Photoassimilation of CO2 by Isolated Bundle-Sheath Strands of Zea mays I. Stimulation of CO2 Assimilation by Adding Various Intermediates of the Photosynthetic Cycle; Evidence for a Deficient Photosystem II Activity

Isolated bundle-sheath (BS) strands from leaves of mature maize plants show enhanced rates of CO₂ fixation in the presence of reduced intermediates of the photosynthetic cycle (R5P, DHAP, FruDP.) 3PGA is the major labelled product of ¹⁴CO₂ fixation whatever the substrate added. CO₂ fixation is much lower with PGA than with reduced intermediates, suggesting a limited capacity of the cells to regenerate RuDP (the CO₂-acceptor) from PGA. These two experimental facts, which are characteristic features of bundle-sheath photosynthesis for maize (a species with agranal bundle-sheath chloroplasts) indicate that phaotosystem II activity is a limiting factor for the evolution of the bundle-sheath photosynthetic process. Nevertheless, a reducing capacity arises as proved by sensitivity of CO₂ fixation to DCMU, particularly when PGA is added to the bundle-sheath. PGA synthesis occurs, in the presence of non-limiting amounts of CO₂, according to the equation: RuDP + CO₂→ 2 PGA; the oxygen effect on ¹⁴CO₂ fixation, at lower CO₂ concentration, is interpreted as a dilution effect of the internal pool of ¹⁴CO₂ by unlabelled CO₂ generated by photorespiration.     

Rates of synthesis and source of glycolate in intact chloroplasts

Summary Intact chloroplasts capable of high rates of CO₂ assimilation completely oxidized 3-phosphoglycerate and dihydroxyacetone phosphate to glycolate when CO₂ concentrations were low. Bicarbonate was converted first into products of the Calvin cycle and then into glycolate. Under high oxygen and at high pH values CO₂ fixation and glycolate formation ceased before bicarbonate was exhausted. This is interpreted as the consequence of a depletion of ribulose diphosphate (RuDP) at the oxygen compensation point, where oxygen consumption by glycolate formation and oxygen evolution by phosphoglycerate reduction balance each other. Depletion of RuDP by glycolate formation is proposed to play a role in the Warburg effect. The maximum rate of glycolate synthesis observed with dihydroxyacetone phosphate as substrate was 35 mol mg^-1 chlorophyll h^-1 at 20°C. This may not reflect the maximum capacity of chloroplasts for glycolate synthesis. Dithiothreitol and catalase, which prevent accumulation of oxygen radicals or H₂O₂ during carbon assimilation, increased glycolate formation. H₂O₂ was inhibitory. Other inhibitors of glycolate formation were glyceraldehyde and carbonylcyanide p-trifluoro-methoxphenylhydrazone. From the sensitivity of glycolate synthesis to uncoupling and the ATP requirement of RuDP formation it is concluded that glycolate originated from RuDP. Different induction periods of carbon fixation and glycolyte formation suggested that glycolate synthesis is not only regulated by the ratio of oxygen to CO₂ but also by another factor.     

C4-acids as the source of carbon dioxide for calvin cycle photosynthesis by bundle sheath cells of the C4-pathway species Atriplex spongiosa

For one group of C₄ species we have proposed that the C₄ acid decarboxylation phase of C₄ photosynthesis proceeds via a NAD ‘malic’ enzyme located in bundle sheath mitochondria. The present studies with Atriplex spongiosa demonstrate the capacity of isolated mitochondria and bundle sheath cell strands to decarboxylate malate at rates commensurate with an integral role in photosynthesis. With bundle sheath cells, rates of H¹⁴CO₃^? fixation into Calvin cycle intermediates and evolution of O₂ when HCO₃^? was added, were above 2 μmoles/min/mg chlorophyll. Similar rates of O₂ evolution resulted from the addition of C₄ acids, and the C-4 carboxyl of malate was rapidly assimilated into photosynthetic intermediates and products.     

Veränderliche Markierungsmuster bei14CO2-Fütterung vonBryophyllum tubiflorum zu verschiedenen Zeitpunkten der Hell/Dunkelperiode

Summary Detached phyllodia ofBryophyllum tubiflorum were fed under illumination with¹⁴CO₂ at different times during the light/dark period (12:12 hours). After photosynthesis in presence of¹⁴CO₂ during the intrinsic dark period the greatest part of soluble radioactivity was found in malate. When the same experiment was repeated during the light period, radioactivity was incorporated mainly into sucrose in the first hours while malate was labelled rather weakly. In the late afternoon (last third of the light period), malate became most heavily labelled again during photosynthesis with¹⁴CO₂.Our results indicate that the synthesis of malate by PEP-carboxylase/malate dehydrogenase is inhibited at certain times during the night/day period by end product inhibition of PEP-carboxylase, as was demonstrated byQueiroz (1967, 1968) andTing (1968) in vitro.During inhibition of the PEP-carboxylase there is no competition between the synthesis of malate and CO₂-fixation by the Calvin cycle. Thus radioactivity can flow into sucrose via the Calvin cycle during this time. When the malate content of the phyllodia is low, CO₂-fixation by PEP-carboxylase is not inhibited. Now this pathway dominates over photosynthesis via the Calvin cycle, for PEP-carboxylase has a higher affinity for CO₂ than carboxydismutase. Therefore malate now becomes more labelled than sucrose.     

Effect of some heterocyclic herbicides on CO2 assimilation by spinach chloroplasts

We have studied the inhibitory effect of heterocyclic herbicides simazine, paraquat, pyrazon and amitrole on photosynthetic CO₂ fixation and on the level of intermediates of the CO₂ assimilation cycle in isolated spinach (Spinacia oleracea) chloroplasts, as well as their in vitro activities on ribulose-1,5-bisphosphate carboxylase and fructose-1,6-bisphosphatase. The half inhibitory concentrations (I₅₀) of CO₂ assimilation were about 1 μM for simazine and paraquat, and 10 μM for pyrazon. Amitrole, with an I₅₀ 100 μM, gave only a weak inhibition. In the presence of simazine or pyrazon the triose-phosphates/phosphoglycerate ratio diminished because of a decrease of the triose-phosphates percentage from 47% to 19%, which means an inhibition of the phosphoglycerate reduction step by a low NADPH synthesis. However, there was not a parallel increase of phosphoglycerate, because of collateral pathways leading to phospho-enolpyruvate, amino acids and other non-identified compounds. Paraquat did not give such a decreased ratio, which could be explained as an inhibition of some step of the Calvin cycle later than triose-phosphates by the H₂O₂ formed in a Mehler reaction. Amitrole did not show any effect on the pattern of intermediates. Simazine and pyrazon at 10 μM concentration promote a 20–30% activation of ribulose-1,5-bisphosphate carboxylase activity, whereas paraquat, pyrazon and simazine showed an I₅₀ about 100 μM for the inhibition of the photosynthetic fructose-1,6-bisphosphatase.     

Fructose-and sedoheptulosebisphosphatase. The sites of a possible control of CO2 fixation by light-dependent changes of the stromal Mg2+ concentration

1. The enzymatic steps of the CO₂ fixation cycle responsible for the overall inhibition of CO₂ fixation caused by the lowering of the Mg²⁺ concentration in the stroma were investigated. For this the Mg²⁺ concentration in the stroma was decreased by addition of the ionophore A 23187, and the levels of the intermediates of the CO₂ fixation cycle in the stroma of intact chloroplasts were assayed by ion exchange chromatography.2. The addition of the ionophore caused an increase of NADPH, ATP, fructose- and sedoheptulosebisphosphate and a dramatic decrease of phosphoglycerate in the stroma. These changes were reversed by the addition of Mg²⁺ and again affected by a subsequent addition of Ca²⁺. Ribulosebisphosphate and pentosemonophosphate levels in the stroma were only a little affected under these different conditions.3. The increase of the NADPH and ATP reflects the decreased utilization of these compounds due to the overall inhibition of CO₂ fixation. As phosphoglycerate and triosephosphate appear to be in near equilibrium with NADPH and ATP, the decrease of phosphoglycerate seems to be a consequence of the changes in the nucleotide levels.4. The rapid increase of fructose- and sedoheptulosebisphosphate after the addition of the ionophore A 23187 clearly demonstrates that the overall inhibition of CO₂ fixation caused by lowering the stromal Mg²⁺ is due to the inhibition of the hydrolysis of these sugar bisphosphates. It is concluded that the activities of fructose- and sedoheptulosebisphosphatase can be controlled by light dependent changes of the stromal Mg²⁺ concentration.     

C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts,mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C₄-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH₄Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C₄-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C₄-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C₄-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C₄-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed ¹⁴CO₂ at high rates and exhibited C₄-acid-dependent O₂ evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.Abbreviations CK carboxykinase - -Kg -ketoglutarate - ME malic enzyme - 3-MPA 3-mercaptopicolinic acid - OAA oxaloacetate - PEP phosphoenolpyruvate - R5P ribose-5-phosphate     

Decarboxylation of malate by isolated bundle-sheath cells of certain plants having the C4-dicarboxylic acid cycle of photosynthesis

Summary Bundle-sheath cells isolated by the grinding and filtration procedure of Edwards and Black (1971b) from species of plants having the C₄-dicarboxylic acid pathway of photosynthesis were tested for the decarboxylation of malate from the C₄-carboxyl position. The bundle-sheath cells, which showed high malic enzyme activity in extracts, decarboxylated 4[¹⁴C]malate at rates sufficient to be involved in photosynthesis. The malate decarboxylation is dependent on the addition of magnesium or manganese and NADP⁺. The activity was increased by raising the temperature from 30 to 50°. The evidence supports the idea that malate may be a carboxyl donor to the reductive pentose-phosphate cycle in bundle-sheath cells in certain C₄-dicarboxylic acid pathway plants such as Zea mays L., Sorghum bicolor L., and Digitaria sanguinalis (L.) Scop.Abbreviations C₄ pathway C₄-dicarboxylic acid pathway - RPP pathway reductive pentose phosphate pathway - C₄ plants plants having the C₄ and the RPP pathways - C₃ plants plants having only the RPP pathway - R5P ribose-5-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Tricine N-tris-(hydroxymethyl)methylglycine     

Photosynthetic activities of isolated bundle sheath cells in relation to differing mechanisms of C-4 pathway photosynthesis.

Photosynthetic activities of bundle sheath cell strands isolated from several C₄ pathway species were examined. These included species that decarboxylate C₄ acids via either NADP-malic enzyme (Zea mays, NADP-malic enzyme-type), NAD-malic enzyme (Atriplex spongiosa and Panicum miliaceum, NAD-malic enzyme-type) or phosphoenolpyruvate carboxykinase (Chloris gayana and Panicum maximum, phosphoenolpyruvate carboxykinase-type). Preparations from each of these species fixed ¹⁴CO₂ at rates ranging between 1.2 and 3.5 μmol min^?1 mg^?1 of chlorophyll, with more than 90% of the ¹⁴C being assimilated into Calvin cycle intermediates. With added HCO₃^? the rate of light-dependent O₂ evolution ranged between 2 and 4 μmol min^?1 mg^?1 of chlorophyll for cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species but with Z. mays cells there was no O₂ evolution detectable. Most of the ¹⁴CO₂ fixed by Z. mays cells provided with H¹⁴CO₃^? plus ribose 5-phosphate accumulated in the C-1 of 3-phosphoglycerate. However, 3-phosphoglycerate reduction was increased several fold when malate was also provided. Cells from all species rapidly decarboxylated C₄ acids under appropriate conditions, and the CO₂ released from the C-4 carboxyl was reassimilated via the Calvin cycle. Malate decarboxylation by Z. mays cells was dependent upon light and an endogenous or exogenous source of 3-phosphoglycerate. Bundle sheath cells of NAD-malic enzyme-type species rapidly decarboxylated [¹⁴C]malate when aspartate and 2-oxoglutarate were also provided, and [¹⁴C]aspartate was decarboxylated at similar rates when 2-oxoglutarate was added. Cells from phosphoenolpyruvate carboxykinase-type species decarboxylated [¹⁴C]aspartate when 2-oxoglutarate was added and they also catalyzed a slower decarboxylation of malate. Cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species evolved O₂ in the light when C₄ acids were added. These results are discussed in relation to proposed mechanisms for photosynthetic metabolism in the bundle sheath cells of species utilizing C₄ pathway photosynthesis.     

Glycerate-3-phosphate, produced by CO2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II

We demonstrated recently that, in intact cells of Chlamydomonas reinhardtii, interruption of CO₂ fixation via the Calvin cycle inhibits the synthesis of proteins in photosystem II (PSII), in particular, synthesis of the D1 protein, during the repair of PSII after photodamage. In the present study, we investigated the mechanism responsible for this phenomenon using intact chloroplasts isolated from spinach leaves. When CO₂ fixation was inhibited by exogenous glycolaldehyde, which inhibits the phosphoribulokinase that synthesizes ribulose-1,5-bisphosphate, the synthesis de novo of the D1 protein was inhibited. However, when glycerate-3-phosphate (3-PGA), which is a product of CO₂ fixation in the Calvin cycle, was supplied exogenously, the inhibitory effect of glycolaldehyde was abolished. A reduced supply of CO₂ also suppressed the synthesis of the D1 protein, and this inhibitory effect was also abolished by exogenous 3-PGA. These findings suggest that the supply of 3-PGA, generated by CO₂ fixation, is important for the synthesis of the D1 Protein. It is likely that 3-PGA accepts electrons from NADPH and decreases the level of reactive oxygen species, which inhibit the synthesis of proteins, such as the D1 protein.     

Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C(4) Plant Zea mays: Capacities for CO(2) Assimilation and Malate Decarboxylation

Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO₂ assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30°C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO₂ assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C₄ photosynthesis by species of the NADP-malic enzyme type.     

The relationship between contents of photosynthetic metabolites and the rate of photosynthetic carbon assimilation in leaves of Amaranthus edulis L.

The relationship between the gas-exchange characteristics of attached leaves of Amaranthus edulis L. and the contents of photosynthetic intermediates was examined in response to changing irradiance and intercellular partial pressure of CO₂. After determination of the rate of CO₂ assimilation at known intercellular CO₂ pressure and irradiance, the leaf was freeze-clamped and the contents of ribulose-1,5-bisphosphate, glycerate-3-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate, triose phosphates, phosphoenolpyruvate, pyruvate, oxaloacetate, aspartate, alanine, malate and glutamate were measured. A comparison between the sizes of metabolite pools and theoretical calculations of metabolite gradients required for transport between the mesophyll and the bundle-sheath cells showed that aspartate, alanine, glycerate-3-phosphate and triose phosphates were present in sufficient quantities to support transport by diffusion, whereas pyruvate and oxaloacetate were not likely to contribute appreciably to the flux of carbon between the two cell types. The amounts of ribulose-1,5-bisphosphate were high at low intercellular partial pressures of CO₂, and fell rapidly as the CO₂-assimilation rate increased with increasing intercellular partial pressures of CO₂, indicating that bundle-sheath CO₂ concentrations fell at low intercellular partial pressures of CO₂. In contrast, the amount of phosphoenolpyruvate and of C₄-cycle intermediates declined at low intercellular partial pressures of CO₂. This behaviour is discussed in relation to the co-ordination of carbon assimilation between the Calvin and C₄ cycles.Abbreviations PEP phosphoenolpyruvate - PGA glycerate-3-phosphate - p _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - triose-P triose phosphates     

Ribulose 1,5-diphosphate carboxylase and Chlorobium thiosulfatophilum

1. Cell-free extracts of the photosynthetic bacterium Chlorobium thiosulfatophilum, strains 8327 and Tassajara, were assayed for ribulose 1,5-diphosphate (RuDP) carboxylase and phosphoribulokinase-the two enzymes peculiar to the reductive pentose phosphate cycle. 2. RuDP carboxylase was consistently absent in strain 8327. The Tassajara strain showed a low RuDP-dependent CO₂ fixation activity that was somewhat higher in cells following transatlantic air shipment than in freshly grown cells. The stability and behaviour of this activity in sucrose density gradients were similar to those described by other workers. 3. The radioactive carboxylation products formed in the presence of RuDP by enzyme preparations from the Tassajara strain did not include 3-phosphoglycerate-the known product of the RuDP carboxylase reaction, but instead consisted of the unrelated acids glutamate, aspartate and malate. 4. Phosphoribulokinase was absent in all preparations of the two Chlorobium strains tested. By contrast, phosphoribulokinase as well as RuDP carboxylase were readily demonstrated in preparations from pea chloroplasts and the photosynthetic bacterium Rhodospirillum rubrum. 5. It is concluded that C. thiosulfatophilum appears to lack RuDP carboxylase, phosphoribulokinase, and hence, the reductive pentose phosphate cycle.Support of a J. S. Guggenheim Fellowship is gratefully acknowledged     

Optimization of CO₂fixation in photosynthetic cells via thermodynamic buffering

Stable operation of photosynthesis is based on the establishment of local equilibria of metabolites in the Calvin cycle. This concerns especially equilibration of stromal contents of adenylates and pyridine nucleotides and buffering of CO₂ concentration to prevent its depletion at the sites of Rubisco. Thermodynamic buffering that controls the homeostatic flux in the Calvin cycle is achieved by equilibrium enzymes such as glyceraldehyde phosphate dehydrogenase, transaldolase and transketolase. Their role is to prevent depletion of ribulose-1,5-bisphosphate, even at high [CO₂], and to maintain conditions where the only control is exerted by the CO₂ supply. Buffering of adenylates is achieved mainly by chloroplastic adenylate kinase, whereas NADPH level is maintained by mechanisms involving alternative sinks for electrons both within the chloroplast (cyclic phosphorylation, chlororespiration, etc.) and shuttling of reductants outside chloroplast (malate valve). This results in optimization of carbon fixation in chloroplasts, illustrating the principle that the energy of light is used to support stable non-equilibrium which drives all living processes in plants.     

Photosynthetic carbon metabolism of isolated corn chloroplasts

Chloroplasts have been isolated from 4- to 6-day-old corn (Zea mays) leaves capable of assimilating 45 micromoles CO₂ per milligram chlorophyll per hour. The effects of various factors such as inorganic phosphate, reducing agents, inhibitors, intermediates of the photosynthetic carbon reduction cycle, organic acids, and oxygen on the photosynthetic rate and on the distribution of ¹⁴C within the products by these chloroplasts were determined. The photosynthetic carbon metabolism of the corn plastids appeared to be similar to that already observed in spinach and pea chloroplasts. It was concluded that the corn plastids can fix CO₂ at meaningful rates via the photosynthetic carbon reduction cycle of Calvin without the operation of a cycle involving the C-4 compounds, malate and aspartate.     

Wide range of metabolic adaptations to the acquisition of the Calvin cycle revealed by comparison of microbial genomes

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO₂ as a potential substrate for biorefining. The most common CO₂ fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO₂ concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indicate yet unknown regulatory mechanisms in Calvin cycle-utilizing microbes. Our gene ranking provides targets for experiments seeking to improve CO₂ fixation, or engineer novel CO₂-fixing organisms.     

Compartmentation and export of 14CO2 fixation products in mesophyll protoplasts from the C4-plant Digitaria sanguinalis

Isolated mesophyll protoplasts, and protoplast extracts containing intact chloroplasts, from the C₄ species Digitaria sanguinalis have been used to study Compartmentation and export of C₄ acids, using different C₃ precursors as substrate for ¹⁴CO₂ fixation. Mg²⁺ was necessary for maximum ¹⁴CO₂ fixation rates with both protoplasts and protoplast extracts, whereas Mg²⁺ was inhibitory for oxaloacetate and phosphoglycerate reduction. This inhibition could be overcome by preincubating the materials in the light with excess of EDTA before addition of Mg²⁺. Under these conditions pyruvate as substrate for ¹⁴CO₂ fixation induced mainly malate formation, whereas phosphoglycerate as substrate induced oxaloacetate formation, indicating competition for available NADPH between oxaloacetate and phosphoglycerate reduction. Oxaloacetate could be exported from the protoplasts at rates comparable to the rates of ¹⁴CO₂ fixation in intact leaves (200 μmol/mg Chl × h). This product probably passed the plasma membrane by simple diffusion, whereas the export of malate and aspartate seemed to be regulated, with the size of the intraprotoplast pool being relatively independent of the export rate. It is concluded that transport via the plasma membrane-cell wall path may play a role in metabolite flow during photosynthesis in C₄ plants.     

Glycine decarboxylase is confined to the bundle-sheath cells of leaves of C3−C4 intermediate species

Immunogold labelling has been used to determine the cellular distribution of glycine decarboxylase in leaves of C₃, C₃–C₄ intermediate and C₄ species in the genera Moricandia, Panicum, Flaveria and Mollugo. In the C₃ species Moricandia foleyi and Panicum laxum, glycine decarboxylase was present in the mitochondria of both mesophyll and bundle-sheath cells. However, in all the C₃–C₄ intermediate (M. arvensis var. garamatum, M. nitens, M. sinaica, M. spinosa, M. suffruticosa, P. milioides, Flaveria floridana, F. linearis, Mollugo verticillata) and C₄ (P. prionitis, F. trinervia) species studied glycine decarboxylase was present in the mitochondria of only the bundle-sheath cells. The bundle-sheath cells of all the C₃–C₄ intermediate species have on their centripetal faces numerous mitochondria which are larger in profile area than those in mesophyll cells and are in close association with chloroplasts and peroxisomes. Confinement of glycine decarboxylase to the bundle-sheath cells is likely to improve the potential for recapture of photorespired CO₂ via the Calvin cycle and could account for the low rate of photorespiration in all C₃–C₄ intermediate species.Abbreviation and symbol kDa kilodaltons - CO₂ compensation point     

Control of photosynthesis in leaves as revealed by rapid gas exchange and measurements of the assimilatory force FA

The rapid transients of CO₂ gas exchange have been measured in leaves ofHelianthus annuus L. In parallel experiments the assimilatory force F_A, which is the product of the phosphorylation potential and the redox ratio NADPH/NADP, has been calculated from measured ratios of dihydroxyacetone phosphate to phosphoglycerate in the chloroplast stroma and in leaves. The following results were obtained: (i) When the light-dependent stroma alkalization was measured under steady-state conditions for photosynthesis in air containing 2000 μl · l^-1 CO₂, alkalization increased with photosynthesis as the quantum flux density (irradiance) was increased. This contrasts to the light-dependent stroma alkalisation measured in dark-adapted leaves during the dark-light transient (Laisk et al. 1989, Planta177, 350–358) which reached a maximum at a quantum flux density far below that necessary to saturate photosynthesis. This maximum was about three times higher than the maximum stroma alkalization at light- and CO₂-saturated photosynthesis. (ii) Accurate calculations of the assimilatory force F_A require a consideration of the stromal pH. However, under many conditions, changes in the stromal pH resulting from changes in photosynthetic flux can be neglected because they are small. (iii) Stromal ratios of dihydroxyacetone phosphate to phosphoglycerate are generally lower than ratios measured in leaf extracts. The value of F_A calculated from stromal metabolites was about 30% lower than F_A calculated from cellular metabolites. Still, it appears sufficient for many purposes to calculate F_A from metabolite measurements in leaf extracts. (iv) In the light, the catalytic capacity of the photosynthetic apparatus is adjusted to the level of irradiance. The response of carbon assimilation to large increases in irradiance is slow because it requires enzyme activation. Deactivation of the Calvin cycle induced by decreases in irradiance is slower than activation. (v) Changes in catalytic capacity and in the availability or level of substrates such as CO₂ alter the flux resistance of the Calvin cycle. A decrease in flux resistance explains why F_A often does not increase by much and may actually decrease when carbon flux is increased. Adjustments of flux resistances in the Calvin cycle and of photosystem-II activity in the electron-transport chain permit varying rates of photosynthesis at low levels of ATP and NADPH. As NADP remains available, the danger of over-reduction which leads to photoinactivation of electron transport is minimized. K.R. und U.H. were guests of the Estonian Academy of Sciences. Support by the Estonian Academy of Sciences, the Sonderforschungsbereich 251 of the University of Würzburg and the Fonds der Chemischen Industrie is gratefully acknowledged.