Senescence-Specific Increase in Cytosolic Glutamine Synthetase and Its mRNA in Radish Cotyledons

Abstract

Changes in the levels of cytosolic and chloroplastic isoforms of glutamine synthetase were examined in senescing radish (Raphanus sativus L. cv Comet) cotyledons by immunoblotting analysis using antibodies raised separately against maize glutamine synthetase isoforms. Translatable mRNAs for these isoforms were also examined by analyzing translation products from poly(A)' RNA in a wheat germ system with the antibodies. The relative content of cytosolic isoform (GS,) increased twofold in the cotyledons that were placed in the dark for 72 hours to accelerate senescence, while that of chloroplastic isoform (GS2) declined to half of its initial level. The dark-treatment also increased the relative level of translatable mRNA for GS, sevenfold after 72 hours, and decreased rapidly that for GS2 and for other nuclear-coded chloroplast proteins as well. Cotyledons also accumulated GS, mRNA when they became senescent after a lengthy growth period under continuous light. These observations suggested that GS, genes were activated, while those for GS2 were repressed, and eventually the population of the enzyme was altered in senescent cotyledonary cells. The role of increased cytosolic enzyme is discussed in relation to the nitrogen metabolism in senescent leaves.Glutamine synthetase plays a central role in nitrogen metabolism of higher plants. It is responsible for the primary assimilation of ammonia in root cells, and for that of ammonia generated by nitrite reduction in chloroplasts. It also works in the assimilation of ammonia produced by nitrogen fixation in root nodules, and in the reassimilation of ammonia released by photorespiration in the leaf cells (14). In many plant species leaf cells contain two isoforms of GS,3 one in the cytosol (GS,) and the other in the chloroplast (GS2). Root nodules of legumes contain another type of enzyme, nodule-specific GS. The accumulation of these isoforms are differentially regulated; the GS2 activity has been shown to increase during the greening of etiolated plants (8,17), and increase in the activity of nodulespecific GS in developing nodules has been shown to accompany the accumulation of its mRNA (5).

Nitrogen Metabolism in the Stalk Tissue of Maize

“…The ratios of GS1:GS2 activities were 6:1 at 3 weeks preanthesis, 1 1:1 at anthesis, 16:1 at 2 to 4 weeks postanthesis, and increased to 24:1 at 6 weeks postanthesis. 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).…”

Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase

“…In the photosynthetic organs of angiosperms, GS1 expression is restricted to vascular bundles, indicating a relevant role for this isoenzyme in the transport of glutamine from the leaf to other organs (Lea, 1997;Cren & Hirel, 1999). In addition, GS1 is expressed in photosynthetic cells of conifers, where GS2 is absent (García-Gutiérrez et al ., 1998), and also in angiosperms during physiological differentiation of plastids (Gallardo et al ., 1988;Kawakami & Watanabe, 1988;Gálvez et al ., 1990) or under stressful situations such as water stress (Bauer et al ., 1997) or pathogen attack (Pérez-García et al ., 1995). GS1 has been found to co-localize with quantitative trait loci (QTL) for yield components in the plant genome (Hirel et al ., 2001;Obara et al ., 2001).…”

Immunolocalization of a Unique Form of Maize Kernel Glutamine Synthetase Using a Monoclonal Antibody

“…moto et al, 1989;Sakakibara et al, 1992;Li et al, 1993). These GS isozymes are active in root and leaf primary ammonia assimilation, in leaf photorespiration, and in storage organ, root nodule, and senscent tissue Gln synthesis for intercellular transport (Kawakami and Watanabe, 1988;Coruzzi, 1991). In developing maize kernels, most of the GS is found in the pedicel region (Muhitch, 1988) as a pair of isozymes, one of which (GS,,) is a physically and kinetically unique form of maize GS (Muhitch, 1989).…”