Almost any nitrogen compound can be considered as a potential end product of NH4+ assimilation, and many act as inhibitor of GS activity in vitro (Stewart et
al., 1980), but their significance in vivo has not been established. There are many examples, however, of nitrogen source influencing GS expression.
Ammonia - In Lemna minor (Rhodes et al., 1976) and in the embryonic axes of Lupinus luteus (Ratajczak et a l , 1981) total GS activity decreased in plants suppUed with N H / as their sole source of nitrogen. This decrease correlated with an increase in free glutamine levels, and a mechanism was proposed whereby the native octameric enzyme is reversibly dissociated into two inactive tetramers
(Stewart et a l, 1980). A tetrameric form of GS has been identified in the leaves of Beta vulgaris (Mack, 1988), although it was found to be active. Hoelzle et al. (1992) have more recently suggested that an inactive form of GS protein might be present in N-limited plants.
Monitoring GS mRNA levels in plants grown in the absence or presence of NH4+, and a comparison of GS mRNA levels in nodules of Fix" mutants and wild-
type Rhizobium has indicated that induction of the ’nodule-specific’ GSl subunits in P. vulgaris (Padilla et a l , 1987; Cock et al., 1990) P. sativum (Walker and Coruzzi, 1989; Brears et a l , 1991) Af. sativa (Dunn et al., 1988) and G. max
1991). Using this GSl promoter to direct GUS expression in transgenic L.
comiculatus, Miao et al. (1991) were able to demonstrate a three-fold increase in GUS activity in plants supplied with 10 mM NH^^, while applications of NO;, asparagine or glutamine had little or no effect. Marsolier et al. (1993) located the NH^+ responsive elements of the gene to the region between 3.5 and 1.3 kb from the transcription start site, although this promoter was unresponsive to
treatment in transgenic N. tabacum.
Nitrate - The reduction of NO3 through NO; to NH4+ is mediated by
cytoplasmically located nitrate reductase (NR) and plastidic nitrite reductase (NiR), and it is now well established that both enzymes are induced as a primary response to NO3 (Redinbaugh and Campbell, 1991; Pelsey and Caboche, 1992). Nitrate is
known to directly affect several other related systems including NO, uptake and transport (Redinbaugh and Campbell, 1991). Nitrate has also recently been shown to increase both ferredoxin and NADPH-dependent ferredoxin-NADP^
oxidoreductase located in the root plastids of P. sativum (Bowsher et al., 1993), both of which are required for the transfer of reductant to NiR. As GS is the next enzyme along the pathway of NO3 reduction, its response to NO3 is of great
interest, especially since it has not been established which isoform is responsible for assimilating NH^"^ from NO/ reduction, although the plastid GS2 seems a likely candidate because of the plastidic location of NiR.
Externally applied NO, has a positive, though minor, effect on GS2 of Sinapsis alba and Helianthus annuus seedlings, and acts synergistically with light (Schmidt and Mohr, 1989; de la Haba et al., 1992), and it was suggested by Weber et al. (1990) that the appearance of S. alba NiR and GS2 in the presence of light and NO, are synchronized. It has been observed that in H. annuus seedlings pre-treated with tungstate, which inhibits NR and thereby prevents the reduction of NO3 to N H / (Deng et al., 1989), NO, still stimulates GS synthesis (de la Haba et
al., 1992).
Barratt (1980) reported the appearance of an extra isoform of root GS in NO3 -grown V. faba, which migrated alongside the leaf GS2 isoform, and which
was absent from NH^^-grown plants. Furthermore Vézina and Langlois (1989) demonstrated a specific increase in pea root plastidic GS2 protein and activity in plants grown on NO3 as compared to NH4+ or nitrogen-fi*ee medium. In neither
instance was it known if the induction was a primary or secondary response to NO3 . In Z. mays roots, mRNA and protein of a plastidic GS was shown to
increase upon nitrate application (Sakakibara et al., 1992a). The GS2 in leaf
mesophyll cells also increased up to 50-fold with nitrate, whereas the bundle sheath GS2 and the cytosolic GS were unaffected. The authors noted that although the photorespiratory enzymes are located in the bundle sheath cells, those of nitrate assimilation are found predominantly in mesophyll cells. The accumulation of GS2 transcripts in the roots of Z, mays has also recently been shown to be a primary response to NO3 (Redinbaugh and Campbell, 1993).
derived from NO, reduction (see Chapter 5), raises the question whether the presence of root plastidic GS2 in legumes is correlated with root nitrate assimilation.
1.5 .1 Root versus shoot nitrate assimilation
Nitrate must be reduced to NH4+ before it can be assimilated into amino
acids, but although most higher plants can assimilate NO, in either the roots or the shoot, the division of the process between the two sites varies greatly among species.
Although many legumes and non-legumes carry out a substantial proportion of their NO, assimilation in the shoot regardless of external NO, concentration, temperate legumes and some non-legumes (including many cereals, grasses and temperate woody species) growing in NO, concentrations expected in non- agricultural soils (less than 1 mM) carry out 30-50% of their assimilation in the root. However, as the NO, concentration increases to that of agricultural soils (1-20 mM) shoot assimilation becomes more important (Andrews, 1986a; Wallace, 1986; Andrews et al., 1992), implying that when an increase in the rate of NO, uptake does not induce a further increase in root activity, more NO, is transported to the shoot for reduction there.
Several factors other than species differences have been found to influence the partitioning of NO, assimilation, including cultivar (Sutherland et at., 1985; Andrews, 1986a), age (Sprent and Thomas, 1984), development of competing
sinks (Selamat and Gardner, 1985; Sung and Sun, 1990), temperature (Deane- Drummond et al., 1980; Sutherland et at., 1985), light intensity (Emes and Bowsher, 1991) and seasonal variation (Sung and Sun, 1990; Oaks, 1992).