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

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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.
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