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

Abstract

Chloroplast and cytosolic isoforms of glutamine synthetase (GS; EC 6.3.1.2) are encoded by separate nuclear genes in plants. Here we report that the promoters for chloroplast GS2 and cytosolic GS3A of Pisum sahvum confer nonoverlapping, cell-specific expression patterns on the j8-glucuronidase (GUS) reporter gene in transgenic tobacco. The promoter for chloroplast GS2 directs GUS expression within photosynthetic cell types (e.g., palisade parenchymal cells of the leaf blade, chlorenchymal cells of the midrib and stem, and photosynthetic cells of tobacco cotyledons). The promoter for chloroplast GS2 retains the ability to confer light-regulated gene expression in the heterologous transgenic tobacco system in a manner analogous to the light-regulated expression of the cognate gene for chloroplast GS2 in pea. These expression patterns reflect the physiological role of the chloroplast GS2 isoform in the assimilation of ammonia generated by nitrite reduction and photorespiration. In contrast, the promoter for cytosolic GS3A directs expression of GUS specifically within the phloem elements in all organs of mature plants. This phloem-speciflic expression pattern suggests that the cytosolic GS3A isoenzyme functions to generate glutamine for intercellular nitrogen transport. In germinating seedlings, the intense expression of the cytosolic GS3A-GUS transgene in the vasculature of cotyledons reveals a role for cytosolic GS in the mobilization of seed storage reserves. The distinct, cell-specific patterns of expression conferred by the promoters for chloroplast GS2 and cytosolic GS3A indicate that the corresponding GS isoforms perform separate metabolic functions.In higher plants, many steps in nitrogen metabolism occur in multiple subcellular compartments. For example, many amino acid biosynthetic isoenzymes are located in the cytosol as well as in the mitochondria or chloroplasts. The relative function of many amino acid biosynthetic isoenzymes has been difficult to assess due to inadequate fractionation of organelle and cytoplasm components, overlapping activity profiles, and immunological cross-reactivity (1, 2). Consequently, it is unclear whether these isoenzymes carry out redundant or distinct roles in plant metabolism.The best-studied example of a plant amino acid biosynthetic enzyme shown to occur as multiple isoforms is glutamine synthetase (GS; EC 6.3.1.2) (3). Early biochemical data revealed that GS functions in the assimilation of ammonia generated by numerous plant processes, which include seed germination (4, 5), photorespiration (6, 7), nitrite reduction (8), nitrogen-fixation in root nodules (9, 10), and primary ammonia assimilation from the soil (11). An analysis of the GS genes in several species has revealed a strong correlation of individual GS gene expression with specific aspects of plant development (12)(13)(14)(15)(16)(17)(18). Recent sequence analysis of GS cDNAs from Pisum sativum and Phaseolus vulgaris has shown that chloroplast and cytosolic GS are encoded by separate but similar nuclea...

Nitrogen assimilation in Citrus based on CitEST data mining

“…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). However, in citrus, no GS sequence was found in root libraries, probably because few root libraries were available.…”

Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene

“…GS 1 is also present in the shoots where it is localized in the vascular tissue Kamachi et al 1992;Pereira et al 1992;Dubois et al 1996;Sakurai et al 1996;Pérez-García et al 1998). The role of GS 1 in the vasculature, however, is not well defined but it has been suggested that it may be involved in the synthesis of glutamine for nitrogen transport (Edwards et al 1990;Dubois et al 1996).…”

Nitrogen Metabolism in the Stalk Tissue of Maize

“…When detached radish cotyledons were placed in the dark to induce senescence, mRNA for GS1 increased severalfold, whereas that for GS2 decreased rapidly (9). Study of the cell-specific expression of GS genes in trans- genic tobacco has provided evidence that the cytosolic form, GS1, is confined to the phloem, where it has a role in the translocation of seed storage reserves during germination (5). GS2, which is present in chloroplasts of green tissue, was reported to reassimilate ammonia released from the photorespiration process (5).…”

“…Study of the cell-specific expression of GS genes in trans- genic tobacco has provided evidence that the cytosolic form, GS1, is confined to the phloem, where it has a role in the translocation of seed storage reserves during germination (5). GS2, which is present in chloroplasts of green tissue, was reported to reassimilate ammonia released from the photorespiration process (5). Levels of GS protein in field-grown maize stalks from 3 weeks preanthesis to physiological maturity were determined by western blot.…”

“…The frozen samples were homogenized in buffer (3 g fresh weight/mL) with a Polytron. For extraction of GS, GOGAT, and GDH, the buffer was 50 mM Tris-HCl at pH 7.6 containing 1 mM EDTA, 10 mM MgC92, and 3 mm DTT; for NR, the buffer contained 0.2 M Tris-HCl at pH 8.2, 1 mM EDTA, 1 gM sodium molybdate, 5 ,uM flavin adenine dinucleotide, 3 mm DTT, and 1.5% BSA. The homogenate was centrifuged at 20,000g for 15 min.…”