Immunocytochemical Localization of Glutamine Synthetase in Organs of Phaseolus vulgaris L.

Abstract

Glutamine synthetase was localized in nodules, roots, stems, and leaves of red kidney bean (Phaseolus vulgaris L.) by immunocytochemistry. Affinity purified antibodies reactive with glutamine synthetase were prepared using purified nodule-enhanced glutamine synthetase. Immunogold labeling was observed in the cell cytoplasm in each plant organ. In nodules, the labeling was more intense in the infected cells than in the uninfected cells. No labeling was observed in nodule bacteroids, peribacteroid spaces, or in peribacteroid membranes, while previous reports of glutamine synthetase immunolabeling of legume nodules showed labeling in the bacteroid fraction. Significant labeling was observed in nodule proplastids which contained starch granules. Substantial labeling was also observed in leaf chloroplasts. No labeling was observed in other organelles including mitochondria, peroxisomes, and endoplasmic reticulum. Preimmune IgGs did not bind to any structure in the tissues examined.GS2 (EC 6.3.1.2) catalyzes the first step in the assimilation of ammonia in higher plants (14). Using differential centrifugation, Suzuki et al. (17) showed that glutamine synthetase was a soluble enzyme in root cells of Phaseolus vuilgaris.Cullimore et al. (6) purified two forms of GS in P. vulgaris root nodules. One form (GS.2) was similar to that found in roots (GSr), while the other isoform (GS,,) was a noduleenhanced form (1, 6, 8).Subunit structure of GS,, and GSn2 was studied extensively by Cai and Wong (4). Both forms of GS are octamers. GS,Iis composed of two subunit polypeptides, 3 and -y, which differ in charge but not size. GSn2 is an octamer of a single A polypeptide. GS,, is a composite of eight isozymes which differ in their ratio of A and y subunits.In leaves, GS is found in both the cytoplasm and the chloroplast (12). Results of mutant analyses suggest that the role of GS in leaves is the reassimilation of ammonia derived from photorespiration (2,20 GS is composed of four subunits, which are dissimilar to a and / (10). More recently, Lightfoot et al. (1 1), using a fulllength cDNA clone, have revealed that the chloroplast-located GS of bean is encoded by a single nuclear gene. Their nucleotide sequence indicated that this cDNA is more similar to a cDNA encoding chloroplast GS of the pea than to cDNAs encoding a, A, and -y GS subunits of the bean. They also showed that poly(A+) RNA specifically related to the chloroplast GS gene is most abundant in bean leaves, but a trace amount is also detectable in the roots and nodules. The polypeptide encoded by this gene is called delta, and a small amount of it is found in the plastids of root nodules (1, 5).There are three previous reports of immunolabeling GS in legume nodules. In soybean, Verma et al. (19) published one electron micrograph of immunolabeling of GS in the infected cell. Significant labeling was observed in the plant cell cytoplasm, but cross-reactivity with bacteroids was also shown. No controls or experimental details were shown or mentioned. Brangeon et ...

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

“…lmmunogold localization studies have shown that common bean nodule-enhanced GSnl is localized to both infected and uninfected cells of bean nodules (Datta et al, 1991). Likewise, recent immunogold localization studies from our laboratory have shown that nodule-enhanced AAT-2 and PEPC proteins, both of which are important for synthesis of Asp, are present in infected and uninfected cells (Robinson et al, , 1996.…”

Localization of tobacco cytosolic glutamine synthetase enzymes and the corresponding transcripts shows organ- and cell-specific patterns of protein synthesis and gene expression

“…It was clearly shown that plastidic GS is located in the chloroplast stroma [4,6,10,42] and may also be present in nodule plastids [6,10]. Cytosolic GS was found in the cytosol of root and root nodules [6,10] and in the leaf cytosol depending on the plant examined [10]. Interestingly, a significant amount of the leaf cytosolic GS was also detected in phloem cells in both monocots and dicots [8,14,26] or in the scutellum and aleurone layers of germinating barley seeds [35].…”

“…It was clearly shown that plastidic GS is located in the chloroplast stroma [4,6,10,42] and may also be present in nodule plastids [6,10]. Cytosolic GS was found in the cytosol of root and root nodules [6,10] and in the leaf cytosol depending on the plant examined [10].…”

A Transposable Partitioning Locus Used To Stabilize Plasmid-Borne Hydrogen Oxidation and Trifolitoxin Production Genes in a Sinorhizobium Strain

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