Biochemical studies clearly show that GS in higher plants is an octameric protein with a native molecular mass of 350-400 kD (Ireland and Lea 1999). Leaves of most plants contain two isoenzymes of GS, which are readily separated by ion-exchange chromatography (McNally et al. 1983). The two forms were termed GS1 and GS2 from their order of elution. Tissue fractionation studies have shown that GS1 is localised in the cytosol while GS2 is in the chloroplasts/plastids (Mann et al. 1979, Hirel and Gadal 1981). Direct evidence to support the sub-cellular localisation of GS 1 and GS2 was obtained by immunogold labelling techniques in several species (Botella et al. 1988, Branjeon et al. 1989, Carvalho et al. 1992, Pereira et al. 1992, Peat and Tobin 1996). There is only one gene encoding GS2. The GS2 protein is first translated into the precursor protein that possesses chloroplast/plastid-targeting sequences at the N-terminus region. On the other hand, GS1 is encoded by a small multigene family, which varies from 2-6 genes, in monocotyledons and dicotyledons. Each gene synthesises a different subunit, which may be assembled into either a homooctametric and heterooctameric form (Walker and Coruzzi 1989, Sechley et al. 1992, Ireland and Lea 1999). The fact that regulation of GS gene families differs with cell type and environmental conditions indicates that each GS could have a non-overlapping function (Tingey et al. 1987, Edwards et al. 1990, Kamachi et al. 1992b, Lam et al. 1996).
GS2, the major isoenzyme in green leaves, is located in chloroplasts of mesophyll cells. Because mutants lacking GS2 were able to grow normally under non-photorespiratory conditions (Blackwell et al. 1988, Wallsgrove et al. 1987), GS1 in leaves could be important in the synthesis of glutamine for normal growth and development. Promoter-deletion analyses revealed that the 323-bp region in pea GS2 was able to express a reporter gene as a mesophyll cell-specific and light-responsive protein in transgenic tobacco and A. thaliana (Tjaden et al. 1995). Cellular localisation studies showed that GS1 is abundant in vascular tissues. For example, immunolocalisation studies indicate that GS1 protein is localised in phloem companion cells and related vascular cells of senescing rice leaf blades (Kamachi et al. 1992b, Sakurai et al. 1996) and sclerenchyma, xylem parenchyma, and guard cells of developing rice leaves (Sakurai et al. 2001). In tobacco, one of two GS1 genes is expressed in the vascular tissues of the stem and leaf midrib (Dubois et al. 1996). Promoter analyses also showed that one of three GS1 genes in pea (Edward et al. 1990) and kidney bean (Watson and Cullimore 1996), respectively, directed vascular-tissue specific expression in the heterologous transformation system. The localisation studies suggest that GS1 is important in the synthesis of glutamine, which is the major form of N in phloem sap in rice (Hayashi and Chino 1990), and for export from mature and senescing leaves (Tobin and Yamaya 2001). In developing leaves, GS 1 is probably involved in assimilating NH/ released from the reaction of phenylalanine ammonia lyase during lignin synthesis (Sakurai et al. 2001). Transgenic tobacco expressing antisense RNA for a GS1 (Gln1-5) showed a pronounced stress phenotype associated with a decrease in proline content, suggesting that GS1 in phloem plays a major role in regulating proline production (Brugiere et al. 1999). On the other hand, GS1 protein was detected both in vascular tissues and mesophyll cells of barley primary leaves (Tobin and Yamaya 2001) and in mesophyll cells in tobacco leaves during senescence (Brugiere et al. 2000). There are two GS1 genes in rice, i.e. GS1 preferentially expressed in leaves and GSr, which is mainly expressed in roots (Sakamoto et al. 1989). Promoter analysis of the GS1 gene of rice showed a preferential expression in vascular tissues of leaf blade, leaf sheath, roots and ear, but also expression in the wall-region of anther loculus and embryo in transgenic rice (Hanzawa et al. 2002). One of the five GS1 maize genes is preferentially expressed in the pedicels of developing kernels (Rastogi et al. 1998).
Non-legumes using NH4+ as the major N source, would be expected to have an efficient NH4+ assimilation system. Conversely there is an additional Casparian strip between exodermis and sclerenchyma in rice roots and therefore apoplastic transport of NH4+ is unlikely (Ishiyama et al. 1998). NH4+ taken up by rice roots is transported through the xylem to the shoots, mainly as glutamine (Fukumorita and Chino 1982). Immunolocalisation using GS1-specific IgG, which cross-reacts with both GS1 and GSr, showed that these cytosolic GS forms are distributed throughout the rice roots with apparent homogeneity within the epidermis, exodermis, cortex, and central cylinder (Ishiyama et al. 1998). To distinguish between GS1 and GSr in rice roots, GS1-promoter was fused with a GUS reporter gene and the chimeric gene was introduced into rice. Preliminary data showed that the GS 1 promoter is active in the central cylinder and cortex of the main roots as well as in the central cylinder of lateral roots (Hanzawa et al. 2002). Thus, GSr in epidermis and exodermis is probably responsible for the assimilation of NH4+ taken up by rice roots.
In contrast to rice, most plants growing on well-aerated soils use NO3- as their main N source. In the root NO3- is partly reduced to NH4+ before being assimilated via GS/GOGAT, but the proportion varies with plant species, plant age, and N availability (Tobin and Yamaya 2001). In roots of hydroponically grown barley seedlings, a GS1 (42 kD in subunit molecular mass) was constitutively detected in all root sections. Two additional polypeptides were detected in mature roots of NH4+-grown seedlings and one additional GS 1 in those grown on NO3- (Peat and Tobin 1996). Changes in GS1 polypeptides in response to NH4+ were also observed in barley roots (Mack 1995), although these differ slightly in molecular mass from the observation of Peat and Tobin (1996). Immunogold localisation studies indicated a higher concentration of GS1 in cortical parenchyma than in vascular stele cells (Peat and Tobin 1996). In these cell types the apparent concentration of GS1 was highest in N-deficient plants. In addition, significant labelling of GS protein was detected in plastids of cortical and vascular parenchyma cells of barley root apical cells (Peat and Tobin 1996). When barley plants were grown on 15 mM NO3-, there was an increase in immunogold labelling in the "tubular" and "flat" plastids in the roots (Tobin and Yamaya 2001).
In maize roots, a constitutive GS 1 polypeptide (40 kD) and an N inducible form (GSr, 39 kD) were detected (Sakakibara et al. 1992a). Two genes, GS1a and GS1b, encoded the GS1, while GSr was the product from GS1c and GS1d. Rapid accumulation of mRNA for GS1c and GS1d was observed when NH4+ was supplied, whereas the mRNA for GS1a and GS1b decreased under these conditions (Sakakibara et al. 1996). The GSr enzyme had a higher ratio of synthetase/transferase activities than the GS1 enzyme. Sakakibara et al. (1996) proposed that the GSr isoenzyme was more important in the assimilation of external NH4+. GS2 mRNA was also detected in maize roots exposed to low NO3- concentrations (Redinbaugh and Campbell 1993). Three putative genes for
GS1 and one for GS2 were expressed in roots of A. thaliana (Peterman and Goodman 1991). Gln1-5 from tobacco was preferentially expressed in the root vascular system, whereas Gln1-3 was detected in all root tissues (Dubois et al. 1996). More conclusive evidence, such as the use of knockout mutants, is required to establish the function of individual GS 1s and GS2 isoenzymes in roots.
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