Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules.
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.
Blanco
Lourdes
L
Centro de Ciencias Genómicas, Univrsidad Nacional Autónoma de México, Av Universidad, C.P. 62210, Cuernavaca, Morelos, México.
Reddy
Pallavolu M
PM
Silvente
Sonia
S
Bucciarelli
Bruna
B
Khandual
Sanghamitra
S
Alvarado-Affantranger
Xochitl
X
Sánchez
Federico
F
Miller
Susan
S
Vance
Carroll
C
Lara-Flores
Miguel
M
D000595:Amino Acid Sequence; D002244:Carbon / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D050790:Glutamate Synthase (NADH) / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism / Q000494:pharmacology; D027805:Phaseolus / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053204:Root Nodules, Plant / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*
D018076:DNA, Complementary; D018749:RNA, Plant; D002244:Carbon; D050790:Glutamate Synthase (NADH); D009584:Nitrogen