Leaf-specific overexpression of plastidic glutamine synthetase stimulates the growth of transgenic tobacco seedlings.

Abstract

The impact of increased plastidic glutamine synthetase (GS-2; EC 6.1. 3.2) activity on foliar amino-acid levels and on biomass production was examined in transgenic tobacco. For that, tobacco was transformed via Agrobacterium tumefaciens with a binary vector containing a tobacco GS-2 cDNA downstream of the leaf-specific soybean ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promotor. Two transgenic tobacco lines with 15- to 18-fold higher foliar GS-2 transcript levels than the wild type were obtained. The GS-2 protein pools and the specific GS-2 activities were, however, only 2- to 2.3-fold higher in the leaves of the transgenic plants than in the leaves of the wild type. This discrepancy may reflect a post-transcriptional control of GS-2 protein accumulation. The increased GS-2 activity was correlated with a decrease in the leaf ammonium pool (3.7-fold) and an increase in the levels of some free amino acids, including glutamate (2. 5-fold) and glutamine (2.3-fold). The accumulation of soluble protein per unit fresh weight, however, remained unchanged. This result indicates that a process downstream of the synthesis of the primary organic products of N-assimilation is limiting leaf protein accumulation. Nevertheless, the overexpression of GS-2 stimulated the growth rate of the transgenic tobacco seedlings which, consequently, were larger (20-30% on a fresh-weight basis) than wild-type seedlings grown under identical conditions. This result suggests that GS-2 is the rate-limiting enzyme during biomass production in tobacco seedlings. The requirement for glutamate as the ammonium acceptor in the reaction catalysed by GS-2 may imply that there is co-regulation of GS-2 and ferredoxin dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) gene expression. Increased leaf GS-2 activity had, however, no influence on the foliar Fd-GOGAT protein abundance. This result suggests that in tobacco leaves, more Fd-GOGAT is present than required to meet the demands of primary ammonium assimilation and that there is no strong interdependence between GS-2 and Fd-GOGAT protein expression.