GDH-isoenzymes strongly deaminate glutamate and only the anionic GDH-isoforms exhibit very low aminating activity under normal trophic conditions (Purnell and Botella 2007, Skopelitis et al 2007, for more information see below). Whereas, under stress the aminating activity of GDH is induced (Skope-litis et al 2006).
Numerous studies based on the physical, biochemical and immunological characterization of the ammonia assimilatory enzymes have been performed in a variety of plants, including model and crop species. These studies, revealed important information on the regulation of nitrogen assimilation into amino acids taking into account that each reaction catalyzed by multiple isoenzymes is located in distinct organs, tissues or subcellular compartments. Originally, most of the molecular studies on ammonia assimilation in plants had focused on GS, and consequently there was greater understanding of the molecular expression and regulation of GS than with GOGAT, GDH, Asparagine synthetase (AS) and Aspartate aminotransferase (AspAT; Schultz et al. 1998, Hirel and Lea 2001, Lea et al. 2007).
The products of ammonium assimilation, glutamine and glutamate, serve as nitrogen-transport compounds and nitrogen donors in many cellular reactions, including the biosynthesis of aspartate and asparagine by AspAT and AS, respectively (Lea 1999). It has also recently been demonstrated that glutamine and glutamate play an important role as signalling molecules in a variety of metabolic and developmental processes (Coruzzi and Zhou 2001, Forde and Lea 2007). The four amino acids glutamine, glutamate, aspartate and asparagine, generated by this pathway, can account for more than 60% of the total free amino acid pool in most legumes and crop plants and are transported through the vascular tissues (Lea 1999, Ochs et al. 1999). Aspartate is an amino acid that participates in the malate-aspartate shuttle to transfer reducing equivalents from the mitochondrion and chloroplast into the cytosol and in the transport of carbon from mesophyll to bundle sheath for C4 carbon fixation. Asparagine is thought to be an important compound for transport and storage of nitrogen resources because of its relative stability and high nitrogen to carbon ratio (Lam et al. 1995, 1996, Lea et al. 2007).
The genes involved in ammonium assimilation are regulated by several factors such as nitrogen source, metabolites, light, and cell type (Lam et al. 1996). However, factors such as exogenously supplied nitrogen have been reported to have a variable influence in the synthesis of a number of isoenzymes involved in nitrogen metabolism, depending upon plant material and experimental conditions. Now we have a wider view on the regulation of ammonia assimilating enzymes including GDH, GOGAT, AS, AspAT at least in the model species Arabidopsis and tobacco. Extensive biochemical, genetic and molecular analyses of mutants or transgenic plants have allowed to define the in vivo roles of individual isoenzymes and uncovered the regulatory mechanisms involved (Hirel and Lemaire 2005, Purnell and Botella 2007, Skopelitis et al. 2006) as well as the interactions with carbon metabolism (Foyer et al. 2001).
Plant GS is an octameric enzyme with a native molecular mass of 350400 kD. In size and quaternary structure it strongly resembles the enzyme isolated from mammals, but it is distinct from the bacterial enzyme, which consists of 12 subunits. The subunits are assembled as two tetramers stacked one upon another, with the active site in the interface of subunits (Unno et al. 2006). The reaction catalysed by GS is believed to proceed via a two-step process, the first involving the formation of GS-bound glutamyl phosphate from ATP and glutamate, followed by the addition of ammonia to form a tetrahedral adduct with the subsequent liberation of glutamine, ADP, and Pi (Lea and Ridley 1989, Ishi-maya et al. 2006). GS exists as multiple isoenzymes that are of two types; one type is exclusively localized to the cytosol (GS1) and the other is restricted to the plastids (GS2). Subunits of the various isoenzymes exhibit both, size (37-44 kD) and charge heterogeneity (Bennett and Cullimore 1989, Temple et al.
1995). In general, all plant species examined contain one GS2 isoform encoded by a single nuclear gene (Peterman and Goodman 1991, Stanford et al. 1993) and a number of cytosolic GS1 isoforms encoded by small gene families (up to six genes) (Peterman and Goodman 1991, Sakakibara et al. 1992, Stanford et al. 1993, Temple et al. 1995).
In addition to the dual cellular compartmentalization, the relative proportion of the individual isoforms of GS may vary according to the plant species, the physiological status of the plant, the developmental stage and the organ examined (Cren and Hirel 1999). In photosynthetic organs, light, possibly via phy-tochrome and a blue light receptor, and also ammonium induce the expression of the plastid forms of the enzyme (Edwards and Coruzzi 1989, Migge et al.
1996). This form is also preferentially degraded during plant senescence in several plant species (Masclaux et al. 2001).
In shoots and leaves, chloroplastic GS is thought to respond for the assimilation of primary ammonium reduced from nitrate and also for the reassimilation of ammonium released during photorespiration. The GS in root plastids generates amide nitrogen for local consumption (Peat and Tobin 1996). The GS1 isoforms are expressed in germinating seeds or in the vascular bundles of roots and shoots and seem to generate glutamine required as a substrate for several biosynthetic pathways and for transport of organic nitrogen throughout the plant (Miflin and Lea 1980, Lea et al. 1990, Lam et al. 1996, Oliveira and Coruzzi 1999). GS gene families have been relatively well characterized in leguminous plant species and in some non-legume annual plants, and now in per ennial plant species, especially at the molecular level (Cánovas et al. 2007).
In grapevine GS activity has been detected in several tissues such as leaves, shoots, roots, berries, calluses and cell suspensions (Roubelakis-Angelakis and Kliewer 1983, Ghisi et al. 1984, Loulakakis and Roubelakis-Angelakis 1996b, 2000). Grapevine leaf GS was analyzed by ion exchange chromatography into two distinct classes of isoenzymes, the cytosolic and the chloroplastic. By analogy with the corresponding chromatographic forms of other higher plants, they were designated as GS1 and GS2, respectively. The GS activity of root extracts was eluted as a single peak at position identical with the lower ionic strength-eluted GS1 peak of the leaf enzyme (Loulakakis and Roubelakis-Angelakis 1996b).
Western blot analysis revealed that grapevine GS consists of three types of polypeptides of distinct size (44, 43 and 39 kD, respectively) and differential tissue specificity. The 39 kD class of polypeptides was present in all tissues examined, the 43 kD band showed leaf-specificity, whereas the 44 kD band was detected in leaf and in lower amounts in shoot and cell suspension but not in root tissue (Loulakakis and Roubelakis-Angelakis 1996b). These bands were further analyzed into several polypeptides using 2-D electrophoresis (Fig. 3). Thus, grapevine GS seems to have similar isoenzyme and polypeptide patterns with the enzyme from other higher plants (Bennett and Cullimore 1989, Peterman and Goodman 1991, Sakakibara et al. 1992). The 39 kD class of polypeptides is cytosolic and the 43 and 44 kD class of grapevine GS polypep-tides are chloroplastic that assemble into active GS1 and GS2 isoforms, respectively. The size divergence of the GS2 subunits could be attributed to post-translational modification of the product of a single nuclear gene such as glyco-sylation (Nato et al. 1984) or phosphorylation (Lima et al. 2006). On the other hand, cytosolic GS consists of at least 3 or 4 polypeptides of similar size but with different charge.
The existence of small gene families encoding for different cytosolic isoenzymes has been described for several plant species. For example, up to five putative cytosolic cDNA clones have been characterized in Arabidopsis thaliana (Peterson and Goodman 1991), and maize (Sakakibara et al. 1992, Li et al. 1993). In grapevine, three homologous but structurally distinct cDNA clones, pGS1;1, pGS1;2 and pGS1;3, encoding GS were isolated and characterized (Loulakakis and Roubelakis-Angelakis 1996b, 2000 and our unpublished results). Their structural characteristics and the expression patterns of the respective genes supported that the three clones are derived from mRNAs encoding cytosolic polypeptides.
Each clone contains an open reading frame of 1068 nucleotides that encodes a protein of 356 amino acids with a predicted molecular weight of 39 kD. The coding regions of the three grapevine GS clones show 84 to 93% nucleotide and amino acid identity, whereas, their 5'- and 3'-untranslated sequences are quite divergent showing 40% nucleotide identity. Grapevine GS clones, as other cytosolic GS forms, lack the characteristic N-terminal and C-terminal amino acid extensions of the chloroplastic polypeptides. At the amino acid level, pGS1;1, pGS1;2 and pGS1;3 were 82-92% identical to the cytosolic GS sequences, whereas only 76-80% identity to the chloroplastic GS sequences of other higher plants was observed. Although sequence conservation among GS genes with common functions has been reported between species (Temple et al. 1995), comparison of the coding sequences and the 3' untranslated sequences of grapevine GS cDNA clones to each member of GS1 gene families from other plants failed to reveal any difference in homologies (Loulakakis and Roube-lakis-Angelakis 1996b).
Was this article helpful?
Acai, Maqui And Many Other Popular Berries That Will Change Your Life And Health. Berries have been demonstrated to be some of the healthiest foods on the planet. Each month or so it seems fresh research is being brought out and new berries are being exposed and analyzed for their health giving attributes.