Isoenzymes of Glutamine Synthetase in Roots of Pea (Pisum sativum L. cv Little Marvel) and Alfalfa (Medicago media Pers. cv Saranac)

Abstract

Cell organelles have been isolated from protoplast lysates and total homogenates obtained from root tips of Pisum sativum L. (cv Little Marvel) and Medicago media Pers. (cv Saranac) grown in hydroponics with nitrate nutrient solutions. Density-gradient and differential centrifugation procedures have been used to prepare mitochondria-and plastidenriched fractions in which glutamine synthetase (GS) activity was estimated. Even when purified protoplasts were gently ruptured, significant breakage of plastids occurred during preparation as shown by the high proportion of nitrite reductase recovered in the soluble fraction. Of the total GS activity recovered, up to 20% was associated with the plastid fraction, depending on the source of plant material and the GS assay utilized; when corrected for recovery of the plastid marker nitrite reductase, it was calculated that 15 to 57% of alfalfa and 14 to 64% of pea root GS was located in the plastids. A true biosynthetic assay in which glutamine production was monitored by high performance liquid chromatography was devised to estimate the physiological significance of the transferase and the semibiosynthetic assays currently used for activity measurements. When compared with the true and semibiosynthetic assays, the transferase assay for GS appeared to underestimate the root plastid enzyme. Root plastid GS was partially purified by ion-exchange chromatography, and results show that the isoenzyme found in root plastids is different from chloroplastic or cytosolic GS.In most plants, free ammonium ions are assimilated through the action of the glutamine synthetase/glutamate synthase cycle ( 17). In relation to its key role in nitrogen metabolism, GS2 has been intensively studied in leaves, roots, and nodules of higher plants (5, 10, 11, 13

Tissue and Cellular Distribution of Glutamine Synthetase in Roots of Pea (Pisum sativum) Seedlings

“…When nitrite reductase was not present in plastids, all fractions from sucrose gradients were assayed for triosephosphate isomerase activity as described in Vezina et al (17). GS activity was determined by an ADP-dependent transferase assay (13).…”

“…Although it has been shown that nitrite reductase is also strictly associated with plastids in root cells, there are still uncertainties as to how and where the ammonium ions produced by its activity are incorporated into active amino acid biosynthesis (1 1, 12). Cytosolic-and plastid-isoforms of root GS have been partially characterized in peas ( 17), and it has been shown that plastids isolated from dark-grown calli have the ability to synthesize glutamine from glutamate and ammonium (20 the plastids without being released to the cytosol. However, several other reports have suggested that GS is wholly cytosolic in roots (6,15,16) and that only one ammonium pool exists within root cells (6).…”

GLUTAMINE SYNTHETASE AND AMMONIUM ASSIMILATION IN ROOTS OF ZINC‐TOLERANT AND NON‐TOLERANT CLONES OF DESCHAMPSIA CESPITOSA (L.) BEAUV. AND ANTHOXANTHUM ODORATUM L.

“…Closer examination of the roots revealed a vesicular-arbuscular-like fungal infection (J. Faull & N. Smirnoff, unpublished observations), and the first peak of activity could be tentatively ascribed to this. However, root nodules of Phaseolus vulgaris have two plant-derived glutamine synthetase isoforms (CuUimore et al, 1983), and recently two isoforms have been resolved in Pisum sativum and Medicago media roots (Vezina, Hope & Joy, 1987). The identity of the first peak of activity in A. odoratum therefore requires further study.…”

Induction of Glutamine Synthetase Activity in Nonnodulated Roots of Glycine max, Phaseolus vulgaris, and Pisum sativum

“…Considerable research effort has focused on GS in nodulated roots and it has been shown that nodule GS activity is high relative to root activity, and that nodule GS activity is the result of a pool of GS isozymes with at least one isozyme identical to that found in nonnodulated roots. Although some reports indicate that the increased GS activity in nodules is due to induction of one or more GS isozymes by NH4' (7, 11,21,22), other research shows increased nodule GS prior to NH4' production by nitrogenase, the source of nodule NH4' (2, 3,23 …”

“…Several GS isozymes have been identified in different plant tissues (4, 16,19,21). Considerable research effort has focused on GS in nodulated roots and it has been shown that nodule GS activity is high relative to root activity, and that nodule GS activity is the result of a pool of GS isozymes with at least one isozyme identical to that found in nonnodulated roots.…”