¹⁵N-Ammonia Assimilation, 2-Oxoglutarate Transport, and Glutamate Export in Spinach Chloroplasts in the Presence of Dicarboxylates in the Light

Abstract

The direct incorporation of`5NH4Cl into amino acids in illuminated spinach (Spinacia okracea L.) chloroplasts in the presence of 2-oxoglutarate plus malate was determined. The amido-N of glutamine was the most highly labeled N-atom during "5NH4 assimilation in the presence of malate. In 4 minutes the '5N-label of the amido-N of glutamine was 37% enriched. In contrast, values obtained for both the N-atom of glutamate and the amino-N of glutamine were only about 20% while that of the Natom of aspartate was only 3%. The addition of malate during the assimilation of '5N11CI and Nal5N02 greatly increased the '5N-label into glutamine but did not qualitatively change the order of the incorporation of '5N-label into all the amino acids examined. This evidence indicates the direct involvement of the glutamine synthetase/glutamate synthase pathway for ammonia and nitrite assimilation in isolated chloroplasts. The addition of malate or succinate during ammonia assimilation also led to more than 3-fold increase in ['4q2-oxoglutarate In leaves of higher plants the GS/GOGAT2 pathway is the primary route for the assimilation of ammonia (8,13,16,18,24). High rates of ammonia assimilation have been observed in spinach and pea chloroplasts and this activity appears to involve the GS/GOGAT pathway (1,4,14,19,22), even though the chloroplast also contains a NADP-specific GDH (9). Since the chloroplastic isoform of GS appears to be predominant in leaves of C3 plants (12) the assimilation of ammonia during photorespiration (7,18,23,24) would therefore be expected to take place predominantly in the chloroplast (2,23).In recent studies we have shown that the rates of (NH3, 19,22). These dicarboxylates were also found to partially relieve the inhibition of 2-OG transport by NH4Cl in the light (21). It is now possible to explain the above discrepancies by a two-translocator model for 2-OG and glutamate transport during NH3 assimilation in chloroplasts (5, 20). However, the fate of the assimilated N during ammonia assimilation in isolated chloroplasts under these conditions has never been established. It is not known whether the observed stimulation of 2-OG transport by dicarboxylates in the presence of NH4Cl is directly coupled to ammonia assimilation via GS/ GOGAT and glutamate export in the chloroplast. The present study examines and provides direct evidence for the involvement ofthe GS/GOGAT pathway for the assimilation of '5NH4Cl and Na'5N02 in spinach chloroplasts in the presence and absence of malate. Malate also increased the rate of 15N incorporation into glutamine and glutamate. The results further show that the increase in 2-OG transport in the presence of a dicarboxylate also led to increased amino acid synthesis and glutamate export from the chloroplast. MATERIALS AND METHODSMeasurements of "5N-Incorporation. The isolation of intact chloroplasts from spinach (Spinacia oleracea L.) leaves was as described (3). '5N-Labeling studies (in duplicates) were carried out in an O2 electrode reaction chamber in an assay mediu...

Glutamine Transport and the Role of the Glutamine Translocator in Chloroplasts

“…Thus it ensures that nitrogen is not drained out of the photorespiratory NH3 pathway as a result of cytosolic GS activity. In species like spinach and tobacco which do not have a cytosolic GS (11) the recycling of photorespiratory NH3 will only involve a two-translocator model consisting ofjust the dicarboxylate and 2-OG translocators (4,22,23). Like our earlier two-translocator model the three-translocator model in Figure 6 also leads to an imbalance in charge distribution across the chloroplast envelope.…”

“…We have proposed a two-translocator model for photorespiratory NH3 assimilation in chloroplasts of spinach which does not have a cytosolic GS (11). In this model the transport of 2-OG on the 2-OG translocator and that of glutamate on the dicarboxylate translocator are stoichiometrically coupled to malate transport in a cascade-like manner (4,22,23).…”

“…These stimulated values are comparable to in vivo rates of photorespiration. Our recent studies (23) show that the stimulation by malate is directly linked to increased '5NH4Cl assimilation and increased transport of 2-OG into and glutamate out ofthe chloroplast. We have proposed a two-translocator model for photorespiratory NH3 assimilation in chloroplasts of spinach which does not have a cytosolic GS (11).…”

“…We have proposed a two-translocator model for photorespiratory NH3 assimilation in chloroplasts of spinach which does not have a cytosolic GS (11). In this model the transport of 2-OG on the 2-OG translocator and that of glutamate on the dicarboxylate translocator are stoichiometrically coupled to malate transport in a cascade-like manner (4,22,23).On the other hand, the GS in the cytosol must play a role, albeit a minor one, in photorespiratory NH3 assimilation in many C3 species (17). The proportion of the photorespiratory NH3 fixed by the cytosolic GS is presumably similar to its relative distribution in the cytosol, i.e.…”

A Metabolic Control Analysis of the Glutamine Synthetase/Glutamate Synthase Cycle in Isolated Barley (Hordeum vulgare L.) Chloroplasts

“…The influx of ammonia into the chloroplast has not been studied in detail in higher plants, although it is generally accepteld that free ammonia (NH,) diffuses across the envelope artd into the stroma without the involvement of a membrane translocator (Kleiner, 1981). 2-Oxciglutarate is transported into the chloroplast via a dicarboxylate translocator, which counter-exchanges 1:l with stromal dicarboxylates (Dry and Wiskich, 1983;Somerville and Ogren, 1983;Wallsgrove et al, 1986;Woo et al, 1987). There is evidence that more than one translocator is involved in 2-oxoglutarate uptake.…”