Oct 19, 1983 - 3Abbreviations: GOGAT, glutamate synthase EC 1.4.1.13; CMF, chloroform:methanol:formic acid mixture; GS, glutamine synthetase EC. 6.3.1.2 ...
Plant Physiol. (1984) 75, 60-66 0032-0889/84/75/0060/07/$01.00/0
Glutamic Acid Metabolism and the Photorespiratory Nitrogen Cycle in Wheat Leaves METABOLIC CONSEQUENCES OF ELEVATED AMMONIA CONCENTRATIONS AND OF BLOCKING AMMONIA ASSIMILATION Received for publication October 19, 1983 and in revised form January 5, 1984
KEVIN A. WALKER', CURTIS V. GIVAN*, AND ALFRED J. KEYS2 Department of Plant Biology, University of Newcastle upon Tyne, Newcastle upon Tyne, NEJ 7RU, United Kingdom; and Botany Department, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, United Kingdom investigations using conditional mutants of Arabidopsis viable only under nonphotorespiratory conditions have provided powerful confirmation of the existence of this cycle and the importance of the reassimilation of NH3 released during photorespiration (25, 26). The amount of NH3 generated by photorespiratory oxidation of glycine is several-fold greater than the amount produced through reduction of NO3- (5, 14, 25). Thus, the principal function of the chloroplastic GOGAT cycle is the reassimilation of NH3 generated by photorespiration (3, 31). The chloroplastic GOGAT cycle involves operation of the ferredoxin-dependent GOGAT and the GS isoenzyme located in the chloroplast stroma (15, 19). Several previous studies have examined ['4C]glutamate metabolism in leaves. Jordan and Givan (12) studied the role played by light in controlling the metabolism of ['4C]glutamate in leaves of Viciafaba and showed that the glutamine-synthetase inhibitor MSO not only prevented glutamine synthesis but also led to rapid metabolism of glutamate by decarboxylation, deamination, and oxidation via the tricarboxylic acid cycle (cf. 17). Franz et al. (5) subsequently found that total levels of glutamate declined markedly in oat leaves when GS was inhibited, but they provided no information on the products or pathway of glutamate utilization. The inhibition of GS with MSO leads to large accumulations of NH3 in plant tissues (e.g. 22, 27). Elsewhere K. A. Walker, A. J. Keys, and C. V. Givan (in preparation) describe the effects of ammonium and MSO on rates and products of '4C02 assimilation in wheat leaves. A major goal of the present investigation was to distinguish between those effects of MSO that result principally from elevated tissue levels of NH3 per se and those that are specifically the result of blocked NH3 assimilation. Particular attention has been paid to metabolic interactions between glutamate and the tricarboxylic acid cycle and to the effects of light on operation of the tricarboxylic acid cycle.
ABSTRACT
The effects of methionine sulfoximine and ammonium chloride on ll4q glutamate metabolism in excised leaves of Triticum aesti'um were investipted. Glutamine was the principal product derived from [U"Cjglutamate in the light and in the absence of inhibitor or NH4Cl. Other amino acids, orgnic acids, sugars, sugar phosphates, and CO2 became slightly radioactive. Ammonium chloride (10 mM) increased formation of 114C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and '4C02. Methionine sulfoximine (1 mM) suppressed glutamine synthesis, caused NH3 to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the "C from IU-'4C]glutamate to be incorporated into malate and succinate, whereas most of the '4C from 11-'4Clglutamate was metabolized to CO2 and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through "dark" flxation of CO2 released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-'4q glutamate without inhibitor or NH4CI were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and "4C02. Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.
Glutamate occupies a position of central importance in the N metabolism of leaves. It is a major amino donor for transamination reactions (cf. 7) and is the NH3-acceptor compound in the GOGAT3 cycle, which mediates primary assimilation of NH3 and reassimilation of NH3 generated during photorespiration (14). The essential features of the photorespiratory N cycle, originally proposed by Keys and collaborators (14) have been confirmed by subsequent studies (e.g. 5, 16, 22, 29). Elegant
MATERIALS AND METHODS Plant Growth Conditions. Wheat plants, Triticum aestivum cv Timmo were grown at 20 ± 2C in a regime of 16 h light, 8 h dark in pots of soilless peat-based compost and irrigated with 0.78 g x dm3 Chempak No. 3 solution (Chempak Products, Brew House Lane, Hertford, U.K.) Irradiance of 850 uE/mi2. s' (400 to 700 nm range) was provided by Phillips GroLux light bulbs. Tissue Preparation and Incubation Procedures. Three-weekold plants were darkened for about 16 h at 15 to 20C. Segments
' Supported by a Science and Engineering Research Council CASE Research Studentship. Present address: Department of Biochemistry, University College, Cardiff, P.O. Box 78, Cardiff CFI I XL, Wales, U.K. 2 Present address: Biochemistry Department, Rothamsted Experimental station, Harpenden, Hertfordshire ALS 2JQ, England, U.K. 3Abbreviations: GOGAT, glutamate synthase EC 1.4.1.13; CMF, chloroform:methanol:formic acid mixture; GS, glutamine synthetase EC 6.3.1.2; MSO, methionine sulfoximine.
60
GLUTAMATE METABOLISM AND PHOTORESPIRATION
61
Radioactive compounds were located on chromatograms using a radiochromatogram spark chamber (Birchover Instruments, Letchworth, Hertfordshire, U.K.) and identified by elution and a thin wire frame were placed with their proximal ends in a co-chromatography with authentic standards. Amino acid stanperspex well containing water plus the appropriate substrates dards were located with ninhydrin, and organic acids with a with or without inhibitor. The total fresh weight of tissue was bromocresol purple spray followed by exposure of the paper to approximately 150 mg for the 5 segments. ['4CJglutamate was gaseous NH3. Areas of paper containing radioactive compounds supplied at a concentration of 0.5 mm, specific radioactivity 10 were cut out and the 14C measured by scintillation counting using Ci/mol. The perspex well containing the leaf segments was placed a toluene based scintillant. Quantification of Amino Acids. Amino acid amounts were in a 20-cm3-volume perspex chamber and sealed from the atmosphere outside. Four identical chambers were surrounded by measured by the method of Atfield and Morris (1). Paper chromatograms dried for 24 h to remove all traces of the phenola common cooling jacket through which water was circulated at 25 ± 1°C. Illumination (1200 E/m-2 s-' was supplied through developing solvent were dipped in a solution of 50 mg cadmium acetate dissolved in 5 cm3 water with 1 cm3 glacial acetic acid the water jacket by a quartz-iodine projector lamp. Air was drawn through the leaf chamber by an aspirator pump and mixed with 50 cm3 acetone and 0.5 g ninhydrin. The at a flow rate of 500 cm3/min. "4CO2 evolved by the tissue was chromatograms were dried briefly in air and placed in a darkened collected by passing the effluent air through scintered glass airtight glass vessel containing a beaker of concentrated H2SO4. bubblers in Dreschel bottles containing 2.5 N NaOH which After 24 h the red amino acid spots on the chromatograms were cut out and eluted in 4 cm3 methanol. Absorbance of the eluate retained approximately 98% of the '4CO2 evolved. Deproteinization and Extraction Procedures. Leaf segments was measured at 500 nm (352 nm fo. proline) and amino acid quantities estimated on the basis of a calibration curve prepared were rinsed in cold water, immediately frozen in liquid N2, and then extracted with 4 cm3 CMF solution (5:12:1, v/v/v) at -20°C with glycine standards. Chemicals. All radioactive chemicals were purchased from the overnight in a deep freeze. The extract was decanted and the leaf tissue was then reextracted with 2 cm3 cold CMF. This extract Radiochemical Centre, Amersham, Bucks, England. Other chemicals were obtained from British Drug Houses and Sigma was combined with the original to give a total of 6 cm3 solution. An additional 4.5 cm3 chloroform and 3 cm3 water were added. Chemical Co. Seeds were obtained from the U.K. Seed Executive. After phase separation the upper aqueous layer was pigment-free RESULTS and contained the extracted water-soluble compounds. Ammonium Determinations. Ammonium was assayed in 0.5 Table I shows the radioactive products derived from L-[U-'4C] cm3 samples of the aqueous phase from the CMF extraction by glutamate in excised wheat leaves illuminated for periods of 30 the colorimetric method of McCullough (18) by reference to a to 120 min in the absence of NH4C1 and MSO. The total calibration curve prepared with NH4CI solutions. Ammonium Table I. Products of[U-'4CJGlutamate Metabolism in Illuminated recoveries were checked for known amounts of NH4Cl added to Wheat Leaves leaf tissue and taken through the extraction procedure. Measured recoveries were 96 to 101 % of values expected for the amounts 150 mg fresh weight of leaf segments were supplied with [U-_4C] added. glutamate (0.5 mm, 10 MCi/umol). No additional substrates or inhibitors Chromatographic Methods. Ion-Exchange Fractionation. The present. aqueous extracts were dried in a flash evaporator under reduced Minutes Illumination pressure at 35°C and redissolved in distilled H20. Samples of the Labeled Compound solutions were fractionated on ion-exchange columns (23) giving 30 60 90 120 four fractions: (a) a basic fraction containing amino acids, (b) an dpm x io-O acidic fraction containing organic acids, (c) a second acidic 360.3 567.1 944.5 1473.8 fraction containing phosphate esters, and (d) a neutral fraction, Glutamate 16.6 54.5 128.1 269.2 containing soluble sugars. The columns of the cation and anion Glutamine Aspartate 3.5 13.5 25.7 39.2 and were SP-C-25 exchangers Sephadex Sephadex QAE-A-25 ND 1.8 3.1 4.9 prepared in lengths of Pasteur pipette tubing and contained 0.4 Glycine ND ND ND Serine trace The cm3 bed volume of the ion exchanger. fractions, collected 5.9 15.9 28.6 56.4 in glass vials, were dried overnight in vacuo over anhydrous Alanine GABAb 8.0 11.5 14.8 21.5 CaCl2 (anhydrous CaCI2 and KOH pellets for the phosphateester fraction). Fractions were then dissolved in 0.2 cm3 water Total amino acids 394.2 664.3 1029.4 1865.0 for paper chromatography. Recoveries of 14C standard com1.5 1.7 7.2 18.5 pounds from the columns were estimated at 95 to 103%, indi- Citrate 2.2 2.6 20.6 cating negligible losses to other fractions or to the column Succinate 21.8 4.6 2-Oxoglutarate material. 15.0 19.0 37.8 6.1 23.2 Paper Chromatography Procedures. Amino-acid constituents Malate 26.5 27.5 of the basic fraction were separated by ascending 2-dimensional Total organic acids 14.3 42.5 73.3 105.7 paper chromatography on 25 x 25 cm sheets of Whatman No. I chromatography paper using n-butanol:acetone:diethylam- '4CO2 13.8 22.3 33.9 44.6 ine:water (10:10:1:5 v/v/v) in the first dimension (5 to 6 h) and Phosphate esters 5.4 8.9 18.2 28.2 80% (w/v phenol:ammonia (specific gravity 0.88) (200:1) (12 to Neutrals 11.5 28.2 48.2 87.7 14 h) in the second dimension. total 439.2 766.2 1203.1 2131.3 Sample Organic acids were separated by descending 2-dimensional chromatography using 88% (w/v) phenol:water:acetic acid (84:16:1, v/v/v) for the first dimension (18 to 20 h) and n- NH4+-N content Amol g-' fresh wt 2.16 2.04 2.04 1.78 butanol:acetic acid:water (12:3:1, v/v/v) (14 to 16 h) for the a second dimension. ND, not detected. b Identification and Measurement of Radioactive Compounds. -y-aminobutyrate. 4
cm
long were cut (under water to maintain the transpiration
stream intact) from first leaves immediately distal to a 2-cm basal segment which was discarded. Five segments supported by
62
WALKFER ET AL.
Plant Physiol. Vol. 75, 1984
radioactivity in soluble products increased progressively during Table III. Products of[U-"CJGlutamate Metabolism in Illuminated the 2-h incubation period; this reflects the continuing uptake and Wheat Leaves Supplied with MSO transport of the radioactive precursor from the cut base of the 150 mg fiesh weight of leaf segments were supplied with [U-"4C] leaf segment. Glutamate contained more than half the radioac- glutamate (0.5 mM) and MSO (1 mM). tivity. The principal radioactive product was glutamine formed Minutes Illumination during assimilation of NH3 generated from endogenous sources Labeled Compound in the leaf cell (14). Other minor radioactive products included 30 60 90 120 amino acids, organic acids, C02, and sugar phosphates. The NH3 dpm x 10-J concentration was near 2 gmol/g fresh weight during the 2-h Glutamate 301.4 411.2 248.2 218.9 illumination period and only slightly higher than that of dark Glutamine 10.1 ND 2.9 ND tissue (Table VI). Aspartate 2.3 5.7 2.9 trace Table I} shows the radioactive products when [U-"4C]gluta3.7 Glycine 2.1 1.6 1.6 mate was supplied to the tissue with 10 mM NH4C1. The total Serine 7.4 1.2 1.1 0.6 radioactivity recovered from the tissue increased by about 20% Alanine 4.9 3.2 2.5 2.5 in the presence of NH4C1 (cf. Table II and Table I). NH4C1 GABA 8.2 23.8 24.3 28.4 increased the accumulation of ["4C]glutamine to about 35% of the total radioactivity recovered, and the NH3 concentration Total amino acids 337.9 450.1 280.6 252.1 present was 2 to 3 times higher than in the control tissue (Table I). NH4CI also increased the radioactivity in aspartate, malate Citrate 0.9 0.3 3.3 10.7 and citrate but decreased the formation of radioactive CO2, 2Succinate 1.5 36.5 91.0 198.4 oxoglutarate and alanine. About 1% of the radioactivity retr 2-Oxoglutarate 1.8 5.4 6.9 covered was in y-aminobutyrate in both NH4Cl treated and Malate 30.5 289.7 453.2 664.1 control tissue (Tables I and II). Total organic acids 32.9 328.3 552.8 880.1 Table III shows the radioactive products derived from [U-14C] glutamate supplied to illuminated leaftissue in the presence of 1 15.5 30.4 14CO2 56.2 113.4 mM MSO. The total radioactivity recovered per sample, includesters Phosphate 5.6 15.0 69.8 181.6 ing that lost as "CO2, was usually less in MSO-treated samples Neutrals 6.3 52.5 82.0 140.8 than in control tissue, but MSO does not decrease transpiration rates (28). MSO had several very striking effects on the metabo398.1 876.3 1041.3 1568.0 Sample total lism of glutamate. MSO completely eliminated the formation of ND, not detected. radioactive glutamine by 90 min, presumably owing to the irreversible inhibition of GS caused by this inhibitor. The MSO also caused a corresponding increase in ammonia concentration (5, 14, 16, 22) and a large increase in the accumulation of radioactivity in organic acids (Table III), especially malate and This increased radioactivity in organic acids was succinate. Table II. Products of[U-"qC Metabolism in Illuminated largely accounted for by the increased metabolism of the [14C] glutamate. Accumulation of "CO2 and 'C-sugar-phosphate esWheat Leaver ss upplied with NH4Cl 150 mg fresh weight of lealf segments were supplied with [U-"C] ters was significantly increased in the presence of MSO (compare Table III and Table I). glutamate (0.5 mM) and NH4 Cl1(10 mM). The decreased 14C in glutamate was associated with a decline Minutes Illumination in the total pool sizes of the major soluble amino-acid compoLabeled Compound 90 60 120 30 nents (Fig. 1). Glutamate declined sharply in MSO-treated tissue during the 2-h illumination period, whereas it rose significantly dpm x 10-3 in the control tissue. MSO also decreased glutamine, aspartate, 403.4 573.7 899.3 1233.7 Glutamate serine, and glycine levels. 42.2 313.1 594.2 893.8 Glutamine Table IV shows the radioactive products of [U-"4Cglutamate 7.6 16.7 40.6 81.5 Aspartate metabolism in the presence of both 1 mM MSO and 10 mM 1.2 2.2 3.3 8.2 Glycine ND NH4CL. The labeled products are for the most part similar to Serine 0.9 ND; 1.2 17.1 6.1 2.7 10.0 those observed with MSO without added NH4Cl (Table III) and Alanine are very different from those found when NH4Cl was added 16.7 22.6 GABA 10.3 25.1 alone (Table II). However, glutamine synthesis was not com467.4 928.4 1570.7 2260.6 Total amino acids pletely suppressed, indicating incomplete inhibition of GS. As with MSO treatment alone, much of the added ["4C]glutamate 11.4 13.4 37.6 5.2 Citrate was converted to organic acids, especially ['4C]malate. 7.5 10.0 11.9 18.4 Succinate The conversion of glutamate to malate is initiated either by a 2.6 1.9 4.2 6.9 decarboxylation of the C-1 of glutamate to produce 7-amino2-Oxoglutarate 6.4 34.1 52.1 Malate 66.9 butyrate, or by decarboxylation ofthe C-I of 2-oxoglutarate after deamination (12, 17). Malate is also a product of other reactions 20.9 58.1 81.6 129.9 Total organic acids in which phosphopyruvate is carboxylated to yield oxaloacetate, which, in turn, is reduced to malate (cf. 9). In order to estimate 21.8 16.2 24.1 30.4 14CO2 the contribution of refixed CO2 to the MSO-induced organic 8.2 5.2 20.1 23.9 Phosphate esters acid synthesis, the leaftissue was supplied with [l-"4C]glutamate. Neutrals 14.2 33.8 54.9 106.6 of the C-I atom leaves the remaincase,atoms decarboxylation In this 523.9 1050.4 1751.4 2551.4 5391. Sample total ing carbon of glutamate unlabeled. Therefore, any radioactive malate from produced [1-_4CJglutamate would be ascribNtI4+-N content Mmol gable to refixation of "4CO2 produced by decarboxylation of the fresh wt 2.7 3.94 4.6 5.45 C-I of glutamate or its keto analog, 2-oxoglutarate. Table V shows the radioactive products derived from [I -'4C] * ND, not detected. a
sIlwamate
63
GLUTAMATE METABOLISM AND PHOTORESPIRATION 5
5
serine
glycine 4
4
3
3
2
2
I
I
I
I
I
8-
glutamine
T. 7 40
2
6
--
50
4
31+0 c
2 _
2
1
threonine
Phosphate esters Neutrals Sample total NH4+-N content 'ND, not detected.
alanine
1 I
I
I
I
0
20
40
60
I 80 100 120 Time (r
0
20
40 60
80 loo 120
FIG. 1. Effect of MSO upon amino acid pool sizes. Leaf segments supplied with I mM MSO (-U*-) or H20 (--) in the light.
were
Citrate Succinate 2-Oxoglutarate Malate Total organic acids
'4CO2
E_
aspartic acid
1
Table IV. Products of[U-14"CGlutamate Metabolism in Illuminated Wheat Leaves Supplied with NH4Cl (10 mM) and MSO (I mM) Minutes Illumination Labeled Compound 30 60 90 120 Glutamate 259.0 656.5 561.2 256.7 Glutamine 21.6 21.9 48.6 1.9 Aspartate 4.6 17.0 11.6 8.1 Glycine NDa ND 3.4 3.7 Serine ND ND ND ND Alanine 1.6 3.8 7.0 3.1 GABA 8.7 17.6 28.6 32.3 Total amino acids 295.5 716.9 660.3 305.7
6.3 3.7 1.6 4.9 16.5
26.2 35.5 6.7 43.4 111.7
49.2 400.5 14.9 481.8 946.4
40.5 343.6 17.9 592.5 994.6
10.3 5.7 14.3 342.3
62.4 35.3 51.4 977.7
146.3 224.4 99.1 2076.5
190.8 291.2 166.2 1948.5
3.04
8.96
21.2
26.39
Table V. Effect ofMSO on Metabolism of[1-'4CJGlutamate in Illuminated Wheat Leaves Leaf segments were supplied with [1-'4C]glutamate (10 Ci/mol) in presence and absence of MSO (1 mM). Minutes Illumination Labeled Compound Control +MSO
glutamate in the presence and absence of I mM MSO. As in the experiments with [U-'4C]glutamate, ['4C]glutamine was the major product in the control, with much of the radioactivity re60 120 60 120 maining as unmetabolized precursor. MSO caused a 90% drop dpm x 10-3 in radioactive glutamate, and there was no detectable radioactive Glutamate 490.7 1341.9 385.6 108.2 glutamine or other amino acids. '4CO2 accumulation was inGlutamine 103.4 268.4 NDa 4.5 creased by more than five-fold, and it accounted for 38% of the 3.9 Aspartate ND 15.8 ND total radioactivity recovered after 2 h. Formation of radioactive Glycine ND ND ND ND malate and succinate was increased only slightly by MSO comSerine ND ND ND ND pared to the increase seen with [U-'4C]glutamate as precursor. Alanine ND ND ND ND Thus, there is no significant stimulation by MSO of carboxylaGABA ND ND ND ND tion reactions yielding malate. With [1-'4C]glutamate as the precursor, MSO increased the accumulation of radioactive comTotal amino acids 598.0 1686.4 390.2 108.2 pounds in neutral and phosphate-ester fractions (Table V); radioactivity in the phosphate-esters was much higher than the Citrate 5.0 9.6 5.1 16.2 amount derived from [U-'4C]glutamate (compare Tables I-IV). Succinate 5.9 1.3 4.6 13.0 Because both the ammonia-producing and ammonia-assimiND 2-Oxoglutarate ND ND ND lating reactions of the photorespiratory nitrogen cycle are directly Malate 6.8 19.5 8.4 48.3 or indirectly light-dependent, the effect of light on ['4Cjglutamate Total organic acids 17.6 41.7 18.0 77.6 metabolism in wheat leaves was studied. The uptake of radioactive substrate is via the transpiration stream, so that experiments 40.5 103.2 113.5 811.0 14C02 done wholly in darkness were not technically feasible. The role esters Phosphate 18.4 51.2 220.4 1048.8 of light was therefore studied by darkening leaves that had been Neutrals 15.7 32.2 29.0 98.7 given ['4C]glutamate in the light for 2 h. total 690.2 1914.7 Sample 771.0 2144.2 Table VI shows results of a light:dark transition experiment carried out on leaf tissue preincubated with [U-'4CJglutamate. In aND, not detected. the absence of MSO (Table VI) the distribution of radioactivity between products after 2 h in the light was as described previously increased markedly. '4C in succinate increased early in the dark (cf. Table I). On darkening, a pronounced decrease in radioactive period; this increase slowed as malate increased concomitant glutamate took place, accompanied by a steady rise of 14C in with increases in '4C02 and phosphate esters. Ammonia fell from glutamine and aspartate, and a more than 5-fold increase in 2.06 to 1.56 gmol/g fresh weight during the hour of darkness. A organic acids, predominantly malate and succinate. 4CO2 also small amount of ['4C]threonine appeared after 40 and 60 min
64
Table VI. Products of[U-'4CJGlutamate Metabolism in Dark Period following 2 Hours Preillumination Leaf segments were incubated in light for 2 h with [U-'4C]glutamate (0.5 mM, 10 MCi/,Amol) then transferred to darkness. Minutes Darkness Labeled Compound 0 20 40 60 dpm x 10-3 1505.4 1189.3 847.6 799.0 Glutamate 426.2 280.8 575.5 809.9 Glutamine 47.7 113.8 157.0 148.0 Aspartate 8.5 12.1 7.6 9.7 Glycine 2.0 3.7 4.0 4.6 Serine 41.7 41.3 58.1 30.8 Alanine 25.4 28.6 20.8 18.6 GABA ND ND 0.4 1.9 Threonine 1913.9 1808.2 1673.0 1824.8 Total amino acids
Citrate Succinate 2-Oxoglutarate Malate Total organic acids Phosphate esters Neutrals Sample total
NH4'-N content Amol g-' fresh wt a ND, not detected.
Plant Physiol. Vol. 75, 1984
WALKER ET AL.
14.0 25.6
26.0 132.8
37.0 173.0
39.2 173.7
58.5 98.2
89.6 248.2
204.8 414.8
317.5 530.4
49.7 30.7 83.6 2176.1
94.3 44.9 50.4 2246.0
192.0 52.0 47.1 2379.0
249.0 74.1 43.0 2721.4
2.06
1.89
1.82
1.56
darkness. In the presence of MSO (Table VII) the distribution of 14C in tissue illuminated for 120 min was as seen previously, with no ['4C]glutamine, only a small amount of ('4Cjglutamate, and large amounts of radioactive malate, succinate, C02, and phosphate esters. The ammonia is high owing to blockage of glutamine synthetase by MSO. There was no major change in 14C distribution upon darkening. The loss of glutamate and the 4C(O2 accumulation merely represented a continuation of the trends established in the light (cf. Table III). There was no further accumulation of ammonia in the dark.
DISCUSSION This investigation has provided information about the metabolic role played by glutamate both in recycling photorespiratory ammonia and in maintaining normal patterns of carbon metabolism in the illuminated leaf. A substantial pool of soluble glutamate is normally maintained in the leaves and this is increased in the light (Fig. 1). ['4C]Glutamate added to illuminated tissue persists mainly as glutamate but some glutamine accumulates (Tables I and II). Glutamate and glutamine are the two compounds directly involved in the GOGAT cycle, which is responsible for reassimilation of ammonia generated during photorespiration (29). As long as NH3 assimilation is actively proceeding, there is little conversion of ["4Cjglutamate to other products outside the GOGAT cycle. The maintenance of substantial levels of glutamate and glutamine must be an essential aspect of reassimilation of photorespiratory NH3. The reassimilation process clearly operates efficiently to prevent any marked increase in NH3 concentrations over levels found in darkness, even though a substantial generation of ammonia is taking place (cf. 5, 14, 16).
Table VII. Products of[U-'4CJGlutamate Metabolism in Dark Period following 2 Hours Preillumination in the Presence ofMSO Minutes Darkness Labeled Compound 0 20 40 60 dpm x 10-i Glutamate 220.2 177.0 122.9 41.3 Glutamine 1.0 NDa trace ND Apartate 1.0 16.2 4.4 1.7 1.8 0.6 0.7 Glycine 0.8 Serine 0.4 ND ND ND Alanine 1.6 1.9 1.9 3.2 GABA 26.0 26.6 31.0 24.8 ND ND Threonine ND ND Total amino acids 251.9 222.2 160.9 71.8
Citrate Succinate 2-Oxoglutarate Malate Total organic acids
'4C02 Phosphate esters Neutrals Sample total
NH41-N content Mmol g7' fresh wt a ND, not detected.
8.6 194.1
10.0 288.3
14.3 288.2
ND
620.8 823.5
ND 559.0 857.3
650.7 953.2
20.1 244.8 ND 522.0 786.9
116.4 230.2 119.7 1541.6
189.2 173.8 116.9 1559.2
266.2 146.2 100.5 1626.9
293.5 176.0 96.2 1424.4
20.03
20.45
ND
21.27
21.93
When 10 mm NH4Cl was supplied to the leaves (Table II), extra ammonium was assimilated to glutamine, and the tissue NH3 levels therefore rose only modestly. The GOGAT cycle clearly had sufficient excess capacity to assimilate considerably more NH3 than the amount generated by nitrate reduction and photorespiratory oxidation of glycine. The glutamine accumulation observed in the presence of NH4Cl is a typical response of plant tissue to elevated NH4' supply (cf.6). The results obtained with the GS inhibitor MSO demonstrate that there are numerous and far-ranging consequences of blocking the reassimilation of photorespiratory NH3. MSO causes a virtually complete disappearance of glutamine and a large accumulation of NH3 directly as the result of GS inhibition (14, 25). Tables III, IV, and V show that a large loss of glutamate takes place owing to rapid catabolism of glutamate. This involves decarboxylation and deamination reactions so that the carbon skeleton of glutamate is converted to succinate, malate, and CO2. The conversion of glutamate to CO2 and organic acids in the presence of MSO reduces the availability of glutamate as an amino donor for transamination reactions; this is manifest in the decreased radioactivity in glycine, alanine, and aspartate when MSO is present (Tables III and IV). The deficiency of amino donor, in retarding the transamination of glyoxylate to glycine, must interfere also with the movement of carbon through the glycolate pathway back into the Calvin cycle (cf. 26). Metabolism of[ l-'4C]glutamate in the presence of MSO (Table V) shows that when the GOGAT cycle is blocked the C-l is rapidly converted to CO2. This decarboxylation may be in part by direct decarboxylation of glutamate catalyzed by glutamate decarboxylase (12) as well as by decarboxylation of the keto analog 2-oxoglutarate. [14C]-y-Aminobutyrate was detected as a minor product in wheat leaves but not to the same extent as in Vicia faba leaves (12). The decarboxylation of [1-'4C]glutamate generates a large
GLUTAMATE METABOLISM AND PHOTORESPIRATION amount of '4CO2, but the present data indicate very little formation of ['4C]malate compared to that derived from [U-'4C] glutamate. Thus, there is no evidence for any enhancement of anaplerotic ,3-carboxylation reactions. The internal ammonia concentration is high in the presence of MSO, but assimilation is blocked. Several investigators (10, 21, 30) have observed that elevated NH4' supply leads to accelerated carboxylation of phosphopyruvate to yield organic acids. This increased synthesis of organic acids by anaplerotic CO2 fixation could be due to elevated levels of NH4+ per se, with NH4 itself directly regulating carboxylation (cf. 13), or alternatively it may be a product of NH3 assimilation that is the regulator (cf. 6). The present data are
entirely consistent with the view (20) that elevated NH4' levels lead to enhanced anaplerotic synthesis of organic acids only when the NH3 is actually being assimilated. Thus the MSOinduced accumulation of [14C]malate derived from [U-'4C]glutamate is not due primarily to refixation of '4CO2. The substantial labeling of neutral and phosphate-ester compounds from [1-'4C]glutamate in the presence of MSO reflects the large amounts of '4C02 available from decarboxylation for reassimilation by photosynthesis. There is severe inhibition of photosynthetic CO2 assimilation in the presence of MSO (22), but the onset of this inhibition is gradual. In work to be presented elsewhere (Walker, Keys, and Givan, in preparation) we have confirmed that considerable photosynthetic CO2 assimilation persists during the first 60- to 90-min treatment with 1 to 2 mm MSO (cf. also 16). Light clearly affects the metabolism of glutamate in wheat leaves. As discussed above, total glutamate levels normally rise after the onset of illumination, and supplied ['4C]glutamate is largely retained as glutamate and glutamine. Upon darkening, ['4C]glutamate is rapidly metabolized to glutamine, organic acids, and CO2 (Table VI). The increased ['4C]glutamine accumulation in the dark takes place in the absence of photophosphorylation and of photorespiratory NH3 production. Previous experiments with Vicia faba leaves indicated that photophosphorylation appeared to be the principal source of ATP supporting the GS reaction (12). NH3 required for glutamine synthesis
in the dark may be derived from mitochondrial deamination of the glutamate that is catabolized (cf. 17). In the present instance ATP generated by dark respiration is evidently able to support glutamine synthesis in the dark period following extensive preillumination. In accordance with this view, Azcon-Bieto and coworkers (2) have recently demonstrated that dark respiration of wheat leaves is enhanced after a period of illumination. The increased accumulation of radioactivity in glutamine in the dark period is probably not due to an acceleration of glutamine synthesis, but to a reduced rate of utilization. Glutamine utilization proceeds largely via the ferredoxin-dependent GOGAT enzyme and is therefore light dependent (15). Ito and collaborators (I 1) have confirmed that transfer of the amide N from '5Nlabeled glutamine to other amino acids is light dependent. Therefore, glutamine utilization in the dark is very slow. The results of the light:dark transition experiments are also relevant to the question whether mitochondrial respiratory metabolism is active in illuminated leaves (cf. 8). Reed and Canvin (24) have pointed out that any appreciable retardation of mitochondrial electron transport in the light would be expected to cause inhibition of succinate oxidation, since succinate dehydrogenase turnover is directly linked to the electron transport chain. The conversion of ['4C]glutamate to [14C]malate in the light proceeds via succinate dehydrogenase (12). The present data demonstrate active transfer of radioactivity through succinate to malate in the light. Thus, succinic dehydrogenase activity and mitochondrial electron transport are evidently active in the light. In the presence of MSO (Table VII) there was very little increase in the rate of '4C02 evolution from ['4C]glutamate upon trans-
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ferring illuminated leaf tissue to darkness, nor did dark conditions appear to accelerate the oxidation of succinate to produce malate and citrate. This observation is in harmony with other independent evidence (e.g. 4, 8) suggesting that electron-transport and tricarboxylic-acid-cycle activities persist to a substantial extent in illuminated leaves. In conclusion, the active functioning of the GOGAT cycle is essential not only for prevention of NH3 accumulation, but also, and perhaps more importantly, to maintain adequate levels of glutamate in illuminated leaves. When glutamine synthesis is blocked by MSO, the synthesis of glutamate via the GOGAT cycle soon ceases, and much of the tissue glutamate is metabolized. The resulting decline in the availability of glutamate limits the transamination steps in the glycolate pathway and retards the recirculation of carbon from glycolate to the Calvin cycle. This will, in turn, reduce net carbon assimilation. LITERATURE CITED 1. ATFIELD GN, CJOR MORRIS 1961 Analytical separations by high voltage paper electrophoresis. Amino acids in protein hydrolysates. Biochem J 81: 606614 2. AzcoN-BIETo J, H LAMBERS, DA DAY 1983 Effect of photosynthesis and carbohydrate status on respiratory rates and the involvement of the alternative pathway in leaf respiration. Plant Physiol 72: 598-603 3. BERGMAN A, P GARDESTROM, I ERICSON 1981 Release and refixation of ammonia during photorespiration. Physiol Plant 53: 528-532 4. DRY IB, JT WISKICH 1982 Role of the external adenosine triphosphate/ adenosine diphosphate ratio in the control of plant mitochondrial respiration. Arch Biochem Biophys 217: 72-79 5. FRANTZ TA, DM PETERSON, RD DURBIN 1982 Sources of ammonium in oat leaves treated with methionine sulfoximine. Plant Physiol 69: 345-348 6. GIVAN CV 1979 Metabolic detoxification of ammonia in tissues of higher plants. Phytochemistry 18: 375-382 7. GIVAN CV 1980 Aminotransferases in higher plants. In BJ Miflin, ed, The Biochemistry of Plants, A Comprehensive Treatise, Vol 5. Academic Press, New York, pp 329-357 8. GRAHAM D 1980 Effects of light on "dark" respiration. In DD Davies, ed, The Biochemistry of Plants, A Comprehensive Treatise, Vol 2. Academic Press, New York, pp 525-579 9. HALLIWELL B 1981 Chloroplast Metabolism. The Structure and Function of Chloroplasts in Green Leaf Cells. Oxford University Press, Oxford, 257 pp 10. HAMMEL KE, KL CORNWELL, JA BASSHAM 1979 Stimulation of dark CO2
fixation by ammonia in isolated mesophyll cells of Papaver somniferum L. Plant Cell Physiol 20: 1523-1529 1 1. ITo 0, T YONEYAMA, K KUMAZAWA 1978 Amino acid metabolism in plant leaf. IV. The effect of light on ammonium assimilation and glutamine metabolism in the cells isolated from spinach leaves. Plant Cell Physiol 19: 1109-1119
12. JORDAN BR, CV GIVAN 1979 Effects of light and inhibitors on glutamate metabolism in leaf discs of Vicia faba L. Sources of ATP for glutamine synthesis and photoregulation of tricarboxylic acid cycle metabolism. Plant Physiol 64: 1043-1047 13. KANAZAWA T, M DISTEFANO, JA BASSHAM 1983 Ammonia regulation of intermediary metabolism in photosynthesizing and respiring Chlorella pyrenoidosa: comparative effects of methylamine. Plant Cell Physiol 24: 979986 14. KEYS AJ, IF BIRD, MJ CORNELIUS, PJ LEA, RM WALLSGROVE, BJ MIFLIN 1978 Photorespiratory nitrogen cycle. Nature 275: 741-743 15. LEA PJ, BJ MIFLIN 1974 Alternative route for nitrogen assimilation in higher plants. Nature 251: 614-616 16. MARTIN F, MJ WINSPEAR, JD MAcFARLANE, A OAKS 1983 Effect of methionine sulfoximine on the accumulation of ammonia in C3 and C4 leaves. Plant Physiol 71: 177-181 17. MAZELIS M 1980 Amino acid catabolism. In BJ Miflin, ed, The Biochemistry of Plants, A Comprehensive Treatise, Vol 5. Academic Press, New York, pp 541-567 18. MCCULLOUGH H 1967 The determination of ammonia in whole blood by a direct colorimetric method. Clin Chim Acta 17: 297-304 19. McNALLY SF, B HIREL, P GADAL, AF MANN, GR STEWART 1983 Glutamine synthetases of higher plants. Evidence for a specific isoform content related to their possible physiological role and their compartmentation in the leaf. Plant Physiol 72: 22-25 20. OHMORI M 1981 Effect of ammonia on dark CO2 fixation by Anabaena cells treated with methionine sulfoximine. Plant Cell Physiol 22: 709-716 21. PAUL JS, KL CORNWELL, JA BASSHAM 1978 Effects of ammonia on carbon metabolism in photosynthesizing isolated mesophyll cells from Papaver somniferum L. Planta 142: 49-54 22. PLATT SG, GE ANTHON 1981 Ammonia accumulation and inhibition of
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509-513 23. REDGWELL RJ 1980 Fractionation of plant extracts using ion-exchange sephadex. Anal Biochem 107: 44-50 24. REED AJ, DT CANVIN 1982 Light and dark controls of nitrate reduction in wheat (Trilicum aeslivum L.) protoplas. Plant Physiol 69: 508-513 25. SOMERVILLE CR, WL OGREN 1980 Inhibition of photosynthesis in Arabidopsis mutants lacking leafglutamate synthase activity. Nature 286: 257-259 26. SOMERVILLE SC, WL OGREN 1983 An Arabidopsis thaliana mutant defective in chloroplast dicarboxylate transport. Proc Natl Acad Sci USA 80: 12901294 27. STEWART GR, D RHODES 1976 Evidence for the assimilation of ammonia via the glutamine pathway in nitrate-grown Lemna minor L. FEBS Lett 64: 296-299
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28. WALKER KA 1983 Metabolic response of wheat leaves to exogenous ammonium and to prevention ofammonium assimilation. PhD Thesis. University of Newcastle upon Tyne, Newcastle upon Tyne, England 29. WALLSGROVE RM, AJ KEYs, IF BIRD, MJ CORNELIUS, PJ LEA, BJ MIFLIN 1980 The location of glutamine synthetase in leaf cells and its role in the reassimilation ofammonia released in photorespiration. J Exp Bot 31: 10051017 30. Woo KC, DT CANVIN 1980 Effect of ammonia, nitrite, glutamate, and inhibitors of N metabolism on photosynthetic carbon fixation in isolated spinach leaf cells. Can J Bot 58: 511-516 31. Woo KC, CB OSMOND 1982 Evidence for the glutamine synthetase/glutamate synthase pathway during the photorespiratory nitrogen cycle in spinach leaves. Plant Physiol 70: 1514-1517
