Glycolate pathway in green algae

By three criteria, the glycolate pathway of metabolism is present in unicellular green algae. Exogenous glycolate-1-¹⁴C was assimilated and metabolized to glycine-1-¹⁴C and serine-1-¹⁴C. During photosynthetic ¹⁴CO₂ fixation the distributions of ¹⁴C in glycolate and glycine were similar enough to suggest a product-precursor relationship. Five enzymes associated with the glycolate pathway were present in algae grown on air. These were P-glycolate phosphatase, glycolate dehydrogenase (glycolate:dichloroindophenol oxidoreductase), l-glutamate:glyoxylate aminotransferase, serine hydroxymethylase, and glycerate dehydrogenase. Properties of glycerate dehydrogenase and the aminotransferase were similar to those from leaf peroxisomes. The specific activity of glycolate dehydrogenase and serine hydroxymethylase in algae was 1/5 to 1/10 that of the other enzymes, and both these enzymes appear ratelimiting for the glycolate pathway.     

Sequence of Formation of Phosphoglycolate and Glycolate in Photosynthesizing Chlorella pyrenoidosa

In Chlorella pyrenoidosa which have been photosynthesizing in either 1.5% ¹⁴CO₂ or 0.05% ¹⁴CO₂ in air, gassing with 100% O₂ results in rapid formation of phosphoglycolate which is apparently converted to glycolate. However, only about one-third to one-half of the rate of glycolate formation can be accounted for by this route. The remaining glycolate formation may be the result of the oxidation of sugar monophosphates. The rates of formation of both glycolate and phosphoglycolate are about four times greater with algae that have been photosynthesizing in 1.5% ¹⁴CO₂ than with algae which have been photosynthesizing with air, when the algae are then gassed with 100% O₂.     

Oxygen effect on photosynthetic and glycolate pathways in young maize leaves

To study the effect of O₂ on the photosynthetic and glycolate pathways, maize leaves were exposed to ¹⁴CO₂ during steady-state photosynthesis in 21 or 1% O₂. At the two O₂ concentrations after a ¹⁴CO₂ pulse (4 seconds) followed by a ¹²CO₂ chase, there was a slight difference in CO₂ uptake and in the total amount of ¹⁴C fixed, but there were marked changes in ¹⁴C distribution especially in phosphoglycerate, ribulose bisphosphate, glycine, and serine. The kinetics of ¹⁴C incorporation into glycine and serine indicated that the glycolate pathway is inhibited at low O₂ concentrations. In 1% O₂, labeling of glycine was reduced by 90% and that of serine was reduced by 70%, relative to the control in 21% O₂. A similar effect has been observed in C₃ plants, except that, in maize leaves, only 5 to 6% of the total ¹⁴C fixed under 21% O₂ was found in glycolate pathway intermediates after 60 seconds chase. This figure is 20% in C₃ plants. Isonicotinyl hydrazide did not completely block the conversion of glycine to serine in 21% O₂, and the first carbon atom of serine was preferentially labeled during the first seconds of the chase. These results supported the hypothesis that the labeled serine not only derives from glycine but also could be formed from phosphoglycerate, labeled in the first carbon atom during the first seconds of photosynthesis.     

Control of photorespiratory glycolate metabolism in an oxygen-resistant mutant of Chlorella sorokiniana

Under a gas atmosphere of 99% O2/1% CO2, wild-type cells of Chlorella sorokiniana excreted 12% of their dry weight as glycolate during photolithotrophic growth, whereas mutant cells excreted glycolate at only 3% of the cellular dry weight. The observed difference in glycolate excretion by the two cell types appears to be due to a different capacity for the metabolism of glycolate, rather than to a different glycolate formation rate. This was concluded from experiments in which the metabolism of glycolate via the glycine-serine pathway was inhibited by the addition of isoniazid. Under such conditions, glycolate excretion rates for both cell types were identical. The mutant appeared to have significantly higher specific activities of glycine decarboxylase, serine hydroxymethyltransferase, serine-glyoxylate aminotransferase, glycerate kinase, and phosphoglycolate phosphatase than did the wild type. The specific activities of D-ribulose-1,5-bisphosphate carboxylase/oxygenase, glycolate dehydrogenase, glyoxylate-aminotransferase, and hydroxypyruvate reductase were the same for wild-type and mutant cells. The internal pool sizes of ammonia and amino acids increased in wild-type cells grown under high-oxygen concentrations but were hardly affected by high oxygen tensions in the mutant cells. Our results indicate that, under the growth conditions applied, the decarboxylation of glycine becomes the rate-limiting step of the glycine-serine pathway for the wild-type cells of C. sorokiniana.     

The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria

The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO(2)-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO(2). Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO(2). These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO(2), glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.     

Formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis

The formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis was investigated and the activities of enzymes of glycolate metabolism assayed. Photosynthetic ¹⁴CO₂ incorporation was O₂ insensitive and no labelled glycolate could be detected in cells incubated at 2 and 21% O₂. Under conditions of 100% O₂ glycolate comprised less than 1% of the acid-stable products indicating ribulose 1,5 bisphosphate (RuBP) oxidation only occurs under conditions of extreme O₂ stress. Metabolism of [1-¹⁴C] glycolate indicated that as much as 62% of ¹⁴C metabolized was released as ¹⁴CO₂ in the dark. Metabolism of labelled glycolate, particularly incorporation of ¹⁴C into glycine, was inhibited by the amino-transferase inhibitor amino-oxyacetate. Metabolism of [2-¹⁴C] glycine was not inhibited by the serine hydroxymethyltransferase inhibitor isonicotinic acid hydrazide and little or no labelled serine was detected as a result of ¹⁴C-glycolate metabolism. These findings indicate that a significant amount of metabolized glycolate is totally oxidized to CO₂ via formate. The remainder is converted to glycine or metabolized via a glyoxylate cycle. The conversion of glycine to serine contributes little to glycolate metabolism and the absence of hydroxypyruvate reductase confirms that the glycolate pathway is incomplete in this cyanobacterium.Abbreviations AAN aminoacetonitrile - AOA aminooxyacetate - DIC dissolved inorganic carbon - INH isonicotinic acid hydrazide - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PG phosphoglycolate - PGA phosphoglyceric acid - PGPase phosphoglycolate phosphatase - PR photorespiration - Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase - TCA trichloroacetic acid - RuBP ribulose-1,5-bisphosphate     

A study of the control of glycolate excretion in chlorella

Chlorella pyrenoidosa cells grown on 5% CO(2) excreted glycolate when incubated in light with 10 mm bicarbonate, but excreted no glycolate under the same conditions when they were maintained on air for 7 hours prior to the assay. Incubation of 5% CO(2)-grown and air-grown cells with 10 mm isonicotinyl hydrazide or 10 mm alpha-hydroxypyridinemethane sulfonate during the assay stimulated the excretion of glycolate by CO(2)-grown cells severalfold that of air-grown cells.Adaptation of CO(2)-grown Chlorella to growth on air did not affect the levels of glycolate dehydrogenase in the cells and did not affect the rate of dark oxidation and metabolism of exogeneous (14)C-glycolate by the cells. These results indicate that the lack of glycolate excretion by air-grown or air-adapted cells of Chlorella cannot be explained by a concomitant change in the level of glycolate dehydrogenase.     

Fixation of O(2) during Photorespiration: Kinetic and Steady-State Studies of the Photorespiratory Carbon Oxidation Cycle with Intact Leaves and Isolated Chloroplasts of C(3) Plants

Mass spectrometric techniques were used to trace the incorporation of [¹⁸O]oxygen into metabolites of the photorespiratory pathway. Glycolate, glycine, and serine extracted from leaves of the C₃ plants, Spinacia oleracea L., Atriplex hastata, and Helianthus annuus which had been exposed to [¹⁸O]oxygen at the CO₂ compensation point were heavily labeled with ¹⁸O. In each case one, and only one of the carboxyl oxygens was labeled. The abundance of ¹⁸O in this oxygen of glycolate reached 50 to 70% of that of the oxygen provided after only 5 to 10 seconds exposure to [¹⁸O]oxygen. Glycine and serine attained the same final enrichment after 40 and 180 seconds, respectively. This confirms that glycine and serine are synthesized from glycolate.

The labeling of photorespiratory intermediates in intact leaves reached a mean of 59% of that of the oxygen provided in the feedings. This indicates that at least 59% of the glycolate photorespired is synthesized with the fixation of molecular oxygen. This estimate is certainly conservative owing to the dilution of labeled oxygen at the site of glycolate synthesis by photosynthetic oxygen. We examined the yield of ¹⁸O in glycolate synthesized in vitro by isolated intact spinach chloroplasts in a system which permitted direct sampling of the isotopic composition of the oxygen at the site of synthesis. The isotopic enrichment of glycolate from such experiments was 90 to 95% of that of the oxygen present during the incubation.

The carboxyl oxygens of 3-phosphoglycerate also became labeled with ¹⁸O in 20- and 40-minute feedings with [¹⁸O]oxygen to intact leaves at the CO₂ compensation point. Control experiments indicated that this label was probably due to direct synthesis of 3-phosphoglycerate from glycolate during photorespiration. The mean enrichment of 3-phosphoglycerate was 14 ± 4% of that of glycine or serine, its precursors of the photorespiratory pathway, in 10 separate feeding experiments. It is argued that this constant dilution of label indicates a constant stoichiometric balance between photorespiratory and photosynthetic sources of 3-phosphoglycerate at the CO₂ compensation point.

Oxygen uptake sufficient to account for about half of the rate of ¹⁸O fixation into glycine in the intact leaves was observed with intact spinach chloroplasts. Oxygen uptake and production by intact leaves at the CO₂ compensation point indicate about 1.9 oxygen exchanged per glycolate photorespired. The fixation of molecular oxygen into glycolate plus the peroxisomal oxidation of glycolate to glyoxylate and the mitochondrial conversion of glycine to serine can account for up to 1.75 oxygen taken up per glycolate.

These studies provide new evidence which supports the current formulation of the pathway of photorespiration and its relation to photosynthetic metabolism. The experiments described also suggest new approaches using stable isotope techniques to study the rate of photorespiration and the balance between photorespiration and photosynthesis in vivo.

     

PHOTOSYNTHESIS OF GLYCINE AND SERINE IN GREEN PLANTS

1. The effects of "carbonyl" reagents on the photosyntheticin-corporation of ¹⁴CO₂ into the assimilation products of tobaccoand spinach leaves were studied. The presence of "carbonyl"reagents causes an increase in the ratio of ¹⁴CO₂ incorporatedin glycine and a decrease in serine. The incorporation of ¹⁴Cfrom glycolate-1-¹⁴C and glycolaldehyde-2-¹⁴C into glycine andserine was also affected by "carbonyl" reagents, as in the caseof ¹⁴CO₂-experiment. 2. The feeding experiments of glycine-1-¹⁴C and serine-1-¹⁴Cin the presence and in the absence of "carbonyl" reagents revealedthat these reagents inhibit the conversion of glycine to serine. 3. The results obtained above, together with the effects ofthiols on ¹⁴CO₂ incorporation presented in this paper, supportthe assumption that glycine and serine are formed via glycolateand glyoxylate during photosynthesis in green plants. 4. Comparison of ¹⁴C incorporation in malate from ¹⁴CO₂, glycolate-1-¹⁴C,glycine-1-¹⁴C and serine-1-¹⁴C in the presence and in the absenceof "carbonyl" reagents suggested the occurrence of the pathwayof the malate formation via glycolate and glyoxylate, not passingthrough glycine and serine, during photosynthesis. ¹ A part of this paper was presented at the Symposium on "Nitrogenand Plant" by the Japanese Society of Plant Physiologists, inOctober, 1963 ² Present address: Radiation Center of Osaka Prefecture, Sakai,Osaka     

The C4-dicarboxylic acid pathway of photosynthesis. Identification of intermediates and products and quantitative evidence for the route of carbon flow

1. When leaves with the C(4)-dicarboxylic acid pathway of photosynthesis are exposed to (14)CO(2) the major labelled compounds formed, in order of labelling, are dicarboxylic acids, 3-phosphoglycerate, bexose phosphates and sucrose. During the present studies several quantitatively minor intermediates were identified and their labelling behaviour is described. 2. The pattern of labelling of dihydroxyacetone phosphate, fructose 1,6-diphosphate and ribulose di- and mono-phosphates during radiotracer pulse-chase experiments was consistent with their operation as intermediates in the pathway of carbon dioxide fixation. 3. Serine, glycine, alanine and glutamate had labelling patterns typical of products secondary to the main flow of carbon. 4. The mechanism of the transfer of label from C-4 of dicarboxylic acids to C-1 of 3-phosphoglycerate was also examined. Evidence consistent with pyruvate being derived from C-1, C-2 and C-3 of oxaloacetate, and for a relationship between ribulose 1,5-diphosphate and the acceptor for the C-4 carboxyl group, was obtained. 5. Evidence is provided that, under steady-state conditions, essentially all the label incorporated from (14)CO(2) into C-1 of 3 phosphoglycerate enters via C-4 of the dicarboxylic acids. These and other studies indicated that the route via dicarboxylic acids is essentially the sole route for entry of carbon into 3-phosphoglycerate.     

Rate of Glycolate Formation During Photosynthesis at High pH

The products of C¹⁴O₂ fixation by Chlamydomonas and Chlorella were studied under conditions most favorable for glycolate synthesis. The highest percentage of the C¹⁴ was incorporated into glycolate in the pH range of 8 to 9. After 1 to 2 minutes as much as 40% of the C¹⁴ was found in glycolate products and only a trace of C¹⁴ was present as phosphoglycerate. Below pH 8 the rate of photosynthesis was much faster, but only a small percent of the C¹⁴ was incorporated into glycolate in 1 or 2 minutes, while a high percent of the C¹⁴ accumulated in phosphoglycerate. C¹⁴ labeling of glycolate even at pH 8 or above did not occur at times shorter than 10 seconds. During the first seconds of photosynthesis, nearly all of the C¹⁴ was found in phosphoglycerate and sugar phosphates. Thus glycolate appears to be formed after the phosphate esters of the photosynthetic carbon cycle.

Washing Chlamydomonas with water 2 or 3 times resulted in the loss of most of their free phosphate. When a small aliquot of NaHC¹⁴O₃ was added to washed algae in the absence of this buffering capacity, the pH of the algal medium became 8 or above and much of the fixed C¹⁴ accumulated in glycolate.

     

3-Phosphoglycerate Phosphatase in Plants: II. Distribution, Physiological Considerations, and Comparison with P-Glycolate Phosphatase

3-Phosphoglycerate phosphatase and phosphoglycolate phosphatase were found in leaves of all 52 plants examined. Activities of both phosphatases varied widely between 1 to 20 micromoles per minute per milligram chlorophyll. Plants were grouped into two categories based upon the relative ratio of activity of 3-phosphoglycerate phosphatase to phosphoglycolate phosphatase. This ratio varied between 2:1 to 4:1 in the C₄-plants except corn leaves which had a low level of 3-phosphoglycerate phosphatase. This ratio was reversed and varied between 1:2 to 1:6 in all C₃-plants except one bean variety which had large amounts of both phosphatases. By differential grinding procedures for C₄ plants a major part of the 3-phosphoglycerate phosphatase was found in the mesophyll cells and P-glycolate phosphatase in the bundle sheath cells. Phosphoglycolate phosphatase, but not 3-phosphoglycerate phosphatase, was located in chloroplasts of C₃- and C₄- plants. Formation of 3-phosphoglycerate phosphatase increased 4- to 12-fold during greening of etiolated sugarcane leaves. This cytosol phosphatase displayed a diurnal variation in sugarcane leaves by increasing 50% during late daylight hours and early evening. It is proposed that the soluble form of 3-phosphoglycerate phosphatase is necessary for carbon transport between the bundle sheath and mesophyll cells during photosynthesis by C₄-plants. In C₃- and C₄-plants this phosphatase initiates the conversion of 3-phosphoglycerate to serine which is an alternate metabolic pathway to glycolate metabolism and photorespiration.     

Mitochondrial glycolate oxidation contributes to photorespiration in higher plants

The oxidation of glycolate to glyoxylate is an important reaction step in photorespiration. Land plants and charophycean green algae oxidize glycolate in the peroxisome using oxygen as a co-factor, whereas chlorophycean green algae use a mitochondrial glycolate dehydrogenase (GDH) with organic co-factors. Previous analyses revealed the existence of a GDH in the mitochondria of Arabidopsis thaliana (AtGDH). In this study, the contribution of AtGDH to photorespiration was characterized. Both RNA abundance and mitochondrial GDH activity were up-regulated under photorespiratory growth conditions. Labelling experiments indicated that glycolate oxidation in mitochondrial extracts is coupled to CO(2) release. This effect could be enhanced by adding co-factors for aminotransferases, but is inhibited by the addition of glycine. T-DNA insertion lines for AtGDH show a drastic reduction in mitochondrial GDH activity and CO(2) release from glycolate. Furthermore, photorespiration is reduced in these mutant lines compared with the wild type, as revealed by determination of the post-illumination CO(2) burst and the glycine/serine ratio under photorespiratory growth conditions. The data show that mitochondrial glycolate oxidation contributes to photorespiration in higher plants. This indicates the conservation of chlorophycean photorespiration in streptophytes despite the evolution of leaf-type peroxisomes.     

Stoichiometry in the assay of ribulose bisphosphate oxygenase and carboxylase

Complete stoichiometry of the reaction catalyzed by ribulose 1,5-bisphosphate (RuBP) oxygenase from spinach and Rhodospirillum rubrum has been determined. Before initiation and after termination, RuBP has been measured either by release of equimolar orthophosphate at 25°C in the presence of 1 n NaOH or by complete carboxylation using ¹⁴CO₂ and RuBP carboxylase. The RuBP-dependent oxygen consumption has been measured continuously with an oxygen electrode. After termination of catalysis, 3-phosphoglycerate production has been determined spectrophotometrically using phosphoglycerokinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, α-glycerophosphate dehydrogenase, ATP, and NADH. To measure phosphoglycolate, this product was first hydrolyzed with alkaline phosphatase and the resultant glycolate oxidized by glycolate oxidase. Attendant H₂O₂ formation catalyzed by peroxidase has then been measured colorimetrically. Interference by ribulose in the measurement of glycolate can be easily corrected. Procedures are rapid and do not require separation of reactants and products. Results are in excellent accord with the expected stoichiometry for catalysis by RuBP oxygenase and also enable an estimate of competing catalysis by RuBP carboxylase.     

The pathway of glycollate utilization in Chlorella pyrenoidosa

1. Exogenous glycollate was rapidly metabolized in both the light and the dark by photoautotrophically grown Chlorella pyrenoidosa. 2. The incorporation of (14)C from [1-(14)C]glycollate by these cells was inhibited by the tricarboxylic acid-cycle inhibitors monofluoroacetate, diethylmalonate and arsenite, and also by alpha-hydroxypyrid-2-ylmethanesulphonate and isonicotinylhydrazine. 3. Short-term kinetic experiments showed over 80% of the total (14)C present in the soluble fraction from the cells to be in glycine and serine after 10s. This percentage decreased with time whereas the percentage radioactivity in glycerate increased for up to 30s then remained steady. The percentage of the total radioactivity present in citrate increased over the experimental period. Malate was the only other tricarboxylic acid-cycle intermediate to become labelled. 4. The kinetic and inhibitor experiments supported the following pathway of glycollate incorporation: glycollate --> glyoxylate --> glycine --> serine --> hydroxypyruvate --> glycerate --> 3-phosphoglycerate --> 2-phosphoglycerate --> phosphoenolpyruvate --> pyruvate --> acetyl-CoA. 5. The specific activities of the enzymes catalysing this metabolic sequence in cell-free extracts were great enough to account for the observed rate of glycollate metabolism of 0.25mumol/h per mg dry wt. of cells in the light.     

Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana

We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C(3) plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO(2) concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C(3) photosynthesis.     

Stimulation of Malate Synthesis by Glycolate in Tomato Leaves in Relation to CO2 Concentration

The mechanism by which malate synthesis from CO2 is increasedunder low concentrations of CO₂ was investigated in C₃ plants.A number of metabolites were administered to illuminated tomatoleaves, and their effects on the incorporation of ¹⁴CO₂ intomalate were determined. Compared with water as a control, glycolate,glyoxylate, D,L-glycerate, glycine, phosphoglycolate and L-serineincreased malate synthesis by factors of 6.8, 3.8, 3.3, 2.5,2.3 and 2.2, respectively. The effect of exogenous glycolateon malate synthesis from CO₂ was dependent on its concentrationup to 100 mu, but was independent of ambient CO₂ concentration.The feeding of l-¹⁴C-glycolate in the light indicated that glycolatestimulated the carbon flow from CO₂ to malate. The analysis of the products of ¹⁴CO₂ fixation in illuminatedleaves supplied with glycolate showed increases in malate andsugar and decreases in serine and phosphate esters. However,this stimulated malate synthesis ceased when malonate was suppliedsimultaneously with glycolate. Treatment with glycolate didnot affect the dark ¹⁴CO₂-fixation, but increased the ¹⁴C-malatesynthesis, with a corresponding decrease in ¹⁴C-aspartate and14^C-glutamate. These results suggest that exogenous glycolateactivates malate dehydrogenase in leaves, and that the increasedglycolate formation at low CO₂ concentrations is associatedwith the increased malate synthesis from CO₂. (Received January 12, 1981; Accepted May 20, 1981)     

Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana

Summary. Serine biosynthesis in plants proceeds by two pathways; the glycolate pathway which is associated with photorespiration and the pathway from 3-phosphoglycerate which is presumed to take place in the plastids. The 3-phosphoglycerate pathway (phosphorylated pathway) involves three enzymes catalyzing three sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase (PSAT) and 3-phosphoserine phosphatase (PSP). cDNA and genomic clones encoding these three enzymes from spinach and Arabidopsis thaliana were isolated by means of heterologous probe screening, homologous EST clones and genetic complementation in an Escherichia coli mutant. The identity of the isolated cDNAs was confirmed by functional complementation of serine auxotrophy in E. coli mutants and/or the detection of catalytic activity in the recombinant enzymes produced in E. coli. Northern blot analyses indicated the most preferential expression of these three genes in light-grown roots. In contrast, the mRNAs of two proteins involved in the glycolate pathway (H-protein of glycine decarboxylase multienzyme complex and serine hydroxymethyltransferase) accumulated to high levels in light-grown shoots. Environmental stresses, such as high salinity, flooding and low temperature, induced changes in mRNA levels of enzymes in the plastidic phosphorylated serine biosynthetic pathway but not in that of the glycolate pathway. These results indicate that the plastidic 3-phosphoglycerate pathway plays an important role in supplying serine in non-photosynthetic tissues in plants and under environmental stresses. Received December 9, 1999 Accepted February 2, 2000     

Excretion of Glycolate, Mesotartrate and Isocitrate Lactone by Synchronized Cultures of Ankistrodesmus braunii

Fixation of ¹⁴CO₂ by synchronized cultures of Ankistrodesmus braunii was highest for young growing cells, low for mature cells, and lowest for dividing cells. The amount of ¹⁴C excreted during photosynthesis followed the same trend. Cells at the end of the growing phase, after 10 hours of a 16-hour light phase, excreted nearly 35% of the total ¹⁴C fixed as one product, glycolate. Dividing cells from the dark phase, when tested in the light, excreted only 4% as much glycolate-¹⁴C as the young growing cells. Dividing cells also excreted as much mesotartrate as glycolate and also some isocitrate lactone and an unidentified acid. None of these excreted acids were found inside the cells in significant amounts. Methods for isolation and identification of the excreted acids are present. With ¹⁴C-labeled algae, it was shown that the excretion of glycolate was light-dependent and inhibited by 1,1-dimethyl-3-(p-chlorophenyl) urea. The excretion of labeled mesotartrate, isocitrate lactone, and an unknown acid, but not glycolate, also occurred in the dark. The excreted mesotartrate was predominantly carboxyl-labeled even after long periods of ¹⁴CO₂ fixation. Since glycolate is known to be uniformly labeled, glycolate could not be the precursor of the carboxyl-labeled mesotartrate. The reason for the specific excretion of glycolate, mesotartrate, and isocitrate lactone is not known, but the metabolism of all three acids by the algae may be limited and each can form dilactides or lactones by dehydration. In this context isocitrate lactone was excreted rather than the free acid.     

A transport system for phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate in Salmonella typhimurium.

Salmonella typhimurium strain LT-2 was found to utilize phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate as sole sources of carbon and energy for growth, but Escherichia coli strains did not. The following evidence suggests that this growth difference was due to the presence in Salmonella cells of an inducible phosphoglycerate permease distinct from previously studied transport systems: (a) The ability of cells to take up 3-phospho[14-C]glycerate was induced by growth in the presence of phosphoenolpyruvate, 2-phosphoglycerate, or 3-phosphoglycerate, but not glycerate, alpha-glycerophosphate, or other carbon sources tested. (b) Uptake of 3-phospho[14-C]glycerate was strongly inhibited by the three nonradioactive inducers of 3-phosphoglycerate uptake, but not by glycerate or alpha-glycerophosphate. (c) Mutants which lost the ability to utilize and take up 3-phosphoglycerate simultaneously lost the ability to utilize 2-phosphoglycerate and phosphoenolpyruvate, but not other compounds tested. (d) Mutant strains which constitutively synthesized the phosphoglycerate transport system could use both phosphoglycerates and phosphoenolpyruvate as sole sources of phosphate at low substrate concentrations. (e) A strain lacking alkaline and acid phosphatases could still grow with 3-phosphoglycerate as sole carbon source. Maximal rates of 3-phospho[14-C]glycerate uptake occurred at pH 6 in the presence of an exogenous energy source. The apparent Km for 3-phosphoglycerate uptake under these conditions was about 10-minus 4 M. The maximal uptake rate (but not the Km) was dependent on potassium ions. Although synthesis of the phosphoglycerate transport system appeared to be under adenosine 3:5-monophosphate control, glucose repressed induction only slightly. The genes controlling synthesis of the phosphoglycerate transport system (pgt genes) appeared to map at about 74 min on the Salmonella chromosome.