Kinetic Properties and Ammonium-dependent Regulation of Cytosolic Isoenzymes of Glutamine Synthetase in Arabidopsis

Abstract

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, cytosolic GS (GS1) was accumulated in roots when plants were excessively supplied with ammonium; however, the GS activity was controlled at a constant level. The discrepancy between the protein content and enzyme activity of GS1 was attributable to the kinetic properties and expression of four distinct isoenzymes encoded by GLN1;1, GLN1;2, GLN1;3 and GLN1;4, genes that function complementary to each other in Arabidopsis roots. GLN1;2 was the only isoenzyme significantly up-regulated by ammonium, which correlated with the rapid increase in total GS1 protein. GLN1;2 was localized in the vasculature and exhibited low affinities to ammonium (K m ‫؍‬ 2450 ؎ 150 M) and glutamate (K m ‫؍‬ 3.8 ؎ 0.2 mM). The expression of the counterpart vascular tissue-localizing low affinity isoenzyme, GLN1;3, was not stimulated by ammonium; however, the enzyme activity of GLN1;3 was significantly inhibited by a high concentration of glutamate. By contrast, the high affinity isoenzyme, GLN1;1 (K m for ammonium < 10 M; K m for glutamate ‫؍‬ 1.1 ؎ 0.4 mM) was abundantly accumulated in the surface layers of roots during nitrogen limitation and was down-regulated by ammonium excess. GLN1;4 was another high affinity-type GS1 expressed in nitrogen-starved plants but was 10-fold less abundant than GLN1;1. These results suggested that dynamic regulations of high and low affinity GS1 isoenzymes at the levels of mRNA and enzyme activities are dependent on nitrogen availabilities and may contribute to the homeostatic control of glutamine synthesis in Arabidopsis roots.Glutamine synthetase (GS 1 ; EC 6.3.1.2) is responsible for the primary assimilation of ammonium in higher plants (1-4). Ammonium is assimilated into glutamine and glutamate through a consecutive reaction of GS and glutamate synthase (GOGAT), the so-called GS/GOGAT cycle. Plants have two types of GS isoenzymes that localize in different compartments: one located in the cytosol (GS1) and the other in the plastid/chloroplasts (GS2) (1-4). GS1 is the major form of GS in plant roots, and the ammonium taken up from the soil is directly converted to Gln by its reaction. Molecular biological studies have identified a number of genes encoding GS1 from various plant species (5-9). The presence of multiple GS1 isoenzymes complicates the overall understanding of their physiological functions. The isoenzymes of GS1 show organ-and cell-specific patterns of expression and are developmentally regulated (10 -17). In addition, the expression of GS1 is metabolically regulated by the availability of nitrogen and carbon sources (18 -22, 24).In the roots of legumes, GS1 is regulated by ammonium supplied from the environment or the symbiotic nitrogen fixation (18 -21). Transgenic studies with the soybean GS1 promoter suggested that ammonium-dependent regulation is specific for nitrogen assimilation in leguminous plants; the soybean-derived GS1...

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