Role of Glycine and Glyoxylate Decarboxylation in Photorespiratory CO2 Release' Received for Publication February 5, 1981 and in Revised Form May 12, 1981

Plant Physiol. (1981) 68,1031-1034 0032-0889/81/68/103 1/04/$00.50/0

Role of Glycine and Glyoxylate Decarboxylation in Photorespiratory CO2 Release' Received for publication February 5, 1981 and in revised form May 12, 1981

DAVID J. OLIVER Department ofBacteriology and Biochemistry, University ofIdaho, Moscow, Idaho 83843

ABSTRACT anism was shared with added glycine, was sensitive to INH2 (14) and KCN, and therefore involved the mitochondrial glycine de- Mechanicafly isolated soybean leafcells metabolized added glycolate by carboxylation reaction. The second mechanism of CO2 release two mechanisms, the direct oxidation ofglyoxylate and the decarboxylation from glycolate was insensitive to INH and KCN, not inhibited by of glycine. The rate of glyoxylate oxidation was dependent on the cellular added glycine, insensitive to glycidate, an inhibitor of the gluta- glyoxylate concentratn and was linear between 0.58 and 2.66 micromoles mate:glyoxylate amino transferase (9), and inhibited by the gly- glyoxylate per milligam chlorophyll Tbe rate extrapolated to zero at a colate oxidase inhibitor methylhydroxybutynoate (5). It appar- concentration of zero. The concentration and, therefore, the rate of oxi- ently resulted from the direct decarboxylation of glyoxylate. The dation of glyoxylate could be decreased by adding glutamate or serine to rates of CO2 release from these sites were approximately equal. the cells. These substrates were amino donors for the transamination of Recently, Somerville et al. (17) have shown that Arabidopsis glyoxylate to glycine. In the presence of these amino acids more CO1 was thaliana mutants with no measurable leaf mitochondrial serine released from added glycolate via the glycine decarboxylation reaction and hydroxymethyl transferase activity photorespire. The appearance less by the direct oxidation of glyoxylate. of photorespiratory CO2 follows a lag while glycine accumulates Leaves from soybean plants of various ages grown under different and probably results from the direct oxidation of glyoxylate. This nitrogen regimes had glyoxylate concentrations of about 80 to 100 nano- photorespiration was inhibited by supplying ammonia. The au- moles per millgram chlorophylL Using the isolated cells as a model to thors suggest that direct glyoxylate decarboxylation occurred only determine the relationships between the glyoxylate concentration and rate under conditions of amide depletion. of its decarboxylatfon indicated that about 2.5% of the photorespiratory Data are presented to suggest that the direct decarboxylation of CO2 would arise from this reactio This percentage would not be expected glyoxylate occurs only in isolated soybean leaf cells under condi- to vary greatly with growth conditions. tions ofextreme nitrogen deprivation and that when supplied with adequate amino donors, glyoxylate was transaminated to glycine with little direct decarboxylation. The possible physiological sig- nificance of these observations is discussed.

MATERIALS AND METHODS During photosynthetic carbon fixation in many species, carbon is drawn from intermediates in the Calvin cycle to make glycolate. Soybean mesophyll cells were isolated from greenhouse grown Before the carbon can reenter the cycle it must be processed plants as described earlier (13). These cell preparations had CO2 through the glycolate pathway. During this processing, some part fixation rates of 30 to 40 ,umol/mg Chl-h at 0.5 mm NaHCO3 and of the carbon is lost as CO2 (18, 20). The major part of this 21% 02. The CO2 fixation rate increased about 50%o when the 02 photorespiratory CO2 loss has been associated with the mitochon- concentration was decreased to 0%o. The [1-14C]glycolate (Amer- drial conversion of 2 mol of glycine to one each of serine, C02, sham Corp.) and [1-14CJglycine (Research Products International and NH3 (8, 18). Corp.) were used without further purification. Isonicotinic acid Several authors, however, have presented data which could be hydrazide and aminoacetonitrile were from Sigma. interpreted as suggesting that some CO2 could result from the Decarboxylation reactions were run in 10-ml sidearm flasks that direct oxidation of glyoxylate. Zelitch (21) showed that oxidants were equipped with serum stoppers and removable centerwells produced by illuminated chloroplasts were able to oxidize added (Kontes Glass Co.). The reactions were terminated and any 14CO2 glyoxylate to CO2 and formate. In isolated peroxisomes, excess was released by injecting 0.1 ml of 2 N H2SO4 (12). The 1'CO2 was H202 produced by the glycolate oxidase reaction results in glyox- trapped in a filter paper wick that was dampened with 25 ul of 5 ylate decarboxylation (2-4). Kinetic studies with intact leaves (1) N monoethanolamine, and quantitated by liquid scintillation and isolated cells (16) have suggested that the rate of glycolate counting in a toluene cocktail containing 1% Protosol (New Eng- synthesis can readily exceed the rate of glycine synthesis, suggest- land Nuclear). Unless otherwise indicated the reactions were run ing that carbon is lost (probably as C02) between glycolate and in the dark. glycine. The NH3 produced during the reaction was determined by using Inhibitor studies with mechanically isolated soybean leaf cells a modified Seligson apparatus (15) constructed from a 20-ml have shown conclusively that with these preparations added gly- scintillation vial, a no. 2 one-hole rubber stopper, and a short glass colate was metabolized by two mechanisms (11). The first mech- rod. The NH3 was released from 0.5 ml of the acid-terminated reaction mix by adding an equal volume of saturated K2CO3 to ' This work was supported by United States Department ofAgriculture, the scintillation vial and was trapped on a drop of 10 N H2SO4 on Science and Education Administration, Competitive Research Grants Office Award 59-2161-0-1-490-0 and is publication no. 8154 of the Idaho 2Abbreviations: INH, Isonicotinic acid hydrazide; AAN, aminoaceto- Agricultural Experiment Station. nitrile. 1031 1032 OLIVER Plant Physiol. Vol. 68, 1981 the tip of the glass rod. The amount of NH3 trapped was deter- reaction, could substitute for glutamate (Table I). mined by the Nesslers reaction (6). These data confirm that isolated soybean leafcells, metabolizing Glyoxylate was determined colorimetrically by measuring the added glycolate, release much of the photorespiratory CO2 from ferricyanide oxidized phenylhydrazone derivative (10). Leaves the direct decarboxylation of glyoxylate. When amino donors were plunged into liquid N2 before detaching from plants. After were readily available, however, the glyoxylate was transaminated the leaves were thoroughly ground, the still frozen powder was to glycine and glyoxylate decarboxylation was replaced by INH- extracted with 5 ml of ether to remove Chl and denature proteins. sensitive glycine decarboxylation as the source ofphotorespiratory After 1 h, 5 ml of 4 N phosphate buffer (pH 7.0) was added. Over CO2 loss. 95% of added glyoxylate was found to partition into the aqueous The glycine decarboxylase reaction has a fixed stoichiometry of phase. 2 mol of glycine yielding one each of CO2, NH3, and serine (18). The reassimilation ofNH3 was blocked by the glutamine synthase RESULTS AND DISCUSSION inhibitor, methionine sulfoximide (6), and cells were darkened to prevent the photosynthetic refixation ofthe photorespiratory CO2. Substantial '4CO2 was released from [1-14C] lycolate under Under these conditions, the metabolism of added glycine yielded conditions where the release of 14CO2 from [1- 4C]glycine was equal rates of CO2 and NH3 release (Table II). The addition of blocked. This resulted whether the glycine decarboxylation reac- glutamate did not alter this ratio. When soybean leaf cells metab- tion was inhibited by INH (Fig. 1) or AAN, an alternate inhibitor olized glycolate, the rate of CO2 release was 4 times the rate of ofthe reaction (19) (Table I). The sensitivity of 0CO2 release from NH3 release. This provides additional proof that under these glycolate to INH or AAN could, however, be completely restored conditions, large amounts of CO2 were being released from the by including 20 mm glutamate in the reaction mixture (Fig. 1 and direct oxidation of glyoxylate. Table I). Serine, the alternate substrate for the transamination The addition of 20 mm glutamate to cells metabolizing added glycolate stimulated the rate of CO2 release from 5.4 to 9.2 ,umol/ I 12 0 mg Chl.h. Ammonia release was increased from 1.3 to 8.0,umol/ GLYCINE 0 CONTROL mg Chl-h (Table II). Glutamate addition decreased the ratio of 0 ** GLUTAMATE CO2 release to NH3 release from 4.15 to 1.15. In this latter case, GLYCOLATE CONTROL the site ofphotorespiratory 8 a CO2 release was shifted from the direct - \\ * GLUTAMATE' decarboxylation of glyoxylate to the glycine decarboxylation re- I action by the addition of the substrate for the transaminase reaction, glutamate. ma4 Glutamate and serine stimulated CO2 release from glycolate in EJ cells that were not preincubated with INH (Fig. 2). The increase e in CO2 loss was accompanied by a decrease in the tissue levels of glyoxylate, presumably because of its transamination to di0 I al glycine 0 0 10 20 30 (Fig. 2). If, as indicated, the movement ofcarbon from glyoxylate INH (mM) to glycine increases the rate of CO2 release, then the propensity FIG. 1. The effect of INH and glutamate on '4CO2 release from [1- for photorespiratory CO2 release from glycine must be greater 14Clglycolate and glycine by soybean leaf cells. Cells were preincubated than from glyoxylate. with INH and 20 mm glutamate as indicated for 30 min before the addition The kinetics for the stimulation of CO2 release from glycolate of 10 mm glycine or glycolate. After an additional 15 min at 27 C the by the two amino acids were different. Glutamate increased the reaction was stopped and any "CO2 released by adding H2SO4 to a final decarboxylation rate from 1.28 to 6.39 ,mol/mg Chl.h. Serine concentration of 0.2 N. stimulated CO2 release from a control value of 1.28 to a maximum of 3.87 ,umol/mg Chl.h. Concentrations of serine above 10 mM Table I. Effect ofAminoacetonitrile (AAN) on 14C02 Releasefrom showed a small but reproducible decrease from the maximum rate [1-l4CJglycolate and [1-_4CJGlycine by Isolated Soybean Leaf Cells of CO2 release (Fig. 2). The cells were preincubated with AAN, 20 mm glutamate, or 10 mm The increase in CO2 release was half maximal at about 1.5 mM serine, as indicated for 15 min before the addition of either 10 mm for serine and 4 to 6 mm for glutamate. The Km for glutamate of [1-'4Clglycolate or 10 mm [1l-4CJglycine. After an additional 15 min, the the glutamate:glyoxylate aminotransferase has been reported to reaction was stopped by adding H2SO4 to a final concentration of 0.2 N. be between 3.6 mm (7) and 5.7 mm (9). The Km for serine of the serine:glyoxylate aminotransferase has been reported as 1.5 mM Substrate Amino Donor AAN Release (9). All of these values were for the tobacco enzymes. mM pmol/mg Chl.h Table II. Effect of Glutamate on the Rate of CO2 and NH3 Releasefrom Glycine 0 3.74 Glycolate and Glycine by Soybean Leaf Cells Serine 0 3.58 Cells were preincubated in the dark with 5 mm methionine sulfoximine Glutamate 0 4.38 for 15 min before the addition of 10 mM [1-'4CJglycine, 10 MM _1-'4CJ- glycolate, and 20 mm glutamate as indicated. After 30 min, the cells were 30 0.11 killed by adding acid, and "4CO2 release and NH3 formation were mea- Serine 30 0.09 sured. Glutamate 30 0.08 Rate of Release Glycolate 0 1.60 Substrate C02/NH3 Serine 0 4.94 CO2 NH3 Glutamate 0 8.03 .tmol/mg Chl.h ratio Glycine 11.8 11.0 1.07 30 1.09 Glycine + glutamate 12.5 12.7 0.98 Serine 30 0.11 Glycolate 5.4 1.3 4.15 Glutamate 30 0.10 Glycolate + glutamate 9.2 8.0 1.15 Plant Physiol. Vol. 68,1981 GLYOXYLATE DECARBOXYLATION 1033 0 0 E I I I II i7 : 1.5 0 0 me E / a. Is s II 2 22E2. '3 / *~~~~~~~~~I16 7 a ) 0.5 a0~ 0 0 i3 'U 0 10 JI ml I- N0 i P.I 0 I I 0 0 1 2 3 0 (50 5 10 1S GLYOXYLATE 0 a CONC, AMINO ACID CONC, mU pmol/mg Chi 3B. The between concentration FIG. 2. The effect ofglutamate and serine on the rates of '4CO2 release FIG. relationship cellular glyoxylate and glyoxylate formation from [1-'4Clglycolate by soybean leafcells. Cells and the rate of INH-insensitive CO2 release. The data from Figure 3A has were preincubated at the indicated amino acid concentration for 20 min been replotted. before 10 mm glycolate was added. The reaction was terminated after an Table III. Effect ofNitrogen Deficiency on the Level of Glyoxylate in additional 15 mi. Intact Soybean Leaves Twelve plants were grown under each treatment. After 8 weeks the 0 center leaflets from each of the three newest trifoliates were plunged into liquid N2 before the petiole was cut. Glyoxylate was measured as in (10). 3 L£3 The values shown are the mean of four determination ± SD. In all cases the plants were exposed to 2 h of sunlight before the leaves were sampled. 0 0O GLYOXYLATE 0 GLUTAMATE 0 The plants were grown in sterile sand and were watered weekly with !20 0 SEI E solutions containing 0, 0.1, and 1.0 mm KNO3 for the low, medium, and high N treatments, respectively. Nitrogen Level Glyoxylate Concn I~~~~~~~~~~~~~~~~~~I nmol/mg Chl Low 82 ± 6

0N _+ Medium 90 ± 4 High 85 ± 8 AMIINO ACIDMAT CONC, FIG. 3A. The effect of glutamate and serine on the rates of "'CO2 may be inhibitory because they decrease the maximum rate of release and glyoxylate formation from [1l-"Cglycolate by INH-treated glycolate decarboxylation. The line generated by comparing gly- soybean leaf cells. The cells were preincubated with the indicated concen- oxylate concentration and CO2 release extrapolates to an INH- tration of amino acid and 30 mm INH for 20 min. The remaining assay insensitive glycolate decarboxylation rate of zero at a glyoxylate conditions were as in Figure 2. concentration of zero. Soybean plants were seeded in sterile washed sand and watered CO2 release from added glycolate was also measured in cells weekly with a nutrient solution containing either 0, 0.1, or 1.0 mm where the glycine decarboxylase reaction was inhibited by INH KNO3. After 8 weeks, when the plants without nitrogen first began treatment. As the concentration of added glutamate or serine was showing deficiency symptoms, the leaves were harvested and the increased from 0 to 20 mm, the rate of CO2 release decreased from cellular glyoxylate levels determined (Table III). Plants grown 1.41 to 0.64 lsmol/mg Chl- h for glutamate and 0.50 ,umol/mg ChlM under low, medium, and high nitrogen concentrations show quite h for serine (Fig. 3A). The simultaneous addition of 20 mm consistent foliar glyoxylate concentrations of around 80 to 100 glutamate and 10 mm serine decreased the rate of INH-insensitive nmol/mg Chl. Extensive measurements taken from various aged CO2 release to 0.32,umol/mg Chl.h. leaves grown under widely varying nutrient conditions all yielded The concentration of glyoxylate in the tissue closely paralleled glyoxylate concentrations within the range. the rate of CO2 release from glycolate in the presence of INH. The glyoxylate concentration fell from 2.66 ,umol/mg Chl in CONCLUSIONS untreated cells to 1.17 and 1.43 ,umol/mg Chl in the presence of 20 mm glutamate and serine, respectively. When both amino Isolated soybean leaf cells decarboxylated added glycolate by donors were added (20 mm glutamate and 10 mM serine) the two mechanisms, the glycine decarboxylase reaction and the direct glyoxylate concentration decreased to 0.58 ,umol/mg Chl. Half oxidation of glyoxylate. Adding the amino donors serine and maximal decrease in the glyoxylate pools occurred at 5 mm glutamate greatly decreased the rate of glyoxylate decarboxyla- glutamate and 2 mm serine. These values agree with those deter- tion. This decrease was matched by a parallel decrease in the mined from Figure 2. Illuminating the cells did not significantly cellular glyoxylate concentration, and an increase in the rate of decrease the steady state glyoxylate concentration or the rate of glycine decarboxylation. This suggests that the direct oxidation of INH-insensitive CO2 release (data not shown). glyoxylate was not limited by the availability of H202 but by the When the data from Figure 3A were replotted as the rate of rapid transamination of glyoxylate to glycine. CO2 release against the cellular glyoxylate concentration, a straight The reason for the apparent amino donor deficiency in the line resulted (Fig. 3B). The only points which do not fail on the isolated leaf cells is not immediately apparent. Several lines of line are the values obtained at the three highest serine concentra- study have suggested that the outer membranes of these cells are tions. As noted above (Fig. 2), serine concentrations above 10 mm leaky (unpublished results). The cells were isolated from rapidly 1034 OLIVER Plant Physiol. Vol. 68, 1981 growing plants that were supplied with sufficient nitrogen. 5. JEWESS PJ, MW KERR, DP WHITAER 1975 Inhibition ofglycollate oxidase from pea leaves. FEBS Lett 53: 292-296 A reasonable estimation of the rate of glyoxylate decarboxyla- 6. KEYs AJ, IF BIRD, MJ CoRNELIus, PJ LEA, RM WALLSGROVE, BJ MIFLIN 1978 tion in intact soybean leaves can be made by using an average Photorespiratory nitrogen cycle. Nature 275: 741-743 foliar glyoxylate concentration of 100 nmol/mg Chl and the data 7. Kismx T, NE TOLBERT 1969 Glycolate and glyoxylate metabolism by isolated presented in Figure 3B. This figure shows that soybean cells with peroxisomes or chloroplasts. Plant Physiol 44: 242-250 8. KisAKu T, N YOSHIDA, A ImAI 1971 Glycine decarboxylate and serine formation a glyoxylate concentration of 100 nmol/mg Chl decarboxylate in spinach leaf mitochondrial preparation with reference to photorespiration. glyoxylate at about 5% the rate of control cells. From the data in Plant Cell Physiol 12: 275-288 Figure 1 and (11) it can be estimated that about 50%o of the C02 9. LAWYER AL, I ZELITCH 1978 Inhibition ofglutamate:glyoxylate aminotransferase lost from glycolate metabolism with untreated soybean cells comes activity in tobacco leaves and callus by glycidate an inhibitor of photorespiration. Plant Physiol 61: 242-247 from the direct decarboxylation of glyoxylate. Taking 5% of 50%o 10. NumLA J, K SIVARAMA SASTRY 1972 Modified method for analysis ofglyoxylate would indicate that roughly 2.5% of the photorespiratory CO2 derivatives in biological materials. Anal Biochem 47: 218-227 might arise from glyoxylate decarboxylation. 11. OLIVER D 1979 Mechanism of decarboxylation of glycine and glycolate by There are, of course, many possible sources of error in this isolated soybean cells. Plant Physiol 64: 1048-1052 12. OLIVER D 1981 Mechanism and control ofphotorespiratory glycolate metabolism: estimate. What is apparent from the data presented in Figure 3B CO2 loss from the (C-2) carbon ofglycolate. In G Akoyunoglou, ed, Photosyn- is that there is a direct proportionality between the glyoxylate thesis. Balaban International Science Service, Rehovot, Israel. In press concentration in the tissue and the rate of glyoxylate decarboxyl- 13. OLIVER DJ, TH THORNE, RP POINCELOT 1979 Rapid isolation ofmesophyll cells ation. As long as glyoxylate is present in the tissues, some portion from soybean leaves. Plant Sci Let 16: 149-155 14. PRITCHARD GG, WJ GRIFFIN, CP WHIT-INGHAM 1962 The effect of isonicotinyl of it will be decarboxylated. The amount of glyoxylate decarbox- hydrazide on the photosynthetic incorporation ofradioactive CO2 into ethanol- ylation that occurs in normal tissue, however, appears to be small soluble compounds of Chlorella J. Exp Bot 14: 281-289 when compared to the rate of photorespiratory CO2 loss from 15. RICHTERCH R 1969 Clinical Chemistry Theory and Practice. Academic Press, decarboxylation. It also does not appear to change with New York, pp 86-95 glycine 16. SERVAITES JC, WL OGREN 1977 Chemical inhibition of the glycolate pathway in changes in the age and nutrient status of the plant. soybean leaf cells. Plant Physiol 60: 461-466 17. SommvniaLE CR, WL OGREN 1981 Photorespiration-deficient mutants ofArabi- LITERATURE CITED dopsis thaliana lacking mitochondrial serine hydroxymethylase activity. Plant Physiol 67: 666-671 1. CANVIN DT, NDH LLOYD, H Focy, CP PRZYBYLLA 1976 Glycine and serine 18. TOLBERT NE 1971 Microbodies-peroxisomes and glyoxysomes. Annu Rev Plant metabolism and photorespiration. In RH Burris, CC Black, eds, CO2 Metab- Physiol 22: 45-74 olism and Plant Productivity. University Park Press, Baltimore, pp 161-176 19. USADA H, GP ARRON, GE EDWARDS 1980 Inhibition of glycine decarboxylation 2. GRODZINsU B 1978 Glycolate decarboxylation during photorespiration. Planta by aminoacetonitrile and its effect on photosynthesis in wheat. Plant Physiol 144: 31-37 65: S-70 3. GRODZINsiU B, VS Burr 1976 Hydrogen peroxide production and the release of 20. ZELITCH 1 1971 Photosynthesis, Photorespiration, and Plant Productivity. Aca- carbon dioxide during glycollate oxidation in leaf peroxisomes. Planta 128: demic Press, New York 225-231 21. ZELITCH 1 1972 The photooxidation of glyoxylate by envelope-free spinach 4. GRODZINsIu B, VS Burr 1977 The effect of temperature on glycollate oxidation chloroplasts and its relation to photorespiration. Arch Biochem Biophys 150: in leaf peroxisomes. Planta 133: 261-266 698-707