The overexpression of asparagine synthetase A from E. coli affects the nitrogen status in leaves of lettuce (Lactuca sativa L.) and enhances vegetative growth

Abstract

The asparagine synthetase A (EC 6.3.1.1) of E. coli (AS-A) mainly uses ammonia to produce asparagine, a key nitrogen transporter in plants. The AS-A encoding gene (asnA) was expressed constitutively in lettuce cultivar ‘Cortina’ under the control of pMAC, a chimerical promoter, to induce phenotypical alterations of plant growth and quality as a consequence of nitrogen status changes. Nine fertile transgenic lines harbouring independent T-DNA insertions were recovered. Primary transformants shared new visible traits such as a higher leaf number and wider leaf surface than the wild-type. The progeny of three primary transformants stably maintained these phenotypes, to which the synthesis of both asnA transcript and protein were associated. In pMAC:asnA plants, seed germination, formation and development of leaves, bolting and flowering occurred earlier than non-transformed plants. Twenty-eight days after sowing (das), transgenic plants showed a ca. 1.3 increase of leaf area and dry weight as compared to the wild-type. Moreover, the contents of asparagine, aspartic acid and glutamine, but not that of glutamic acid, of pMAC:asnA young plants (21 das) were greater than the wild-type. The level of total soluble protein was higher in transgenic than in non-transformed leaves borne on plants at 35, 50 and 75 das. A decrease of nitrate was also measured in pMAC:asnA leaves with respect to non-transformed ‘Cortina’, in transgenic populations at 60 das. In pMAC:asnA genotypes, the altered content of nitrogen transport amino acids, the tolerance to increasing doses of ammonium and phosphinothricin indirectly proved the AS-A enzymatic activity in lettuce.