratios in the different subcellular compartments during photosynthesis in nonphotorespiratory conditions (saturating CO₂). In photorespiratory conditions (limiting CO₂), on the other hand, aminoacetonitrile increased the ATP/ADP ratio in the chloroplasts and decreased the ATP/ADP ratios in the mitochondria and the cytosol. These results are consistent with the hypothesis, that during photorespiration glycine oxidation is coupled to oxidative phosphorylation to provide ATP to the cytosol.     

Glycine decarboxylase and serine formation in spinach leaf mitochondrial preparation with reference to photorespiration

Glycine decarboxylation and serine synthesis were investigatedto account for photorespiratory CO₂ evolution in higher plants.Glycine decarboxylase in leaf mitochondria was found to splitglycine into CO₂, NH₃ and a C₁ unit. Free glyoxylic acid wasnot involved in this process as an intermediate. Serine synthesiswas closely related to decarboxylation of glycine. We inferredthat serine is formed from two molecules of glycine by the combinedaction of glcine decarboxylase and serine hydroxymethyltransferase.Glycine decarboxylation and serine synthesis were stimulatedby NAD, PALP and THFA, and were inhibited by detergents, lipase,sonication, mechanical treatment, thyroxine and thiol compounds,suggesting the importance of structural intactness of the mitochondrialmembrane system. Glycine decarboxylase was present in intacttissues in quantities consistent with glycolate production duringphotosynthesis. We concluded that glycine decarboxylase in mitochondriais principally responsible for CO₂ evolution in photorespiration.A control mechanism of photorespiration is discussed based onthe stimulation of glycine decarboxylase by NAD and on inhibitionby NADH. ¹ A part of this work was presented at the Annual Meeting (April,1969) of the Japanese Society of Plant Physiologists, Kanazawa,and at the annual Meeting (April, 1970) of the Japanese AgricultualChemical Society, Fukuoka. (Received August 3, 1970; )