Glycollate oxidase inhibition and its effect on photosynthesis and pigment formation in Zea mays

We have investigated the effect of 2-hydroxy-3-butynoic acid (HBA) and its methyl ester (MeHBA) on photosynthesis and pigment formation in Zea mays, a C₄ photosynthesis-type plant. In the presence of the specific inhibitor of glycollate oxidase, assimilation of CO₂ was decreased significantly. Labelling patterns showed accumulation of glycollate, though not so marked as in C₃ photosynthesis-type plants, and marked decreases in incorporation into glycine, serine and particularly glycerate. This inhibition was specific for the S(+) enantiomers of HBA and MeHBA. In greening maize R,S-MeHBA inhibited formation of chloroplast pigments and this effect could be shown to be due to the S(+) enantiomer; of a range of metabolises tested only supplementations with serine or pyruvate were partly effective in restoring greening.     

Inhibition by isonicotinyl hydrazide of pigment formation in higher plants

The inhibition of greening of illuminated etiolated maize seedlings by isonicotinyl hydrazide can be alleviated by serine or pyruvate. The similar inhibition in barley can be reversed only by pyruvate. In both plants earlier intermediates in the glycollate pathway and other related compounds were ineffective in overcoming the inhibition of greening produced by isonicotinyl hydrazide. In maize seedlings radioactivity from l-serine-[3-¹⁴C] is poorly incorporated into β-carotene, a typical chloroplast terpenoid, unless glycine and formate or, more effectively, glycine together with isonicotinyl hydrazide are supplied. These supplementations may minimize interconversion of serine and glycine, and hence dilution of radioactivity at C-3 of l-serine by unlabelled C-1 units, before incorporation into terpenoids. The results support the view that in young greening tissue the C2-3 fragment of l-serine can give rise to acetyl-CoA, an obligatory precursor of chloroplast terpenoids.     

Glyoxylate decarboxylation during photorespiration

At 25° C under aerobic conditions with or without gluamate 10% of the [1-¹⁴C]glycollate oxidised in spinach leaf peroxisomes was released as ¹⁴CO₂. Without glutamate only 5% of the glycollate was converted to glycine, but with it over 80% of the glycollate was metabolised to glycine. CO₂ release was probably not due to glycine breakdown in these preparations since glycine decarboxylase activity was not detected. Addition of either unlabelled glycine or isonicotinyl hydrazide (INH) did not reduce ¹⁴CO₂ release from either [1-¹⁴C]glycollate or [1-¹⁴C]glyoxylate. Furthermore, the amount of available H₂O₂ (Grodzinski and Butt, 1976) was sufficient to account for all of the CO₂ release by breakdown of glyoxylate. Peroxisomal glycollate metabolism was unaffected by light and isolated leaf chloroplasts alone did not metabolise glycollate. However, in a mixture of peroxisomes and illuminated chloroplasts the rate of glycollate decarboxylation increased three fold while glycine synthesis was reduced by 40%. Although it was not possible to measure available H₂O₂ directly, the data are best explained by glyoxylate decarboxylation. Catalase reduced CO₂ release and enhanced glycine synthesis. In addition, when a model system in which an active preparation of purified glucose oxidase generating H₂O₂ at a known rate was used to replace the chloroplasts, similar rates of ¹⁴CO₂ release and [¹⁴C]glycine synthesis from [1-¹⁴C]glycollate were measured. It is argued that in vivo glyoxylate metabolism in leaf peroxisomes is a key branch point of the glycollate pathway and that a portion of the photorespired CO₂ arises during glyoxylate decarboxylation under the action of H₂O₂. The possibility that peroxisomal catalase exerts a peroxidative function during this process is discussed.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - INH isonicotinylhydrazide - PHMS pyridyl-2-yl--hydroxymethane sulphonic acid     

Inhibition of glycollate metabolism by amino-oxyacetate: Effects on pigment formation in higher plants

In greening leaf segments amino-oxyacetate inhibited both chlorophyll and carotenoid formation by ca 60 % at 0.5 mM inhibitor concentration. In greening tissue serine: glyoxylate aminotransferase was the only enzyme of the glycollate pathway whose activity was markedly decreased after inhibitor treatment. The inhibition of pigment formation in barley and maize could be alleviated by glyoxylate, pyruvate and acetaldehyde; in the latter case there is probably a preferential reaction with inhibitor which displaces it from combination with enzymic pyridoxal 5′-phosphate.     

The utilization of glycollate by Micrococcus denitrificans: the β-hydroxyaspartate pathway

1. Micrococcus denitrificans utilized glycollate as sole carbon source for aerobic growth. Glyoxylate was utilized less well, and though glycine alone did not support growth it enhanced growth on glyoxylate. 2. During growth on glycollate, ¹⁴C was incorporated from [2-¹⁴C]glycollate into glycine and thence into aspartate, malate and glutamate. No phosphoglycerate was labelled at the earliest times. 3. Glyoxylate was the first product of glycollate utilization, and glycollate oxidase was inducibly formed on transfer of the organism to glycollate-containing media. 4. Extracts of glycollate-grown M. denitrificans contained negligible glyoxylate-carboligase activity and only low tartronate semialdehyde-reductase activity. 5. erythro-β-Hydroxyaspartate is a key intermediate in glyoxylate utilization by this organism. Enzymes catalysing (a) the synthesis of erythro-β-hydroxyaspartate from glyoxylate and glycine, and (b) the conversion of erythro-β-hydroxyaspartate into oxaloacetate, were inducibly formed during growth on glycollate and on other substrates yielding glyoxylate. Methods for the assay of these enzymes were developed. 6. It is concluded that in M. denitrificans the biosynthesis of cell materials from glycollate is accomplished by the `β-hydroxyaspartate pathway', a novel metabolic route that may also perform a catabolic role in glyoxylate oxidation.     

Oxygen inhibition of photosynthesis and stimulation of photorespiration in soybean leaf cells

The occurrence of photorespiration in soybean (Glycine max [L.] Merr.) leaf cells was demonstrated by the presence of an O₂-dependent CO₂ compensation concentration, a nonlinear time course for photosynthetic ¹⁴CO₂ uptake at low CO₂ and high O₂ concentrations, and an O₂ stimulation of glycine and serine synthesis which was reversed by high CO₂ concentration. The compensation concentration was a linear function of O₂ concentration and increased as temperature increased. At atmospheric CO₂ concentration, 21% O₂ inhibited photosynthesis at 25 C by 27%. Oxygen inhibition of photosynthesis was competitive with respect to CO₂ and increased with increasing temperature. The Km (CO₂) of photosynthesis was also temperature-dependent, increasing from 12 μm CO₂ at 15 C to 38 μm at 35 C. In contrast, the Ki (O₂) was similar at all temperatures. Oxygen inhibition of photosynthesis was independent of irradiance except at 10 mm bicarbonate and 100% O₂, where inhibition decreased with increasing irradiance up to the point of light saturation of photosynthesis. Concomitant with increasing O₂ inhibition of photosynthesis was an increased incorporation of carbon into glycine and serine, intermediates of the photorespiratory pathway, and a decreased incorporation into starch. The effects of CO₂ and O₂ concentration and temperature on soybean cell photosynthesis and photorespiration provide further evidence that these processes are regulated by the kinetic properties of ribulose-1,5-diphosphate carboxylase with respect to CO₂ and O₂.     

Effect of phosphinothricin (glufosinate) on photosynthesis and photorespiration of C3 and C4 plants

Phosphinothricin (glufosinate), an irreversible inhibitor of glutamine synthetase, causes an inhibition of photosynthesis in C₃ (Sinapis alba) and C₄ (Zea mays) plants under atmospheric conditions (400 ppm CO₂, 21% O₂). This photosynthesis inhibition is proceeding slower in C₄ leaves. Under non-photorespiratory conditions (1000 ppm CO₂, 2% O₂) there is no inhibition of photosynthesis. The inhibition of glutamine synthetase by phosphinothricin results in an accumulation of NH₄ ⁺. The NH₄ ⁺-accumulation is lower in C₄ plants than in C₃ plants. The inhibition of glutamine synthetase through phosphinothricin in mustard leaves results in a decrease in glutamine, glutamate, aspartate, asparagine, serine, and glycine. In contrast to this, a considerable increase in leucine and valine following phosphinothricin treatment is measured. With the addition of either glutamine, glutamate, aspartate, glycine or serine, photosynthesis inhibition by phosphinothricin can be reduced, although the NH₄ ⁺-accumulation is greatly increased. This indicates that NH₄ ⁺-accumulation cannot be the primary cause for photosynthesis inhibition by phosphinothricin. The investigations demonstrate the inhibition of transmination of glyoxylate to glycine in photorespiration through the total lack of amino donors. This could result in a glyoxylate accumulation inhibiting ribulose-1,5-bisphosphate-carboxylase and consequently CO₂-fixation.Abbreviations GOGAT glutamine-2-oxoglutarate-amidotransferase - GS glutamine synthetase - PPT phosphinothricin - MSO methionine sulfoximine - RuBP ribulose-1,5-bisphosphate     

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.

     

Metabolism and decarboxylation of glycollate and serine in leaf peroxisomes

The linked utilization of glycollate and L-serine has been studied in peroxisomal preparations from leaves of spinach beet (Beta vulgaris L.). The generation of glycine from glycollate was found to be balanced by the production of hydroxypyruvate from serine and similarly by 2-oxoglutarate when L-glutamate was substituted for L-serine. In the presence of L-malate and catalytic quantities of NAD⁺, about 40% of the hydroxypyruvate was converted further to glycerate, whereas with substrate quantities of NADH, this conversion was almost quantitative. CO₂ was released from the carboxyl groups of both glycollate and serine. Since the decarboxylation of both substrates was greatly in creased by the catalase inhibitor, 3-amino-1,2,4-triazole, and abolished by bovine liver catalase, it was attributed to the nonenzymic attack of H₂O₂, generated in glycollate oxidation, upon glyoxylate and hydroxypyruvate respectively. At 25–30° C, about 10% of the glyoxylate and hydroxypyruvate accumulated was decarboxylated, and the release of CO₂ from each keto-acid was related to the amounts present. It is suggested that hydroxypyruvate decarboxylation might contribute significantly to photorespiration and provide a metabolic route for the complete oxidation of glycollate, the magnitude of this contribution depending upon the concentrations of glyoxylate and hydroxypyruvate in the peroxisomes.     

Compartmental modelling of photorespiration and carbon metabolism of water stressed leaves

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

Relationships between glycollate and folate metabolism in Euglena gracilis

When division synchronized cultures of Euglena gracilis Klebs (strain Z) were aerated with 5% CO₂ in air the specific activity of glycollate dehydrogenase was only 13% of that in cultures receiving unsupplemented air. The concentrations of 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) and formylfolate derivatives were also lowered by this treatment. In contrast, the specific activity of serine hydroxymethyltransferase (EC 2.1.2.1) and the concentration of methylfolates were raised by supplying CO₂-supplemented air. These effects on enzyme levels were reversed when air was supplied following a period of CO₂ treatment. The levels of glycollate dehydrogenase, 10-formyl-tetrahydrofolate synthetase and formylfolate derivatives were decreased when cells were aerated in media containing 5 mM α-hydroxy-2-pyridinemethane sulphonate. Cell free extracts had the ability to decarboxylate glyoxylate, producing ca equal amounts of CO₂ and formate from C-1 and C-2 respectively. Cells receiving 5% CO₂ in air had a decreased ability to incorporate formate-[¹⁴C] into serine and methionine. It is concluded that during growth at low CO₂ concentrations glycollate metabolism will provide substrate for the formyltetrahydrofolate synthetase reaction.     

Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

Effect of CO(2) Concentration on Glycine and Serine Formation during Photorespiration

Amount and products of photosynthesis during 10 minutes were measured at different ¹⁴CO₂ concentrations in air. With tobacco (Nicotiana tabacum L. cv. Maryland Mammoth) leaves the percentage of ¹⁴C in glycine plus serine was highest (42%) at 0.005% CO₂, and decreased with increasing CO₂ concentration to 7% of the total at 1% CO₂ in air. However, above 0.03% CO₂ the total amount of ¹⁴C incorporated into the glycine and serine pool was about constant. At 0.005% or 0.03% CO₂ the percentage and amount of ¹⁴C in sucrose was small but increased greatly at higher CO₂ levels as sucrose accumulated as an end product. Relatively similar data were obtained with sugar beet (Beta vulgaris L. cv. US H20) leaves. The results suggest that photorespiration at high CO₂ concentration is not inhibited but that CO₂ loss from it becomes less significant.     

Altered glycine decarboxylation inhibition in isonicotinic Acid hydrazide-resistant mutant callus lines and in regenerated plants and seed progeny

Isonicotinic acid hydrazide (INH), an inhibitor of the photorespiratory pathway blocking the conversion of glycine to serine and CO₂, has been used as a selective agent to obtain INH-resistant tobacco (Nicotiana tabacum) callus cells. Of 22 cell lines that were INH-resistant, none were different from wild-type cells in their ability to take up [³H]INH or to oxidize INH to isonicotinic acid. In 7 of the 22 cell lines, INH resistance was associated with decreased inhibition of NAD-dependent glycine decarboxylation activity in isolated mitochondrial preparations. In the cell line that was most extensively investigated (I 24), this biochemical phenotype (exhibiting a 3-fold higher K_i with INH) was observed in leaf mitochondria of regenerated plants and of plants produced from them by self-fertilization. After crosses between resistant and sensitive plants, the decreased inhibition of glycine decarboxylation was observed among F₂ and backcross progeny only in those plants previously identified as INH-resistant by callus growth tests. In contrast, in siblings identified as INH-sensitive, glycine decarboxylation was inhibited by INH at the wild-type level. This demonstration of the transfer of an altered enzyme property from callus to regenerated plants and through seed progeny fulfills an important requirement for the use of somatic cell genetics to produce biochemical mutants of higher plants.     

The Uptake of (+)-S- and (-)-R-Abscisic Acid by Suspension Culture Cells of Hopbush (Dodonaea viscosa)

The uptake of (+)-S- and (−)-R-abscisic acid (ABA) by suspension culture cells of hopbush (Dodonaea viscosa L. Jacqu.) was followed over a range of temperatures, pH values, and time intervals. The natural (+)-S-ABA was taken up about five times faster than the unnatural (−)-R-ABA. Each 10°C rise in temperature from 1 to 31°C increased the rate of uptake (Q₁₀) of (+)-S-ABA about 2.2-fold, whereas that of the (−)-R increased with a Q₁₀ of 1.4. (+)-ABA was taken into the cells by a saturable carrier, but (−)-ABA and both enantiomers of 2-trans-ABA were not; they appeared to enter by passive diffusion. The uptake of (+)-ABA was linear over the first 8 hours but concentrations within the cells decreased after 2 hours to remain constant after 4 hours as rapid metabolism was induced. Electron microscopy of thin sections of the cells, combined with a stereological analysis of their shape, showed that the vacuoles comprised 80% of the cell volume and the cytoplasm plus nucleus comprised 20%. There were no photosynthetically active plastids in the cells. Concentrations of the endogenous ABA in the cytoplasm (pH 7.32) and vacuoles (pH 5.88) were calculated by applying the Henderson-Hasselbalch equation (ABA pK_a 4.7) so that, provided no active metabolic redistribution occurred, the concentration in the cytoplasm was 7.9 micromolar and that in the vacuole was 0.3 micromolar. In vivo pH was measured by ³¹P nuclear magnetic resonance spectroscopy.     

Relationships between glycollate and formate metabolism in greening barley leaves

The activities of enzymes catalysing glycollate oxidation, formate production and folate-dependent formate utilization were examined in the primary leaves of Hordeum vulgare cv Galt. Seedlings were grown for 6 days in darkness and then transferred to continuous light (500 μinsteins/m² per sec) for up to 5 days. Cell-free extracts of the primary leaves contained glycollate oxidase (EC 1.1.3.1), 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5, 10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) and ability to enzymically decarboxylate glyoxylate. These activities increased during greening and at the end of the light treatment were 70–450% higher than etiolated controls. Greened primary leaves also incorporated [¹⁴C]formate at rates that were three- to four-fold higher than shown by etiolated leaves. The specific activity of 10-formyltetrahydrofolate synthetase was decreased by 20–35% when the leaves were greened in the presence of 10 mM hydroxysulphonate. This inhibitor also reduced the incorporation of [¹⁴C]formate by up to 45%. A potential flow of carbon from glycollate to 10-formyltetrahydrofolate via glyoxylate and formate was suggested by the data.     

Effect of chromate on photosynthesis in Cr(VI)-resistant Chlorella

Chromate-resistant Chlorella spp. isolated from effluents of electroplating industry could grow in the presence of 30 μM K₂Cr₂O₇. Since photosynthesis is sensitive to oxidative stress, chromate toxicity to photosynthesis was examined in this algal isolate. Chromate [Cr(VI)] up to 100 μM was found to stimulate photosynthesis, while 90% inhibition was found, when the cells were incubated with 1 mM Cr(VI) for 4 h. Photosystem (PS) II was inhibited by 80% and PSI by 40% after such Cr(VI) treatment. Thermoluminescence studies on cells treated with 1 mM Cr(VI) for 4 h showed that S₂Q_A ^? recombination peak (Q) was shifted to higher temperature, whereas S₂/S₃Q_B ^? recombination peak (B) was shifted to lower temperature. These shifts indicated alga stress response in order to overcome an excitation stress resulting from the inhibition of photosynthesis by Cr(VI). The nontreated Chlorella cells kept in the dark showed periodicity of four for the Q peak (4–8°C) and B peak (34–38°C) after exposure to series of single, turnover, saturating flashes. This periodicity was lost in Cr(VI)-treated cells. Higher concentrations of Cr(VI) inhibited mainly the electron flow in the electron transport chain, inactivated oxygen evolving complex, and affected also Calvin cycle enzymes in the Cr(VI)-resistant isolates of Chlorella.     

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

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

Inhibition of chlorophyll biosynthesis by lead in greeningPisum sativum leaf segments

Supply of 0.01 to 1.0 mM lead acetate to greening pea(Pisum sativum L.) leaf segments either in the absence or in the presence of inorganic nitrogen lowered total chlorophyll (Chl) content. During a time course study, there was not any appreciable effect of Pb²⁺ upto 4 h but thereafter Pb inhibited Chl synthesis. While supply of succinate, cysteine dithiothreitol, 5,5-dithio-bis-2-nitrobenzoic acid and NH₄C1 had no protective action against Pb²⁺ toxicity, and glycine, glutamate 2-oxoglutarate, MgCl₂, KH₂PO₄, CaCl₂, KC1 protected only partially, reduced glutathione (GSH) could completely overcome the inhibition of Chl biosynthesis by the metal. It is suggested that Pb²⁺ interferes with Chl biosynthesis through GSH availability     

The effect of glyoxylate on photosynthesis and photorespiration by isolated soybean mesophyll cells

Incubating isolated soybean leaf mesophyll cells with glyoxylate increased the rates of CO₂ fixation by as much as 150%. In order to cause this stimulation, the glyoxylate must be presented to the cells before the NaHCO₃. Significant stimulation was observed 15 seconds after beginning the glyoxylate treatment. The glyoxylate-dependent stimulation was increased by high O₂ concentrations and decreased by high CO₂ concentrations. Glyoxylate treatment resulted in a 71% inhibition in the rate of CO₂ incorporation into glycolate and glycine. Glyoxylate may be stimulating net photosynthesis solely by decreasing photorespiration or it may be increasing the amount of CO₂ fixed by both decreasing photorespiration and increasing gross photosynthesis. Ribulose bisphosphate carboxylase, when preactivated and assayed in situ, was unaffected by the glyoxylate treatment.