|Miller, Susan - Sue|
Submitted to: Plant Cell and Environment
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2008
Publication Date: 4/1/2008
Publication URL: http://hdl.handle.net/10113/27937
Citation: Blanco, L., Reddy, P.M., Silvente, S., Bucciarelli, B., Khandual, S., Alvarado-Affantrange, X., Sanchez, F., Miller, S.S., Vance, C.P., Lara-Flores, M. 2008. Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules. Plant Cell and Environment. 31:454-472. Interpretive Summary: Nitrogen (N) is a major element requirement for plant yield and quality. Common bean plants acquire N from both symbiotic N fixation and from N fertilizer. The N acquired from symbiosis and fertilizer is used to make the amino acids needed for plant growth. N is made into amino acids through the activity of the enzyme (protein catalyst) glutamate synthase (GOGAT). The GOGAT enzyme is encoded by plant genes. Little information exists on GOGAT genes in any type of bean plant. In this report we describe the GOGAT genes from common bean. Different from many crop species, we found that there are two GOGAT genes in common bean and they encode two separate GOGAT enzymes. The two GOGAT genes are active in different organs. One type of GOGAT is expressed in roots and root nodules. By comparison, the second GOGAT gene is expressed in leaves, stems, and seeds. Having an understanding of the GOGAT gene sequence and where the genes are expressed is useful because the information can provide insight into how bean N metabolism can be improved.
Technical Abstract: NADH-dependent glutamate synthase (NADH-GOGAT; EC 220.127.116.11) is a key enzyme in primary ammonia assimilation in bean (Phaseolus vulgaris L.) nodules. Two different types of cDNA clones of PvNADH-GOGAT were isolated from two independent nodule cDNA libraries. The full-length cDNA clones of PvNADH-GOGAT-I (7.4 Kb) and PvNADH-GOGAT-II (7.0 Kb), which displayed an 83% homology between them, were isolated using cDNA library screening, 'cDNA library walking' and RT-PCR amplification. Southern analysis employing specific 5' cDNA probes derived from PvNADH-GOGAT-I and PvNADH-GOGAT-II indicated the existence of a single copy of each gene in the bean genome. Deduced amino acid sequences of both bean NADH-GOGATs revealed a similarity ranging from 76% to 86% with alfalfa, Arabidopsis, and rice NADH-GOGATs. Both these proteins contain ~100 amino acid sequences theoretically addressing each isoenzyme to different subcellular compartments. RT-PCR analysis indicated that PvNADH-GOGAT-II expression is higher than PvNADH-GOGAT-I during nodule development. Expression analysis of PvNADH-GOGATs in nodules treated with sucrose and different nitrogen compounds revealed that the induction of PvNADH-GOGAT-II increased in the presence of sucrose but remained unaffected with different nitrogen compounds. On the other hand, PvNADH-GOGAT-I expression in nodules increased up to 0.5% sucrose but was drastically inhibited with amide or ureide treatments. In situ hybridization and promoter expression analyses demonstrated that the NADH-GOGAT-I and -II genes are differentially expressed in bean root and nodule tissues. In silico analyses of the NADH-GOGAT promoters revealed the presence of potential cis elements in them that could mediate differential tissue-specific, and sugar and amino acid responsive expression of these genes.