Molecular characterization of NADH-dependent glutamate synthase from alfalfa nodules.

Abstract

Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules. Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants. A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT. Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence. The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT. DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene o r a small gene family. The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules. A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R. meliloti strains. Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis.

Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule‐enhanced MDH

“…cDNA cloning Approximately 500 000 PFU of an alfalfa nodule cDNA library (Gregerson et al, 1993) were screened by hybridization with the inserts from watermelon mMDH (Gietl, 1992) and gMDH (Gietl, 1990) cDNAs. Positively hybridizing phage were purified, DNA was extracted, and inserts large enough to potentially contain the entire MDH coding region were subcloned into pBluescript.…”

“…To obtain additional MDH clones, E. coli 1477 cells were transformed via electroporation with an alfalfa nodule plasmid © Blackwell Science Ltd, The Plant Journal, (1998), 15, 173-184 (pcDNAII, Invitrogen, San Diego, CA, USA) cDNA library (Gregerson et al, 1993). Following phenotypic lag, the cells were washed three times with 500 µl 0.85% NaCl, resuspended in 1 ml 0.85% NaCl, and 100 µl aliquots were plated on M9-succinate plates containing ampicillin.…”

“…A phylogenetic tree was calculated from the aligned sequence using the neighbor-joining method contained in the CLUSTAL W suite of programs (Higgins and Sharp, 1988;Saitou and Nei, 1987 RNA and DNA gel blot analysis Total and poly(A) ϩ RNA was isolated from alfalfa root, stem, leaf, cotyledon and nodule tissue and blotted as described previously (Gregerson et al, 1993). Membranes were hybridized and washed at high stringency as described (Gantt et al, 1992;Gregerson et al, 1993). Filters were exposed to X-ray film, and the amount of hybridizing RNA was also quantitated using an AMBIS Radioanalytic Imaging System (Scanalytics, Billerica, MA, USA).…”

“…Filters were exposed to X-ray film, and the amount of hybridizing RNA was also quantitated using an AMBIS Radioanalytic Imaging System (Scanalytics, Billerica, MA, USA). DNA was extracted from etiolated alfalfa seedlings and Southern blots were run as previously described (Gantt et al, 1992;Gregerson et al, 1993).…”

Nitrogen assimilation in Citrus based on CitEST data mining

“…In root nodules of legumes, NADH-GOGAT is involved in the assimilation of nitrogen fixed by Rhizobium (Anderson et al, 1989). It has been hypothesized that NADH-GOGAT catalyzes the rate-limiting step of ammonia assimilation in these root nodules (Gregerson et al, 1993). In non-leguminous plants, Nitrogen assimilation in Citrus GOGAT may function in the primary assimilation or reassimilation of ammonia released during amino acid catabolism (Miflin and Lea, 1977).…”