The Location of Nitrite Reductase and Other Enzymes Related to Amino Acid Biosynthesis in the Plastids of Root and Leaves

Abstract

Density gradient separation of plastids from leaf and root tissue was carried out. The distribution in the gradients of the activity of the following enzymes was determined: nitrite reductase, glutamine synthetase, acetolactate synthetase, aspartate amninotransferase, catalase, cytochrome oxidase, and triosephosphate isomerase. The distribution of chlorophyll was followed in gradients from leaf tissue. The presence of plastids that have retained their stroma enzymes was denoted by a peak of triosephosphate isomerase activitv. Coincidental with this peak were bands of nitrite reductase, acetolactate synthetase, glutamine synthetase, and aspartate aminotransferase activity. The results suggest that most, if not all, the nitrite reductase and acetolactate synthetase activity of the cell is in the plastids. The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephosphate dehydrogenase activity in the cell. Some evidence was obtained for low levels of glutamate dehydrogenase activity in chloroplasts.A proportion of the nitrite reductase extracted from plant cells is associated with a particulate fraction in both leaves and roots (10,21). While most work has suggested that nitrite reductase is localized in the chloroplast, Lips and Avissar (17) have suggested that the enzyme is present in the peroxisome. Miflin (21) found that the enzyme from barley roots banded in sucrose density gradients below the mitochondria and microbodies but he did not assay for any plastid markers and was unable to identify the organelle in question. Subsequently, Dalling et al. (7) found that nitrite reductase from wheat roots, under isopycnic density gradient centrifugation, was distributed coincidently with only one of two peaks of plastid marker enzymes. Because of the unusual distribution of the plastid markers in the gradient and the lack of complete coincidence between them and nitrite reductase, it is not possible from these results to state unequivocally that nitrite reductase is located in root plastids.If it is accepted that nitrite reductase is present in the plas- (19,20,23). Leech and co-workers (9, 16) and Lea and Thurman (15) have shown that chloroplasts have a light-stimulated glutamate dehydrogenase, which appears to be NADPH-dependent and bound tightly to the chloroplast lamellae. There is also evidence that chloroplasts can incorporate ammonia into glutamine (12,25,27). Further, chloroplasts contain a wide range of transaminases (13). In root tissues, glutamate dehydrogenase seems to be solely in the mitochondria (7,21), but, apart from these observations, little seems to have been done on the location of these enzymes in roots.Studies on "4CO2 fixation in chloroplasts indicate that "4C does not readily appear in amino acids, which suggests that the carbon skeletons for the amino acids are derived from outside the chloroplasts. In agreement with this a-ketoglutarate, pyruvate, and P-enolpyruvate stimulate the formation of a-amino nitrogen from nitrite...

Cell-specific expression in transgenic plants reveals nonoverlapping roles for chloroplast and cytosolic glutamine synthetase.

“…The activity of the promoter for chloroplast GS2 predominantly in photosynthetic cell types is consistent with findings that chloroplast GS2 functions in the reassimilation of photorespiratory ammonia (7,17) and in the assimilation of reduced nitrite in plastids (8). Previous analysis of photorespiratory mutants revealed that plants that lacked chloroplast GS2 were nonviable when grown under photorespiratory conditions even though they contained normal levels of cytosolic GS (7).…”

“…2D). pGS2ct-GUS is expressed at low levels in root tips (data not shown), where GS in plastids functions in ammonia assimilation from the soil (8).…”

Polarographic Study of Dicarboxylic-Acid-dependent Export of Reducing Equivalents from Illuminated Chloroplasts

“…We presume that labeling of aspartate from ['4C]malate proceeds by transamination from OAA (11); labeling of aspartate in the light and dark, but not in the absence of LDH (Table V), is consistent with this proposal. Since amino-oxyacetate, an inhibitor (2).…”

Nitrogen assimilation in Citrus based on CitEST data mining

“…The analysis of barley mutants deficient in GS2 showed that the main role of this enzyme is in the re-assimilation of ammonia from photorespiration and that GS2 gene expression is tightly regulated by light (ZozayaHinchliffe et al, 2005). GS2 mRNA accumulation has been reported to be higher in leaves of plants cultivated under photorespiratory conditions, which is consistent with the proposed function of the re-assimilation of photorespiratory ammonia (Miflin, 1974). Studies in barley (Hordeum vulgare) also revealed that chloroplastic GS2 is expressed predominantly in leaf mesophyll cells, where photorespiration occurs, whereas cytosolic GS1 is expressed exclusively in phloem (Edwards et al, 1990).…”