title: Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa (Medicago sativa L.).
abstract: Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impact of specifically reducing nodule NADH-GOGAT activity on symbiotic performance of alfalfa (Medicago sativa L.). Four independent transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for GOGATantisense), containing an antisense NADH-GOGAT cDNA fragment under the control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA plants were fertile and showed normal growth in non-symbiotic conditions. The NADH-GOGAT antisense transgene was heritable and the T1 plants showed phenotypic alterations - similar to primary transformants. Clonally propagated plants were inoculated with Sinorhizobium meliloti after rooting and the symbiotic phenotype was analyzed 21 days post-inoculation. Nodules of each GA line had reduced NADH-GOGAT activity, ranging from 33 to 87% of control plants, that was accompanied by comparable decreases in RNA and protein. Plants from the GA89 line, with the lowest NADH-GOGAT activity (c. 30%), presented a strikingly altered symbiotic phenotype: concomitantly activities of key enzyme for carbon and nitrogen assimilation decreased; nodule amino acids and amides were reduced while sucrose accumulated. Antisense GOGAT plants were chlorotic, reduced in fresh weight, and had a lower N content than control plants. Photosynthesis was also impaired in antisense plants. Specifically, reducing NADH-GOGAT in nodules resulted in plants having impaired nitrogen assimilation and altered carbon/nitrogen metabolic flux.
authors.affiliation: Centro de Investigación sobre Fijación de Nitrógeno, UNAM, Ap. Postal 565-A, Cuernavaca, Mor. México.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Cordoba
authors.forename: Svetlana
authors.initials: S
authors.lastname: Shishkova
authors.forename: Carroll P
authors.initials: CP
authors.lastname: Vance
authors.forename: Georgina
authors.initials: G
authors.lastname: Hernández
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D016375:Antisense Elements (Genetics) / Q000235:genetics / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D050790:Glutamate Synthase (NADH); D000455:Medicago sativa / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D038821:Mycorrhizae / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism*; D010641:Phenotype; D030821:Plants, Genetically Modified; D013559:Symbiosis
chemical_list: D016375:Antisense Elements (Genetics); D002244:Carbon; D000594:Amino Acid Oxidoreductases; D050790:Glutamate Synthase (NADH); D009584:Nitrogen