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

Plant Cell Environ. 2008 Apr;31(4):454-72. doi: 10.1111/j.1365-3040.2008.01774.x. Epub 2008 Jan 21.

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 approximately 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Carbon / metabolism
  • Carbon / pharmacology
  • Cloning, Molecular
  • DNA, Complementary / genetics*
  • Gene Expression Regulation, Plant* / drug effects
  • Glutamate Synthase (NADH) / chemistry
  • Glutamate Synthase (NADH) / genetics*
  • Molecular Sequence Data
  • Nitrogen / metabolism
  • Nitrogen / pharmacology
  • Phaseolus / enzymology*
  • Phaseolus / genetics*
  • Promoter Regions, Genetic / genetics
  • RNA, Plant / genetics
  • RNA, Plant / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Root Nodules, Plant / cytology
  • Root Nodules, Plant / enzymology*
  • Root Nodules, Plant / genetics*

Substances

  • DNA, Complementary
  • RNA, Plant
  • Carbon
  • Glutamate Synthase (NADH)
  • Nitrogen