Planta (Berl.) 122, 281--291 (1975) 9 by Springer-Verlag 1975

Light-dependent Synthesis of Glutamine in Pea-Chloroplast Preparations * Curtis V. Givan Department of Plant Biology, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U. K. Received 18 November; accepted 9 December, 1974 Summary. Isolated-chloroplast preparations obtained from Pisum sativum (L.) plants synthesized L-[laC]glutamine from L-[14C]glutamate in the light. In the dark appreciable synthesis of glutamine occurred only in the presence of added ATP. Photoconversion of glutamate to glutamine was inhibited by millimolar concentrations of ammonium or nitrite, and by DCMU. The inhibition due to DCM~, NO2-, or NH~+ was markedly relieved by ATP. ATP-dependent synthesis of glutamine in the dark was not inhibited by ammonium or nitrite ions. The data suggest that ATP necessary to support chloroplastio glutamine synthesis may be derived from noneyclic or pseudocyclio phosphorylation. Since high ammonia concentrations appear to inhibit chloroplastic glutamine synthesis by uncoupling photophosphorylation, it is possible that ATP synthesized outside the chloroplast may be required to drive the rapid synthesis of glutamine that occurs in leaves subjected to toxic levels of ammonia.

Introduction Recent evidence (O'Neal and Joy, 1973; Lea and Miflin, 1974) suggests t h a t in leaves of higher plants the glutamine synthetase (GS, EC 6.3.1.2) reaction (Equation 1) is responsible for the bulk of the primary assimilation of nitrogen into organic form. L-glutamate-t- ATP + NI-Is --> L-glutamine -]-ADP ~- Pr

(1)

The belief, held until very recently, t h a t L-glutamate dehydrogenase (EC 1.4.1.3) was the chief nitrogen-assimilating enzyme of leaves therefore requires serious re-assessment (for information on the older interpretation, c]. Givan et al., 1970 ; Tsenova and Vaklinova, 1970; Tsukamoto, 1970; Givan and Leech, 1971; Lea and Thurman, 1972; Tsenova, 1972; Magalhaes etal., 1974). Present evidence does not yet rule out the possibility t h a t glutamate dehydrogenase m a y catalyze nitrogen assimilation under certain conditions, however. The nitrogen-assimilating GS is thought to operate in conjunction with glutamate synthase (EC 2.6.1.53) a newly discovered plant enzyme that forms glutamic acid from glutamine and g-ketoglutarate in a reduetive reaction (Dongall, 1974; Fowler et al., 1974; Lea and Miflin, 1974). The glutamate synthase reaction proceeds according to Equation 2. L-glutamine A-cr A-NAD(P)H (or reduced ferredoxin) --> 2 L-glutamate-~ NAD(P) -t- (or oxidized ferredoxin).

(2)

* Abbreviations: DCMU~3-(3,4-dichlorophenyl)-l,l-dimethylurea; GS=glutamine synthetase (glutamate: NH 3 ligase).

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Several years ago Santarius and Stocking (1969) d e m o n s t r a t e d t h a t Spinacia chloroplasts isolated in an intact state could amidate glutamate to produce glutamine. This reaction was strictly light-dependent. Santarius and Stocking's findings strongly suggested t h a t a ehloroplastie GS existed which could produce glutamine in the presence of A T P generated b y photophosphorylation. I t has subsequently been confirmed b y direct e n z y m e assay t h a t the isolated chloroplast does in fact contain GS. Moreover, there appears to be a strong possibility t h a t the bulk of the GS activity of leaves m a y be localized in this organelle (O'Neal a n d J o y , 1973; H a y s t e a d , 1973). The ferredoxin-dependent glutamate synthase situated in the chloroplast is now believed to mediate the light-dependent conversion of ~-ketoglutarate to g l u t a m a t e (Lea and Miflin, 1974) which had earlier been t h o u g h t to involve the activity of g l u t a m a t e dehydrogenase (Givan et al., 1970). I n view of these recent developments, it seemed urorth while to undertake further s t u d y of the light-dependent glutamine-synthesizing system of chloroplasts. The present report, which confirms and extends the original observation of Santarius and Stocking (1969), deals with some general properties of the system a n d in particular with the relation of glutamine synthesis to A T P availability. A T P availability is possibly affected b y the t y p e of inorganic-nitrogen source as well as b y light. Reference is m a d e to the special problem which the leaf m a y face u n d e r conditions of a m m o n i a toxicity.

Materials and Methods Pisum sativum plants (c.v. Laxton Superb) were grown in vermiculite for 3 to 4 weeks in a controlled-temperature growth room at 19~ under a daily light regime of 12 h light, I2 h dark. Light intensity was approximately 1800 lux. Chloroplast preparations were made by a procedure based on that of Coekburn et al. (1968) as described previously (Sherratt and Givan, 1973). (The use of isoascorbate in the isolation medium was, however, found to be unnecessary, and the use of this compound has been discontinued during the course of the present studies on glutamine synthesis.) After centrifugation from the isolation medium, the chloroplasts were resuspended in a medium containing D (--) sorbitol, 330 re!K; tricine, 50 mlTI; MgCl2, 2 raM; MnC12, 1 raM; Na~tt~ ethylenediamine tetraaoetic acid, 1 m ~ ; pH 7.6, adjusted with KOH. Incubation of the chloroplast suspension with 0.5 ~Ci of L-[U-l~C]glutamic acid (10 ~Ci/ ~mole, Radiochemical Centre, Amersham, Bucks., England) was carried out in air in darkness or in light. Incubation was carried out either in test tubes (12 • 150 ram) illuminated by a single tungsten projection lamp at one side (intensity 165001ux) or alternatively using specimen tubes 22 • 50 mm) illuminated from above by two quartz-iodine projection lamps, white-light intensity 16500 lux (total energy 60 W/m2). Similar results were obtained with either illumination set-up. Incubations were carried out in a total volume of 1.3 or 1.4~ml consisting of 1.1 ml of chloroplasts ~ resuspension buffer as described above plus 0.2 or 0.3 ml of distilled water or solutions of chemicals whose effects were being tested. Further details of reaction mixtures are supplied in legends to tables and figures. Prior to addition of [l~C]glutamate, the chloroplast suspension was added to the remainder of the reaction mixture and pre-incubated for 12 min in darkness. Temperature was 23 ~ 0.5 ~ At the end of the period of incubation in the presence of [14CJglutamate the reaction was terminated by addition of either hot ethanol (final concentration 70% [w/v]) or HC1 (final concentration 0.65 N). When HC1 was used to deproteinize reaction mixtures it was important to keep samples frozen (--18 ~ between the time of deproteinization and subsequent chromatography. Unfrozen HCl-deproteinized samples showed a slow but quite appreciable hydrolysis

Light-dependent Synthesis of Glutamine

283

of glutamine, whereas in frozen samples there appeared to be no more than a trace of glutamine decay for several days. Aliquots (50 ~1) of the deproteinized extract were spotted onto Whatman No. 1 chromatography paper and developed for 28-44 h in butanol:acetic acid :water (12:3:1). Glutamine and glutamate tended to separate better in the aeid-deproteinized extracts, perhaps owing to conversion of glutamine to the ammonium salt of pyrollidone carboxylic acid. The radioactive product was located by scanning the developed ehromatogram in a strip scanner. The product identified as glntamine coehromatographed with glntamine standard in the above solvent system and also in 72% (w/v) phenol and in butanol:propionie acid:water (47 : 23: 30). When HCl-deproteinized samples containing [laC]glntamine were heated for 2 h at 100~ > 90% of the radioactivity that had formerly co-ehromatographed with glutamine in unheated samples now eo-chromatographed with glutamie acid. In agreement with Santarius and Stocking (1969) glutamine was the only product detected when She chloroplasts were incubated with [~4C]glutamate in the light. The radioactivity in the product was measured by cutting out a rectangular portion of the ehromatogram corresponding to the position of glutamine and counting it in toluenebased scintillation fluor. Corrections were made for background plus a small amount of apparent chemiluminescence caused by the developed chromatography paper.

Results and Discussion Fig. 1 shows a time course illustrating t h a t the Pisum-chloroplast preparation was able to convert L-[14C]glutamate to L-[14C]glutamine in the light (Fig. l a). I n the dark very little glutamine was synthesized in the absence of ATP, while in the presence of A T P ghitamine accumulated at about one third to one half the rate seen in the light (Fig. l b). The light-dependent synthesis of glutamine originally described by Santarius and Stocking (1969) in Spinacia chloroplasts is thus confirmed in Pisum-chloroplast preparations. Elimination of sorbitol from the reaction mixture, a treatment which osmotically ruptures the chloroplast envelopes and promotes the escape of soluble enzymes and cofactors (c/. Walker, 1965), significantly decreased ghitamine accumulation in the light (Fig. 1 a and Table 2). This accords with Santarius and Stocking's earlier findings on the Spinacia chloroplasts to the extent of supporting their contention t h a t intact chloroplasts present in the preparation produce glutamine more rapidly t h a n those t h a t have lost their limiting envelopes. However, there was a considerable residual rate of light-dependent glutamine synthesis in the 0smotically-shocked preparation. Phase-contrast inspection of the preparation in hypotonie medium confirmed t h a t there was essentially complete removal of envelopes from the chloroplasts under the conditions used. I n the dark, the rate of glutamine synthesis was significantly improved when the chloroplasts were osmotically broken (Fig. lb), possibly owing to removal of a permeability barrier to A T P (el. Stokes and Walker, 1971). Is the rate at which isotopic label accumulates in glutamine directly and simply related to the rate of glutamine synthesis ? Newly synthesized glutamine could possibly be subsequently degraded or metabolized rapidly. Were this happening, the overall accumulation of label in glutamine might be affected b y changes in the rate of either synthesis or utilization of this compound. Indeed H a y s t e a d (1973) found that in preparations of Vicia ]aba chloroplasts there was a very active glutaminase which caused conversion of glutamine to glutamate. We therefore carried out an experiment designed to reveal whether rapid utili-

284

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Light-dependent Synthesis of Glutamine

285

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Fig. 2. Persistence in d~rkness of [14C]glutaminesynthesized during a prior period ofillumination. Chlorophyll concentration = 290 ~zg/mlreaction mixture When Pisum-chloroplast preparations were allowed to synthesize glutamine in the light for 9 rain and then transferred to darkness, there was no detectable loss of labelled glutamine (Fig. 2). There was therefore no indication of an active glutaminase reaction at the site of glutamine synthesis within the chloroplast nor did any other reaction appear to be utilizing glutamine rapidly. I t seems probable that accumulation of label in glutamine is equivalent to its rate of synthesis. Thus, the factors affecting the accumulation of glutamine in the present experiments are probably doing so principally by controlling the rate of glutamine synthesis via the GS reaction. Table 1 summarizes results of an experiment where the relation between glutamine synthesis and photosynthetic electron transport was briefly examined. DCMU, which suppresses oxygen evolution and inhibits noncyclic electron transport at a point near system I I of photosynthesis (Bishop, 1958), inhibited glutamine synthesis by about 85 %. This inhibition was nullified in the presence of reduced diehlorophenol-indophenol. I t is known that reduced indophenol dyes bypass the DCMU block, restoring noncyclic electron transport plus some phosphorylation (this artificially restored phosphorylation may be at least partly cyclic [c[. Keister, (1963)]. The DOMU-inhibition of glutamine synthesis was also very significantly relieved, albeit to a lesser extent (approximately 31/2-fold), by addition of ATP. These results suggest that ATP generated in association

286

C.V. Givan

Table 1. Effect of DCMU on glutamine synthesis by illuminated chloroplasts. Compounds were supplied at the following concentrations: DCMU, 7 ~M; ATP, 2 raM; sodium isoascorbate, 20 raM; 2,6-dichlorophenol-indophenol, 36 tzM; chl = 215 ~g/ml. Incubation period = 12 min Experimental conditions

laC in glutamine (cpm. • 10-a/ml reaction mixture)

Control DCMU DCMU ~- ATP DCMU + diehlorophenol-indophenol -{-sodium isoascorbate

49.9 7.1 24.7 49.1

Table 2. Effect of ATP on chloroplastic glutamine synthesis in the light in normal and hypotonic reaction media. Concentrations: Chlorophyll, 101 txg/ml; ATP (where indicated, 2 raM). Incubation period = 12 rain Experimental conditions

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with noncyclic or pseudocyclic electron transport drives the synthesis of glutamine. Although one cannot at present rule out the possibility t h a t a DCMU-sensitive cyclic phosphorylation of the t y p e reported by Kaiser and Urbach (1973) m a y be involved, the persistence of a considerable rate of glutamine synthesis after osmotic breakage of the chloroplasts provisionally seems to make it unlikely t h a t glutamine synthesis is supported exclusively by cyclic phosphorylation. Kaiser and Urbach (1973) believe t h a t retention of the chloroplast envelope is an essential prerequisite for endogenous cyclic phosphorylation. One must, of course, bear in mind the importance of distinguishing between chloroplasts broken during preparation and those t h a t break only after being introduced into the reaction vessel (Forti and Rosa, 1971 ; Hall, 1972). The results with DCMU and reduced indophenol also support the idea t h a t the synthesis of glutamine in Chese preparations is really carried out b y the chloroplasts and not b y contaminating bacteria or mitochondria. On the basis of other investigations it has been suggested (with considerable justification) t h a t a light-dependent, ATP-requiring reaction (e.g. protein synthesis) carried out b y chloroplast preparations could in reality be largely carried out b y bacteria whose oxidative metabolism is initiated b y photosynthetically-produced oxygen (c]. App and Jagendoff, 1964). Given an artificial source of electrons, we could observe active synthesis of glutamine even when DCMU was supplied in order to suppress photosynthetic evolution of oxygen. The stimnlatory effect of A T P also militates against the idea t h a t bacterial contaminants are responsible for the synthesis of glutamine here. Failure of A T P to cancel completely the DCMU-

Light-dependent Synthesis of Glutamine

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