Arabidopsis gls Mutants and Distinct Fd-GOGAT Genes: Implications for Photorespiration and Primary Nitrogen Assimilation

Abstract

Ferredoxin-dependent glutamate synthase (Fd-GOGAT) plays a major role in photorespiration in Arabidopsis, as has been determined by the characterization of mutants deficient in Fd-GOGAT enzyme activity ( gls ). Despite genetic evidence for a single Fd-GOGAT locus and gene, we discovered that Arabidopsis contains two expressed genes for Fd-GOGAT ( GLU1 and GLU2 ). Physical and genetic mapping of the gls1 locus and GLU genes indicates that GLU1 is linked to the gls1 locus, whereas GLU2 maps to a different chromosome. Contrasting patterns of GLU1 and GLU2 expression explain why a mutation in only one of the two genes for Fd-GOGAT leads to a photorespiratory phenotype in the gls1 mutants. GLU1 mRNA was expressed at the highest levels in leaves, and its mRNA levels were specifically induced by light or sucrose. In contrast, GLU2 mRNA was expressed at lower constitutive levels in leaves and preferentially accumulated in roots. Although these results suggest a major role for GLU1 in photorespiration, the sucrose induction of GLU1 mRNA in leaves also suggests a role in primary nitrogen assimilation. This possibility is supported by the finding that chlorophyll levels of a gls mutant are significantly lower than those of the wild type when grown under conditions that suppress photorespiration. Both the mutant analysis and gene regulation studies suggest that GLU1 plays a major role in photorespiration and also plays a role in primary nitrogen assimilation in leaves, whereas the GLU2 gene may play a major role in primary nitrogen assimilation in roots. INTRODUCTIONGlutamate synthase (glutamine-oxoglutarate aminotransferase or GOGAT) is a key enzyme involved in the assimilation of inorganic nitrogen in higher plants ( Lea and Miflin, 1974;Keys et al., 1978;Miflin and Lea, 1980;Stewart et al., 1980). Functioning coordinately with glutamine synthetase (GS; EC 6.3.1.2), the GS/GOGAT pair provides the primary port of entry for nitrogen in whole-plant metabolism. Inorganic nitrogen, in the form of ammonia, is assimilated via this glutamate synthase cycle into the organic nitrogen compounds glutamine and glutamate, which are the nitrogen donors in essentially all biosynthetic reactions involving nitrogen (e.g., amino acids, nucleic acids, and chlorophyll). Primary nitrogen assimilation requires cofactors, reducing equivalents, and carbon skeletons generated during photosynthesis. Thus, in most plants, assimilation of inorganic nitrogen into organic form occurs predominantly in leaf chloroplasts where these components are readily available (Sechley et al., 1992). In plant species that are able to efficiently transport photosynthate to roots, such as maize and temperate legumes, nitrogen assimilation also occurs at high rates in root plastids (Oaks, 1992).In addition to its major role in primary nitrogen assimilation, the GS/GOGAT cycle also plays a crucial role in reassimilating the large amount of ammonia released during photorespiration (Somerville and Ogren, 1980;Kendall et al., 1986). The amount of ammonia released during pho...

Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase, and its spatial and temporal changes during leaf development

“…In contrast, Suzuki et al (1982) reported that rice root Fd-GOGAT was immunologically distinct from the leaf enzyme. Also, in Arabidopsis two distinct Fd-GOGAT genes are differentially expressed, one of which is expressed at high levels in leaves, while the other is expressed at constitutively low levels in leaves and at high levels in roots (Lam et al 1996;Coschigano et al 1998). Our previous DNA blot analysis indicated the existence of two Fd-GOGAT genes in the grapevine genome (Loulakakis and Roubelakis-Angelakis 1997).…”

“…The genomes of several plant species seem to contain a single copy of the Fd-GOGAT gene, while in the amphidiploid genome of tobacco there are two genes present (Zehnacker et al 1992). In Arabidopsis, two distinct and apparently functional Fd-GOGAT genes were reported; one was expressed predominantly in leaves and the other in roots (Lam et al 1996;Coschigano et al 1998). …”

“…The enzyme is localized in the chloroplasts and is the major enzymic form in green tissues, representing up to 1% of the protein content of leaves (Matoh and Takahashi 1982;Suzuki et al 1982). In photosynthetic tissues, Fd-GOGAT is essential for both the re-assimilation of ammonium released during photorespiration and the primary ammonium assimilation (Somerville and Ogren 1980;Lam et al 1995Lam et al , 1996Coschigano et al 1998). Fd-GOGAT transcripts and protein were also found in nonphotosynthetic organs, including roots and nodules (Suzuki et al 1982;Sakakibara et al 1992;Coschigano et al 1998).…”

“…In photosynthetic tissues, Fd-GOGAT is essential for both the re-assimilation of ammonium released during photorespiration and the primary ammonium assimilation (Somerville and Ogren 1980;Lam et al 1995Lam et al , 1996Coschigano et al 1998). Fd-GOGAT transcripts and protein were also found in nonphotosynthetic organs, including roots and nodules (Suzuki et al 1982;Sakakibara et al 1992;Coschigano et al 1998). However, the physiological function of the enzyme in these tissues is not fully understood.…”

Photorespiration

“…GS was also detected in the mitochondria, indicating that some photorespiratory ammonia could be fixed within the mitochondria; however, the physiological significance of this process is unproven (Linka and Weber, 2005;Taira et al, 2004). The functioning of this 'photorespiratory nitrogen cycle' (Keys et al, 1978) is essential for photorespiratory metabolism (Coschigano et al, 1998;Somerville, 2001). For example, the widely used herbicide glufosinate inhibits GS and kills plants by ammonia poisoning.…”