Plant Science 149 1999 175 – 182
The plastidic glutamine synthetase activity is directly modulated by means of redox change at two unique cysteine residues
Yang Ae Choi, Sang Gu Kim, Young Myung Kwon
Department of Biology, College of Natural Sciences, Seoul National Uni6ersity, Seoul,
151
-
742
, South Korea Received 31 May 1999; received in revised form 20 July 1999; accepted 27 August 1999
Abstract
Two cDNA clones for glutamine synthetase GS, Clgln
1
and Clgln
2
, were isolated from a Cana6alia lineata cDNA library constructed with polyA + RNA from the mature plant leaves. From a comparison of the primary structures, Clgln
1
was judged to encode a cytosolic isoform and Clgln
2
to encode a plastidic isoform. It was revealed by those sequence comparisons that the two cysteine residues Cys-306 and Cys-371 of Clgln
2
chloroplastic GS encoding gene were substituted by alanine and serine in the Clgln
1
clone cytosolic GS encoding gene, respectively. These cysteine substitutions were found between chloroplastic GS GS2 and cytosolic GS GS1 sequences of all plants. These unique cysteine residues may account for the specific susceptibility
of the plastidic GS by sulfhydryl reagents. To investigate this assumption the two additional cysteine residues of GS2 were mutated combinatorially and the resulting recombinant GS proteins as well as the two wild-type cytosolic GS and chloroplastic
GS were examined in vitro. Only GS2, of the wild-type forms, was significantly activated by dithiotreitol. Moreover, the mutant form, mutated at both of the two additional cysteine residues, was not activated by the reductant, but the mutant forms mutated
at only one of the two were also activated. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Cana6alia lineata; Chloroplastic glutamine synthetase; Immunoscreening; cDNA cloning; Site-directed mutagenesis; Recombinant GS protein
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1. Introduction
Glutamine synthetase GS: EC 6.3.1.2 catalyzes the ATP-dependent formation of glutamine from
glutamate and ammonia. GS catalyzes the reas- similation of ammonia released from a variety of
metabolic pathways such as photorespiration, catabolism of amino acids and metabolism of
phenylpropanoids. It is also involved in nitrate or nitrite assimilation in leaves and roots and in
dinitrogen fixation in root nodules of legumes [1].
In plants GS is encoded by a small multigene family that shows organ-specific patterns of ex-
pression, with separate genes encoding leaf cytoso- lic and chloroplastic GS isoforms GS1 and GS2,
respectively, and root GS and nodule isoforms in legumes [2 – 7]. Based on the site of localization,
the different GS isoforms assimilate ammonia derived from different sources. Analysis of pho-
torespiratory mutants of barley has established that reassimilation of photorespiratory ammonia
is the function of the chloroplast-localized GS2 protein [8]. Furthermore, analysis of transgenic
plants containing GS1 or GS2 promoters, has shown that GS1 genes are only expressed in the
cells around the phloem, suggesting that the GS1 isoform functions in generating glutamine for ni-
trogen transport [9].
The regulation of GS gene expression is not yet fully understood. The genes for GS1 and GS2 are
differentially expressed during plant growth and development [6,10,11], reflecting the different roles
and cellular compartmentalization of the two isozymes [3,12,13]. In addition, it has been shown
that the pea GS2 promoter is light-induced [3,10]
Corresponding author. Tel.: + 82-2-880-6676; fax: + 82-2-872- 6881.
E-mail address
:
kwonymplaza.snu.ac.kr Y.M. Kwon 0168-945299 - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 6 3 - 6
and the accumulation of GS2 mRNA may in part be due to a phytochrome-mediated response [14].
Photorespiratory production of ammonia also reg- ulates pea GS2 expression [13].
In the work on bacteria it has been shown that there is a single gene whose expression is con-
trolled by two regulatory proteins in a manner which is sensitive to the nitrogen status of the cell.
In addition the enzyme itself is regulated by a reversible activationdeactivation mechanism sen-
sitive to the same cellular stimulus [15]. However, the work in higher plants has shown that the
regulation of GS activity is quite different, involv- ing multiple GS genes which are individually con-
trolled [6,7,10]. This complexity may reflect the greater compartmentalization and differentiation
of higher plant cells andor organs. But, at present, very little is known of the components
that regulate GS activity unlike the situation in prokaryotes. In leaves there are the two forms of
GS: GS1, cytosolic; GS2, chloroplastic. GS2 con- tains additional cysteine residues per subunit,
which may account for the specific susceptibility of this isozyme to sulfhydryl reagents [16 – 18].
Cana6alia lineata, used in this study, contains 10 of canavanine in its seed dry weight [19].
Moreover, this
compound, an
analogue of
arginine, is known to have roles in chemical de- fense and nitrogen storage. It is catabolised to
canaline and urea by arginase. The former com- pound is degraded to homoserine and ammonia by
canaline reductase. The latter compound is de- graded to carbon dioxide and ammonia by urease.
Ammonia is then reassimilated by the GS GOGAT cycle to glutamate [20,21]. Thus, the
understanding of the nitrogen metabolism in this plant, C. lineata, is important in the study of
canavanine metabolism.
We previously reported the study of the bio- chemical
and immunological
aspects of
GS isozymes in C. lineata [22]. The three organs each
have a predominant form which is different from the others in their molecular sizes. In the present
work we set out to clone cDNAs encoding cytoso- lic and chloroplastic GSs and to establish the
molecular basis for the different regulation of the isozyme activities. We also confirmed that the two
additional cysteine residues in GS2 are responsible for the specific susceptibility of this isozyme to
sulfhydryl reagents.
2. Materials and methods
