Monitoring cytosolic pH of carboxysome-deficient cells of Synechocystis sp. PCC 6803 using fluorescence analysis

Disruption of the ccmM gene in the cyanobacterium Synechocystis sp. PCC 6803 causes a deficiency of carboxysomes and impairs growth in ambient CO2. The effect of this gene defect on cellular metabolism was investigated using electron microscopy, biochemical and fluorescence analysis. Mutant cells were devoid of the characteristic dense polyhedral bodies called carboxysomes. The photosynthetic oxygen evolution was considerably lower in mutant cells compared to wild type, while Rubisco activity in cell extracts was similar. During photosynthetic CO2-dependent oxygen evolution, Rubisco Vmax dropped from 142 micromol mg-1 chlorophyll h-1 (WT) to 77 micromol mg-1 chlorophyll h-1 in the mutant cells, and the Km for Ci (inorganic carbon) increased from 0.5 mM (WT) to 40 mM. The fluorescent indicator, acridine yellow, was used for non-invasive measurements of cytoplasmic pH changes in whole cells induced by addition of Ci, making use of the decrease in fluorescence yield that accompanies cytoplasmic acidification. The experimental results indicate that control of the cytoplasmic pH is linked to the internal carbon pool (Ci). Both wild-type and ccmM-deficient cells showed a linear response of acridine yellow fluorescence quenching and, thus, of internal acidification, with respect to externally added inorganic carbon. However, the fluorescence analysis of mutant (carboxysome-free) cells indicated slower kinetics of Ci accumulation.     

The Chlamydomonas reinhardtii cia3 mutant lacking a thylakoid lumen-localized carbonic anhydrase is limited by CO2 supply to rubisco and not photosystem II function in vivo

The Chlamydomonas reinhardtii cia3 mutant has a phenotype indicating that it requires high-CO(2) levels for effective photosynthesis and growth. It was initially proposed that this mutant was defective in a carbonic anhydrase (CA) that was a key component of the photosynthetic CO(2)-concentrating mechanism (CCM). However, more recent identification of the genetic lesion as a defect in a lumenal CA associated with photosystem II (PSII) has raised questions about the role of this CA in either the CCM or PSII function. To resolve the role of this lumenal CA, we re-examined the physiology of the cia3 mutant. We confirmed and extended previous gas exchange analyses by using membrane-inlet mass spectrometry to monitor(16)O(2),(18)O(2), and CO(2) fluxes in vivo. The results demonstrate that PSII electron transport is not limited in the cia3 mutant at low inorganic carbon (Ci). We also measured metabolite pools sizes and showed that the RuBP pool does not fall to abnormally low levels at low Ci as might be expected by a photosynthetic electron transport or ATP generation limitation. Overall, the results demonstrate that under low Ci conditions, the mutant lacks the ability to supply Rubisco with adequate CO(2) for effective CO(2) fixation and is not limited directly by any aspect of PSII function. We conclude that the thylakoid CA is primarily required for the proper functioning of the CCM at low Ci by providing an ample supply of CO(2) for Rubisco.     

Absence of light-induced proton extrusion in a cotA-less mutant of Synechocystis sp. strain PCC6803.

cotA of Synechocystis sp. strain PCC6803 was isolated as a gene that complemented a mutant defective in CO2 transport and is homologous to cemA that encodes a chloroplast envelope membrane protein (A. Katoh, K.S. Lee, H. Fukuzawa, K. Ohyama, and T. Ogawa, Proc. Natl. Acad. Sci. USA 93:4006-4010, 1996). A mutant (M29) constructed by replacing cotA in the wild-type (WT) Synechocystis strain with the omega fragment was unable to grow in BG11 medium (approximately 17 mM Na+) at pH 6.4 or at any pH in a low-sodium medium (100 microM Na+) under aeration with 3% (vol/vol) CO2 in air. The WT cells grew well in the pH range between 6.4 and 8.5 in BG11 medium but only at alkaline pH in the low-sodium medium. Illumination of the WT cells resulted in an extrusion followed by an uptake of protons. In contrast, only proton uptake was observed for the M29 mutant in the light without proton extrusion. There was no difference in sodium uptake activity between the WT and mutant. The mutant still possessed 51% of the WT CO2 transport activity in the presence of 15 mM NaCl. On the basis of these results we concluded that cotA has a role in light-induced proton extrusion and that the inhibition of CO2 transport in the M29 mutant is a secondary effect of the inhibition of proton extrusion.     

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (I. Time Course of Induction of the Two Systems)

Changes in the physiological properties of the green alga Chlorella ellipsoidea (UTEX 20) were determined during adaptation from high CO2 to air. Cells of C. ellipsoidea, grown in high CO2, had an extremely low affinity for dissolved inorganic carbon (DIC). However, high-affinity DIC transport was induced rapidly after switching to air, which caused a massive decrease in the DIC concentration in the medium. Rates of O2 evolution without added carbonic anhydrase (CA) were compared with calculated rates of uncatalyzed CO2 formation in the medium as a measure of active HCO3-uptake. Cells were found to be able to use HCO3- after 5 h of adaptation and this capacity increased during the next 17 h. The stimulation of O2 evolution upon CA addition was used as a measurement of active CO2 transport: such stimulation occurred 2 h after transfer and increased during the next 5 h. Increases in O2 evolution rates were correlated closely with an increasing capacity to accumulate intracellular pools of acid-labile DIC and with decreases in K1/2(CO2) and CO2-compensation point of the cells. Treatment of cells with cycloheximide (5 [mu]g mL-1) during adaptation completely inhibited DIC transport induction, whereas treatment with chloramphenicol (400 [mu]g mL-1) had no effect, indicating the requirement for cytoplasmic protein synthesis in the induction. These results suggest that both CO2 and HCO3- transport are induced upon transfer of cells from high CO2 to air and that there is a temporal separation between the induction of the two systems.     

The lack of mitochondrial complex I in a CMSII mutant of Nicotiana sylvestris increases photorespiration through an increased internal resistance to CO2 diffusion

The cytoplasmic male sterile II (CMSII) mutant lacking complex I of the mitochondrial electron transport chain has a lower photosynthetic activity but exhibits higher rates of excess electron transport than the wild type (WT) when grown at high light intensity. In order to examine the cause of the lower photosynthetic activity and to determine whether excess electrons are consumed by photorespiration, light, and intercellular CO(2), molar fraction (c(i)) response curves of carbon assimilation were measured at varying oxygen molar fractions. While oxygen is the major acceptor for excess electrons in CMSII and WT leaves, electron flux to photorespiration is favoured in the mutant as compared with the WT leaves. Isotopic mass spectrometry measurements showed that leaf internal conductance to CO(2) diffusion (g(m)) in mutant leaves was half that of WT leaves, thus decreasing the c(c) and favouring photorespiration in the mutant. The specificity factor of Rubisco did not differ significantly between both types of leaves. Furthermore, carbon assimilation as a function of electrons used for carboxylation processes/electrons used for oxygenation processes (J(C)/J(O)) and as a function of the calculated chloroplastic CO(2) molar fraction (c(c)) values was similar in WT and mutant leaves. Enhanced rates of photorespiration also explain the consumption of excess electrons in CMSII plants and agreed with potential ATP consumption. Furthermore, the lower initial Rubisco activity in CMSII as compared with WT leaves resulted from the lower c(c) in ambient air, since initial Rubisco activity on the basis of equal c(c) values was similar in WT and mutant leaves. The retarded growth and the lower photosynthetic activity of the mutant were largely overcome when plants were grown in high CO(2) concentrations, showing that limiting CO(2) supply for photosynthesis was a major cause of the lower growth rate and photosynthetic activity in CMSII.     

A Mutant Isolated from the Cyanobacterium Synechococcus PCC7942 Is Unable to Adapt to Low Inorganic Carbon Conditions

Using a novel screening procedure, we have selected a new class of mutant from the cyanobacterium Synechococcus PCC7942 that fails to adapt to growth at an extremely low inorganic carbon (Ci) concentration. The mutant (Tm17) reported in this study grows normally at or above air levels of CO2 (340 [mu]L L-1) but does not survive at 20 [mu]L L-1 CO2 in air. Air-grown Tm17 cells showed properties similar to wild-type cells in various aspects of the CO2-concentrating mechanism examined. Following transfer from air levels to 20 [mu]L L-1 CO2, however, the mutant cells failed to increase their photosynthetic affinity for Ci. This results in an approximately 10-fold difference in photosynthetic affinity between the wild-type and Tm17 cells under Ci-limiting conditions [the K0.5(Ci) values were 11 and 136 [mu]M, respectively]. Further examination of factors possibly contributing to this low photosynthetic affinity showed that Tm17 cells have no inducible high-affinity HCO3- transport and do not appear to show induction of increased carboxysomal carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase activities. It appears that a common factor, possibly relating to CO2 detection and/or induction signal, or the HCO3-transport mechanism may have been impaired in the mutant. Complementation results indicate that the mutation responsible for the phenotype has occurred in an 8- to 10-kb EcoRI genomic DNA fragment.     

Involvement of the cynABDS operon and the CO2-concentrating mechanism in the light-dependent transport and metabolism of cyanate by cyanobacteria

The cyanobacteria Synechococcus elongatus strain PCC7942 and Synechococcus sp. strain UTEX625 decomposed exogenously supplied cyanate (NCO-) to CO2 and NH3 through the action of a cytosolic cyanase which required HCO3- as a second substrate. The ability to metabolize NCO- relied on three essential elements: proteins encoded by the cynABDS operon, the biophysical activity of the CO2-concentrating mechanism (CCM), and light. Inactivation of cynS, encoding cyanase, and cynA yielded mutants unable to decompose cyanate. Furthermore, loss of CynA, the periplasmic binding protein of a multicomponent ABC-type transporter, resulted in loss of active cyanate transport. Competition experiments revealed that native transport systems for CO2, HCO3-, NO3-, NO2-, Cl-, PO4(2-), and SO4(2-) did not contribute to the cellular flux of NCO- and that CynABD did not contribute to the flux of these nutrients, implicating CynABD as a novel primary active NCO- transporter. In the S. elongatus strain PCC7942 DeltachpX DeltachpY mutant that is defective in the full expression of the CCM, mass spectrometry revealed that the cellular rate of cyanate decomposition depended upon the size of the internal inorganic carbon (Ci) (HCO3- + CO2) pool. Unlike wild-type cells, the rate of NCO- decomposition by the DeltachpX DeltachpY mutant was severely depressed at low external Ci concentrations, indicating that the CCM was essential in providing HCO3- for cyanase under typical growth conditions. Light was required to activate and/or energize the active transport of both NCO- and Ci. Putative cynABDS operons were identified in the genomes of diverse Proteobacteria, suggesting that CynABDS-mediated cyanate metabolism is not restricted to cyanobacteria.     

Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds

Several genes involved in the ability of Synechococcus sp. PCC 7942 to grow under different CO2 concentrations were mapped in the genomic region of rbcLS (the operon encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase). Insertion of a cartridge encoding kanamycin resistance within open reading frame (ORF) 78, designated ccmJ, located 7 kb upstream of rbcLS, resulted in a kanamycin-resistant, high-CO2-requiring mutant, M3, which does not contain normal carboxysomes. ccmJ shows significant homology to csoS1 encoding a carboxysomal shell polypeptide in Thiobacillus neopolitanus. Analysis of the polypeptide pattern of a carboxysome-enriched fraction indicated several differences between the wild type and the mutant. The amount of the ribulose-1,5-bisphosphate carboxylase/oxygenase subunits was considerably smaller in the carboxysomal fraction of the mutant when compared to the wild type. On the basis of the sequence analyses, ORF286 and ORF466, located downstream of ccmJ, were identified as chlL and chlN, respectively, which are involved in chlorophyll biosynthesis in the dark.     

Low Activation State of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Carboxysome-Defective Synechococcus Mutants

The high-CO2-requiring mutant of Synechococcus sp. PCC 7942, EK6, was obtained after extension of the C terminus of the small subunit of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). The carboxysomes in EK6 were much larger than in the wild type, but the cellular distribution of the large and small sub-units of Rubisco was not affected. The kinetic parameters of in vitro-activated Rubisco were similar in EK6 and in the wild type. On the other hand, Rubisco appeared to be in a low state of activation in situ in EK6 cells pretreated with an air level of CO2. This was deduced from the appearance of a lag phase when carboxylation was followed with time in cells permeabilized by detergent and subsequently supplied with saturating CO2 and RuBP. Pretreatment of the cells with high CO2 virtually abolished the lag. After low-CO2 treatment, the internal RuBP pool was much higher in mutant cells than in the wild-type cells; pretreatment with high CO2 reduced the pool in mutant cells. We suggest that the high-CO2-requiring phenotype in mutants that possess aberrant carboxysomes arises from the inactivated state of Rubisco when the cells are exposed to low CO2.     

Acclimation of wild-type cells and CO2-insensitive mutants of the green alga Chlorella ellipsoidea to elevated [CO2

CO(2)-insensitive mutants of the green alga Chlorella ellipsoidea were previously shown to be unable to repress an inorganic carbon-concentrating mechanism (CCM) when grown under 5% CO(2). When air-grown, wild-type (WT) cells were transferred to 5% CO(2), an abrupt drop of P(max) to 43% the original level of air-grown cells was observed within the initial 12 h. Photosynthetic affinities of WT cells to dissolved inorganic carbon (DIC) were maintained at high levels for the initial 4 d of acclimation, and then decreased gradually to lower levels over the next 6 d. In contrast to WT cells, the CO(2)-insensitive mutant, ENU16, exhibited a constant P(max) at maximum levels and a low K(1/2)[DIC] throughout the acclimation period. The rapid P(max) drop within 12 h of acclimation in WT cells was significantly reduced by treatment with 0.5 mm of 6-ethoxybenzothiazole-2-sulphonamide (EZA), a specific membrane-permeable inhibitor of carbonic anhydrase (CA), suggesting the participation of internal CAs in the temporary drop in P(max) in WT cells. WT and ENU16 cells were grown in controlled equilibrium [CO(2)], and the photosynthetic rate of each acclimated cell type was measured under equilibrated growth [DIC] conditions. In WT cells acclimated to 0.14-0.4% [CO(2)], K(1/2)[DIC] values increased as [CO(2)] increased, and the photosynthetic rates at growth DIC conditions were shown to decrease to about 70% the P(max) level in this intermediate [CO(2)] range. Such decreases in the net photosynthetic rates were not observed in ENU16. These results suggest that algal primary production could be depressed significantly under moderately enriched CO(2) conditions as a result of acquiring intermediate affinities for DIC because of their sensitive responses to changes in the ambient [CO(2)].     

Inorganic carbon acquisition in red tide dinoflagellates

Carbon acquisition was investigated in three marine bloom-forming dinollagellates-Prorocentrum minimum, Heterocapsa triquetra and Ceratium lineatum. In vivo activities of extracellular and intracellular carbonic anhydrase (CA), photosynthetic O2 evolution, CO2 and HCO3- uptake rates were measured by membrane inlet mass spectrometry (MIMS) in cells acclimated to low pH (8.0) and high pH (8.5 or 9.1). A second approach used short-term 14C-disequilibrium incubations to estimate the carbon source utilized by the cells. All three species showed negligible extracellular CA (eCA) activity in cells acclimated to low pH and only slightly higher activity when acclimated to high pH. Intracellular CA (iCA) activity was present in all three species, but it increased only in P. minimum with increasing pH. Half-saturation concentrations (K1/2) for photosynthetic O2 evolution were low compared to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetics. Moreover, apparent affinities for inorganic carbon (Ci) increased with increasing pH in the acclimation, indicating the operation of an efficient CO2 concentration mechanism (CCM) in these dinoflagellates. Rates of CO2 uptake were comparably low and could not support the observed rates of photosynthesis. Consequently, rates of HCO3- uptake were high in the investigated species, contributing more than 80% of the photosynthetic carbon fixation. The affinity for HCO3- and maximum uptake rates increased under higher pH. The strong preference for HCO3- was also confirmed by the 14C-disequilibrium technique. Modes of carbon acquisition were consistent with the 13C-fractionation pattern observed and indicated a strong species-specific difference in leakage. These results suggest that photosynthesis in marine dinoflagellates is not limited by Ci even at high pH, which may occur during red tides in coastal waters.     

Mechanism of CO2 acquisition in an acid-tolerant Chlamydomonas

The acid-tolerant green alga Chlamydomonas (UTCC 121) grows in media ranging in pH from 2.5 to 7.0. Determination of the overall internal pH of the cells, using (14)C-benzoic acid (BA) or [2-(14)C]-5,5-dimethyloxazolidine-2,4-dione (DMO), showed that the cells maintain a neutral pH (6.6 to 7.2) over an external pH range of 3.0-7.0. The cells express an external carbonic anhydrase (CA) when grown in media above pH 5.5, and CA increases to a maximum at pH 7.0. Removal of external CA by trypsin digestion or by acetazolamide (AZA) inhibition indicated that CA was essential for photosynthesis at pH 7.0 and that the cells had no capacity for direct bicarbonate uptake. Monitoring of CO(2) uptake and O(2) evolution by mass spectrometry during photosynthesis did not provide any evidence of active CO(2) uptake. The CO(2) compensation concentration of the cells ranged from 9.4 microM at pH 4.5 to 16.2 microM at pH 7.0. An examination of the kinetics of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), in homogenates of cells grown at pH 7.0, showed that the K(m) (CO(2)) was 16.3 microM. These data indicate that the pH between the cell interior and the external medium was large enough at acid pH to allow the accumulation of inorganic carbon (Ci) by the diffusive uptake of CO(2), and the expression of external CA at neutral pH values would maintain an equilibrium CO(2) concentration at the cell surface. This species does not possess a CO(2)-concentrating mechanism because the whole cell affinity for Ci appears to be determined by the low K(m) (CO(2)) Rubisco of the alga.     

Isolation and Characterization of High CO(2)-Requiring-Mutants of the Cyanobacterium Synechococcus PCC7942 : Two Phenotypes that Accumulate Inorganic Carbon but Are Apparently Unable to Generate CO(2) within the Carboxysome

A total of 24 high CO₂-requiring-mutants of the cyanobacterium Synechococcus PCC7942 have been isolated and partially characterized. These chemically induced mutants are able to grow at 1% CO₂, on agar media, but are incapable of growth at air levels of CO₂. All the mutants were able to accumulate inorganic carbon (C_i) to levels similar to or higher than wild type cells, but were apparently unable to generate intracellular CO₂. On the basis of the rate of C_i release following a light (5 minutes) → dark transition two extreme phenotypes (fast and slow release mutants) and a number of `intermediate' mutants (normal release) were identified. Compared to wild-type cells, Type I mutants had the following characteristics: fast C_i release, normal internal C_i pool, normal carbonic anhydrase (CA) activity in crude extracts, reduced internal exchange of ¹⁸O from ¹⁸O-labeled CO₂, 1% CO₂ requirement for growth in liquid media, normal affinity of carboxylase for CO₂, and long, rod-like carboxysomes. Type II mutants had the following characteristics: slow C_i release, increased internal C_i pool, normal CA activity in crude extracts, normal internal ¹⁸O exchange, a 3% CO₂ requirement for growth in liquid media, high carboxylase activity, normal affinity of carboxylase for CO₂, and normal carboxysome structure but increased in numbers per cell. Both mutant phenotypes appear to have genetic lesions that result in an inability to convert intracellular HCO₃⁻ to CO₂ inside the carboxysome. The features of the type I mutants are consistent with a scenario where carboxysomal CA has been mistargeted to the cytosol. The characteristics of the type II phenotype appear to be most consistent with a scenario where CA activity is totally missing from the cell except for the fact that cell extracts have normal CA activity. Alternatively the type II mutants may have a lesion in their capacity for H⁺ import during photosynthesis.     

Association of Carbonic Anhydrase Activity with Carboxysomes Isolated from the Cyanobacterium Synechococcus PCC7942

The development of a simple method for the isolation of purified carboxysomes from the cyanobacterium Synechococcus PCC7942 has made it possible to identify a specific and inducible, intracellular carbonic anhydrase (CA) activity that is strongly associated with carboxysomes. This was shown, in part, through enzyme recovery experiments that indicated that a clear majority of a CA activity that is sensitive to the CA inhibitor ethoxyzolamide (I₅₀ = 4 μm) copurifies with a majority of the cell's ribulose-1,5-bisphosphate carboxylase/oxygenase activity in a highly purified pelletable fraction. Electron microscopy of this pelletable fraction revealed the presence of carboxysomes that were physically intact. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of carboxysome proteins showed that the large and small subunits of ribulose-1,5-bisphosphate carbosylase/oxygenase were clearly prominent and that several other minor proteins could be distinguished. The specific location of this carboxysomal CA activity is further reinforced by the finding that a previously isolated high CO₂-requiring mutant, Type II/No. 68 (G.D. Price, M.R. Badger [1989] Plant Physiol 91: 514-525), displayed a 30-fold reduction in carboxysome-associated CA activity when tested under optimal conditions. Carboxysomal CA has the unusual property of being inactivated by dithiothreitol. The enzyme also requires 20 mm Mg²⁺ (as MgSO₄) for near maximum activity; other divalent cations, such as Ca²⁺ and Mn²⁺, also stimulate carboxysomal CA activity, but to a lesser extent than Mg²⁺. Results are discussed in relation to the role of carboxysomes in the CO₂-concentrating mechanism in cyanobacteria and the role that carboxysomal CA activity appears to play in this process.     

Inorganic Carbon Accumulation Stimulates Linear Electron Flow to Artificial Electron Acceptors of Photosystem I in Air-Grown Cells of the Cyanobacterium Synechococcus UTEX 625

The effect of inorganic carbon (Ci) transport and accumulation on photosynthetic electron transport was studied in air-grown cells of the cyanobacterium Synechococcus UTEX 625. When the cells were depleted of Ci, linear photosynthetic electron flow was almost completely inhibited in the presence of the photosystem I (PSI) acceptor N,N-dimethyl-p-nitrosoaniline (PNDA). The addition of Ci to these cells, in which CO2 fixation was inhibited with glycolaldehyde, greatly stimulated linear electron flow and resulted in increased levels of photochemical quenching and O2 evolution. In aerobic conditions substantial quenching resulted from methyl viologen (MV) addition and further quenching was not observed upon the addition of Ci. In anaerobic conditions MV addition did not result in quenching until Ci was added. Intracellular Ci pools were formed when MV was present in aerobic or anaerobic conditions or PNDA was present in aerobic conditions. There was no inhibitory effect of Ci depletion on electron flow to 2,6-dimethylbenzoquinone and oxidized diaminodurene, which accept electrons from photosystem II. The degree of stimulation of PNDA-dependent O2 evolution varied with the Ci concentration. The extracellular Ci, concentration required for a half-maximum rate (K1/2) was 3.8 [mu]M and the intracellular K1/2 was 1.4 mM for the stimulation of PNDA reduction. These values agreed closely with the K1/2 values of extracellular and intracellular Ci for O2 photoreduction. Linear electron flow to artificial electron acceptors of PSI was enhanced by intracellular Ci, which appeared to exert an effect on PSI or on the intersystem electron transport chain.     

Functional mitochondrial complex I is required by tobacco leaves for optimal photosynthetic performance in photorespiratory conditions and during transients

The importance of the mitochondrial electron transport chain in photosynthesis was studied using the tobacco (Nicotiana sylvestris) mutant CMSII, which lacks functional complex I. Rubisco activities and oxygen evolution at saturating CO(2) showed that photosynthetic capacity in the mutant was at least as high as in wild-type (WT) leaves. Despite this, steady-state photosynthesis in the mutant was reduced by 20% to 30% at atmospheric CO(2) levels. The inhibition of photosynthesis was alleviated by high CO(2) or low O(2). The mutant showed a prolonged induction of photosynthesis, which was exacerbated in conditions favoring photorespiration and which was accompanied by increased extractable NADP-malate dehydrogenase activity. Feeding experiments with leaf discs demonstrated that CMSII had a lower capacity than the WT for glycine (Gly) oxidation in the dark. Analysis of the postillumination burst in CO(2) evolution showed that this was not because of insufficient Gly decarboxylase capacity. Despite the lower rate of Gly metabolism in CMSII leaves in the dark, the Gly to Ser ratio in the light displayed a similar dependence on photosynthesis to the WT. It is concluded that: (a) Mitochondrial complex I is required for optimal photosynthetic performance, despite the operation of alternative dehydrogenases in CMSII; and (b) complex I is necessary to avoid redox disruption of photosynthesis in conditions where leaf mitochondria must oxidize both respiratory and photorespiratory substrates simultaneously.