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Notes: Abstract Heterozygous plants of barley (Hordeum vulgare L. cv. Maris Mink) with activities of chloroplastic glutamine synthetase (GS) between 47‰ and 97‰ of the wild-type and ferredoxin-dependent glutamate synthase (Fd-GOGAT) activities down to 63‰ of the wild-type have been used to study the control of photosynthetic fluxes. Rates of CO2 assimilation measured over a range of intercellular CO2 concentrations and photon flux densities (PFDs) were little different in the wild-type and a mutant with 47‰ GS, although total activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased by about 20‰ with a decrease in GS to 50‰ of the wild-type. The quantum efficiencies of photosystem II electron transport (ΦPSII and CO2 assimilation ΦCO2) were determined. ΦPSII was lower than expected in mutants with 50‰ less GS under conditions which enhance the photorespiratory flux, but were identical to the wild-type under non-photorespiratory conditions, suggesting that at high rates of photorespiration the electron requirement for net CO2 assimilation declines in plants with decreased GS. This discrepancy in the electron requirement between the wild-type and the 47‰ GS mutant was enhanced at high temperatures and low CO2, conditions which favour oxygenation by Rubisco. Photochemical and non-photochemical chlorophyll a fluorescence quenching as well as the quantum efficiency of excitation-energy capture by open photosystem II reaction centres were differentially affected in mutants with less GS relative to the wild-type when CO2 was lowered or the PFD was varied. The quantum efficiencies of electron transport in photosystems I and II were closely correlated under a range of PFDs and CO2 concentrations, confirming that the rate of linear electron transport was much lower in plants with less GS. It is shown that GS exerts considerable control (flux control coefficients between 0.5 and 1.0) on the electron requirement for CO2 assimilation at high fluxes of photorespiration relative to CO2 assimilation. Apart from the control of GS on protein and Rubisco contents, GS in the wild-type has also some direct positive control on CO2 assimilation. However, negative control on CO2 assimilation was found in mutants with 50‰ less GS. These data, taken with the data on electron requirements for CO2 assimilation, suggest that CO2-fixing processes other than that catalysed by Rubisco, such as carboxylation of phosphoenolpyruvate, or an inhibition of photorespiration (e.g. glycine decarboxylation), may contribute to the observed CO2 exchange and photosystem II electron transport in plants with less GS. In the 63‰-Fd-GOGAT mutant, rates of CO2 assimilation were appreciably lower than in the wild-type under a range of PFDs and CO2 concentrations, which largely reflected lower contents of Rubisco in the Fd-GOGAT mutants. Assimilation of CO2 was inhibited appreciably at high CO2 concentrations. There was little difference in the electron requirement for CO2 assimilation between the wild-type and mutants with less Fd-GOGAT, although there were indications that a triose-phosphate/glycerate-3-phosphate shuttle or cyclic electron transport operates to balance ATP generation and NADP reduction. The latter was supported by a curvilinear relationship of photosystem I and II electron transport in the 63‰ Fd-GOGAT mutant. A positive control is exerted by Fd-GOGAT on the amounts of protein and Rubisco and on CO2 assimilation.

Notes: Abstract. 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.

Notes: Abstract Heterozygous mutants of barley (Hordeum vulgare L. cv. Maris Mink) with decreased activities of chloroplastic glutamine synthetase (GS) between 97 and 47% of the wild type and ferredoxin dependent glutamate synthase (Fd-GOGAT) down to 64% of the wild type have been used to study aspects of glyoxylate metabolism and the effect of glyoxylate on the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vivo. In the leaf, the extractable activities of serine:glyoxylate aminotransferase decreased with a decrease in GS whereas activities of glutamate and alanine:glyoxylate aminotransferase increased, pointing to a re direction of amino donors from serine to glutamate and alanine. Under ambient conditions, the leaf contents of glutamate and alanine declined continuously with a decrease in GS, in parallel with the decrease in total amino acids. Glycine, serine and asparagine contents decreased with a decrease in GS to approximately 70% of the wild type, but increased again with a further decrease in GS. At high irradiances and at low CO2 concentrations, glyoxylate contents exhibited a pronounced minimum between 60% and 80% GS. With a further decrease in GS, glyoxylate contents recovered and approached values similar to the wild type. The activation state of Rubisco showed a negative correlation with glyoxylate contents, indicating that a decrease in GS feeds back on the first step of carbon assimilation and photorespiration. The activation state of stromal fructose-1,6-bisphosphatase was unaffected by a decrease in GS or Fd-GOGAT, whereas the activation state of NADP dependent malate dehydrogenase changed in a complex manner. The CO2photocompensation point, Γ*, was appreciably increased in mutants with 47% GS. ‘Mitochondrial respiration’ in the light (Rd) was reduced with a decrease in GS. Relative rates of CO2 release into CO2-free air between the wild type and the 47%-GS mutant correlated with determinations of Γ*. These data are consistent with the view that when GS is decreased there is an increased oxidative decarboxylation of glyoxylate resulting from a decreased availability of amino donors for the transamination of glyoxylate to glycine, and that when GS activities are lower than 70% of the wild type an additional mechanism operates to reduce the photorespiratory loss of ammonia.

Notes: Abstract A mutant line of barley, R(othamsted)-Pr 79/4, has been isolated which grows poorly in natural air, but normally in air enriched to 0.2% CO2. Analysis of the products of 14CO2 fixation showed that there was no major block in photosynthetic or photorespiratory carbon metabolism in the mutant and that rates of CO2 fixation were only slightly lower than those measured in the wild type (c.v. Maris Mink). Leaves of the mutant line contained only 10% of the catalase (EC 1.11.1.6) activity found in the wild type; and the two major bands of catalase activity detected after starch-gel electrophoresis of extracts of normal leaves were missing from similar extracts of RPr 79/4. Peroxisomes isolated from mutant leaves contained negligible catalase activity, but normal levels of other enzymes involved in photorespiration. Genetic analysis has shown that the mutation is recessive and that both air-sensitivity and catalase-deficiency segregate together in F2 plants derived from a cross between the mutant and the cultivar Golden Promise. [1-14C]Glycollate was not converted to 14CO2 faster in the mutant leaves than in the normal leaves. Thus there was no evidence that photorespiratory CO2 may be obtained by the chemical action of H2O2 on glyoxylate or hydroxypyruvate.

Notes: Abstract Guard-cell protoplasts were isolated by enzymic digestion of the epidermis peeled from the abaxial surface of leaves from Commelina communis L. The protoplasts were separated from mesophyll-cell protoplasts and other contaminants by density-gradient centrifugation, and the purity of the preparations carefully and quantitatively assessed by light microscopy. The preparations of guard-cell protoplasts were then compared with mesophyll-cell protoplasts in terms of the activity of photosystem II as assessed by a) the light-induced evolution of oxygen under both steady-state and flashing light and b) the characteristics of photosystem-II chlorophyll fluorescence. In all experiments, clear photosystem-II activity was found in guard-cell protoplasts, although some subtle distinctions between guard-cell and mesophyll-cell protoplasts were found. The contribution of any contaimination by mesophyll-cell chlorophyll to guard-cell-protoplast signals was estimated to be less than 3% in all cases. The results indicate that photosystem II is present and active in guard cells of Commelina.

Notes: Abstract Wild-type and mutant plants of barley (Hordeum vulgare L. cv. Maris Mink) lacking activities of chloroplastic glutamine synthetase (GS) and of ferredox-in-dependent glutamate synthase (Fd-GOGAT) were crossed to generate heterozygous plants. Crosses of the F2 generation containing GS activities between 47‰ and 97‰ of the wild-type and Fd-GOGAT activities down to 63‰ of the wild-type have been selected to study the control of both enzymes on photorespiratory carbon and nitrogen metabolism. There were no major pleiotropic effects. Decreased GS had a small impact on leaf protein and the total activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). The activation state of Rubisco was unaffected in air, but a decrease in GS influenced the activation state of Rubisco in low CO2. In illuminated leaves, the amino-acid content decreased with decreasing GS, while the content of ammonium rose, showing that even small reductions in GS limit ammonium re-assimilation and may bring about a loss of nitrogen from the plants, and hence a reduction in protein and Rubisco. Leaf amino-acid contents were restored, and ammonium and nitrate contents decreased, by leaving plants in the dark for 24 h. The ratios of serine to glycine decreased with a decrease in GS when plants were kept at moderate photon flux densities in air, suggesting a possible feedback on glycine decarboxylation. This effect was absent in high light and low CO2. Under these conditions ammonium contents exhibited an optimum and amino-acid contents a minimum at a GS activity of 65‰ of the wild-type, suggesting an inhibition of ammonium release in mutants with less than 65‰ GS. The leaf contents of glutamate, glutamine, aspartate, asparagine, and alanine largely followed changes in the total amino-acid contents determined under different environmental conditions. Decreased Fd-GOGAT resulted in a decrease in leaf protein, chlorophyll, Rubisco and nitrate contents. Chlorophyll a/b ratios and specific leaf fresh weight were lower than in the wild-type. Leaf ammonium contents were similar to the wild-type and total leaf amino-acid contents were only affected in low CO2 at high photon flux densities, but mutants with decreased Fd-GOGAT accumulated glutamine and contained less glutamate.

Notes: Abstract. Glycine-accumulating mutants of barley (Hordeum vulgare L.) and Amaranthus edulis (Speg.), which lack the ability to decarboxylate glycine by glycine decarboxylase (GDC; EC 2.1.2.10), were used to study the significance of an alternative photorespiratory pathway of serine formation. In the normal photorespiratory pathway, 5,10-methylenetetrahydrofolate is formed in the reaction catalysed by GDC and transferred to serine by serine hydroxymethyltransferase. In an alternative pathway, glyoxylate could be decarboxylated to formate and formate could be converted into 5,10-methylenetetrahydrofolate in the C1-tetrahydrofolate synthase pathway. In contrast to wild-type plants, the mutants showed a light-dependent accumulation of glyoxylate and formate, which was suppressed by elevated (0.7%) CO2 concentrations. After growth in air, the activity and amount of 10-formyltetrahydrofolate synthetase (FTHF synthetase; EC 6.3.4.4), the first enzyme of the conversion of formate into 5,10-methylenetetrahydrofolate, were increased in the mutants compared to the wild types. A similar increase in FTHF synthetase could be induced by incubating leaves of wild-type plants with glycine under illumination, but not in the dark. Experiments with 14C showed that the barley mutants incorporated [14C]formate and [2-14C]glycollate into serine. Together, the accumulation of glyoxylate and formate under photorespiratory conditions, the increase in FTHF synthetase and the ability to utilise formate and glycollate for the formation of serine indicate that the mutants are able partially to compensate for the lack of GDC activity by bypassing the normal photorespiratory pathway.

Notes: Abstract Flash-induced redox reactions in spinach PS II core particles were investigated with absorbance difference spectroscopy in the UV-region and EPR spectroscopy. In the absence of artificial electron acceptors, electron transport was limited to a single turnover. Addition of the electron acceptors DCBQ and ferricyanide restored the characteristic period-four oscillation in the UV absorbance associated with the S-state cycle, but not the period-two oscillation indicative of the alternating appearance and disappearance of a semiquinone at the QB-site. In contrast to PS II membranes, all active centers were in state S1 after dark adaptation. The absorbance increase associated with the S-state transitions on the first two flashes, attributed to the Z+S1→ZS2 and Z+S2→ZS3 transitions, respectively, had half-times of 95 and 380 μs, similar to those reported for PS II membrane fragments. The decrease due to the Z+S3→ZS0 transition on the third flash had a half-time of 4.5 ms, as in salt-washed PS II membrane fragments. On the fourth flash a small, unresolved, increase of less than 3 μs was observed, which might be due to the Z+S0→ZS1 transition. The deactivation of the higher S-states was unusually fast and occurred within a few seconds and so was the oxidation of S0 to S1 in the dark, which had a half-time of 2–3 min. The same lifetime was found for tyrosine D+, which appeared to be formed within milliseconds after the first flash in about 10% inactive centers and after the third and later flashes by active centers in Z+S3.

Notes: Abstract The turnover of the D1 and D2 proteins of Photosystem II (PSII) has been investigated by pulse-chase radiolabeling in several strains of the cyanobacterium Synechocystis PCC 6803 containing different types and levels of the psbA transcript. Strains lacking psbA1 and psbA3 gene and containing high levels of the psbA2 transcript showed the selective synthesis of D1 whose degradation could be slowed down by the protein synthesis inhibitor lincomycin. In contrast, in strains containing just the psbA3 gene, the intensity of the D1 protein labeling was lower and labeling of the D2 and CP43 proteins was stimulated in comparison to the psbA2-containing strains. In addition, the rate and selectivity of the D1 degradation and its dependence on the presence of lincomycin was proportional to the level of the psbA3 transcript in the particular strain. Consequently, there was parallel, lincomycin-independent and slowed-down breakdown of the D1 and D2 proteins in strains with the lowest level of psbA3 transcript. These results are discussed in terms of a model in which the rate of D1 and D2 degradation in cyanobacteria is affected not only by the rate of PSII photodamage, but also by the availability of newly synthesized D1 protein. Moreover, the comparison of the non-oxygen-evolving D1 mutants D170A** and Y161F*** differing by the presence of tyrosine Z has indicated a minor role of the oxidized form of this secondary PSII electron donor in the donor side mechanism of D1 and D2 protein breakdown.

Notes: Abstract The CO2 dependence of rates of CO2 fixation (A) and photochemistry of PS II at 5, 15 and 30% O2 were analyzed in the C4 plant Amaranthus edulis having a C4 cycle deficiency [phosphoenolpyruvate carboxylase (PEPC) mutants], and in the C4 plant Flaveria bidentis having a C3 cycle deficiency [Rubisco small subunit antisense (αSSU)]. In the wild type (WT) A. edulis and its heterozygous mutant having less than 50% WT PEPC activity there was a similar dependence of A and PS II photochemistry on varying CO2, although the CO2 saturated rates were 25% lower in heterozygous plants. The homozygous plants having less than 2% PEPC of the WT had significant levels of photorespiration at ambient levels of CO2 and required about 30 times ambient levels for maximum rates of A. Despite variation in the capacity of the C4 cycle, more than 91% of PS II activity was linearly associated with A under varying CO2 at 5, 15 and 30% O2. However, the WT plant had a higher PS II activity per CO2 fixed under saturating CO2 than the homozygous mutant, which is suggested to be due to elimination of the C4 cycle and its associated requirement for ATP from a Mehler reaction. In the αSSU F. bidentis plants, a decreased rate of A (35%) and PS II activity (33%) accompanied a decrease in Rubisco capacity. There was some increase in alternative electron sinks at high CO2 when the C3 cycle was constrained, which may be due to increased flux through the C4 cycle via an ATP generating Mehler reaction. Nevertheless, even with constraints on the function of the C4 or C3 cycle by genetic modifications, analyses of CO2 response curves under varying levels of O2 indicate that CO2 assimilation is the main determinant of PS II activity in C4 plants.