Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules

Lourdes Blanco, Pallavolu M. Reddy, Sonia Silvente, Bruna Bucciarelli, Sanghamitra Khandual, Xochitl Alvarado-Affantranger, Federico Sánchez, Susan Miller, Carroll Vance, Miguel Lara-Flores

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

NADH-dependent glutamate synthase (NADH-GOGAT) is a key enzyme in primary ammonia assimilation in Phaseolus vulgaris nodules. Two different types of cDNA clones of PvNADH-GOGAT were isolated from the 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. 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 by RT-PCR also revealed that both of these genes are differentially regulated by sucrose. On the other hand, the expression of PvNADH-GOGAT-I, but not PvNADH-GOGAT-II, was inhibited with nitrogen compounds. 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.

Original languageEnglish (US)
Pages (from-to)454-472
Number of pages19
JournalPlant, Cell and Environment
Volume31
Issue number4
DOIs
StatePublished - Apr 1 2008

Keywords

  • Carbon and nitrogen compounds
  • Gene expression
  • Phaseolus vulgaris
  • Promoter analysis
  • PvNADH-GOGAT

Fingerprint Dive into the research topics of 'Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules'. Together they form a unique fingerprint.

Cite this