Fig. 17.2. Grain yields of transgenic rice at T0 generation, introduced either with a cDNA fragment for NADH-GOGAT in the antisense orientation (A1 to A5) or with the pIG121Hm-vector alone (G1 and G2).
intracellular localization studies indicated that NADH-GOGAT protein is located in the plastids of rice roots.29 The localization in plastids might be expected in developing leaf blades and young grains, since NADH-GOGAT occurs as a single gene in rice.23
The presumed transcribed region (11.7 kbp) of the NADH-GOGAT gene consists of twenty-three exons separated by twenty-two introns with a range in exon size of from 65 bp to 1,530 bp.23 This gene structure is slightly different from that found in alfalfa nodules,30 which comprises twenty-two exons and twenty-one introns, and apparently lacks the first exon seen in the rice gene. Most of the corresponding exons show more than 60% similarity in the nucleotide sequence between the two species. Sequences for a putative amide transfer, FMN binding, [3Fe-4S] cluster, and NADH binding regions were detected,23 as in the NADH-GOGAT gene from alfalfa nodules.30 The genomic clone for rice NADH-GOGAT covered a 3.7 kbp 5'-upstream region from the first methionine. The transcriptional start sites were identified with primer extension analysis and S1 nuclease protection experiments. Rice NADH-GOGAT is synthesized as a 2,166 amino acid protein that includes a 99 amino acid presequence.23
By using the 3.7 kbp 5'-upstream region as a promoter for NADH-GOGAT, a chimeric gene construct having this promoter and the p-glucuronidase (GUS) structural gene as a reporter was introduced into rice calli that had been generated from germinating seeds, and transgenic rice plants were produced. Expression of the NADH-GOGAT gene is now monitored by GUS activity staining in various organs of the regenerated rice at the T0 generation. Preliminary results show that GUS activity is detected in vascular bundles of various organs of the T0 rice plants (Kojima et al, unpublished results). The 3.7 kbp upstream region from the translation start possesses the promoter activity for the NADH-GOGAT gene, with element(s) which determine the cell type specificity for gene expression in rice plants. Further transformation with a series of deletion clones of this region is now in progress to identify cis-elements related to the age-specific, cell-specific, and nitrogen-responsive expression of NADH-GOGAT in rice plants.
In the roots of rice seedlings, the mRNA for NADH-GOGAT was markedly accumulated within 3 h after the supply of a low concentration of ammonium ions.26 Cycloheximide in the presence of ammonium ions had no effect on the increase in mRNA, suggesting that protein synthesis is not required for the accumulation of mRNA. This could be related to the fast response of the accumulation after the supply of ammonium ions. Methionine sulfoximine, an inhibitor of GS, completely inhibited the accumulation of NADH-GOGAT mRNA and the supply of glutamine replaced the effect of ammonium ions.26 These results show that ammonium ions are not a direct inducer of this process. Glutamine or its metabolite might be the signaling factor for the inducible accumulation of NADH-GOGAT mRNA in rice roots. A similar response would be expected in the young developing organs to regulate the expression of a NADH-GOGAT gene caused by glutamine or its metabolite in solute transported through vascular tissues.
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