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United States Department of Agriculture

Agricultural Research Service

Title: Alfalfa Malate Dehydrogenase (Mdh): Molecular Cloning and Characterizationof Five Different Forms Reveals a Unique Nodule Enhanced Mdh

item Driscoll, Brian - MCGILL UNIVERSITY
item Gregerson, Robert - LYON COLLEGE
item Vance, Carroll

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 8, 1998
Publication Date: N/A

Interpretive Summary: Legumes like alfalfa living in symbiosis with the soil bacterium Rhizobium can make their nitrogen (N) fertilizer through a process called symbiotic N2 fixation. This process occurs on roots in wart like structures called nodules. The plant gives the bacterium large amounts of carbon for growth and N2 fixation, while the bacterium gives the plant nitrogen fertilizer. The type of carbon which the plant gives to the Rhizobium bacteria is called malate. Plant synthesis of malate is controlled by the enzyme (protein catalyst) malate dehydrogenase (MDH). The number and type of MDHs that occur in N2 fixing root nodules are unknown. Efforts to improve N2 fixation and N use require that we know how malate is controlled and which MDH is important in nodules. In this report we document the discovery of five different MDH enzymes in alfalfa and the genes which control them. One of the five MDHs is highly enhanced in N2 fixing root nodules. This enzyme and the gene which makes it have not been previously found in any organism. The nodule enhanced MDH has an extraordinary capacity to produce malates, much higher than any other MDH enzyme. The discovery of nodule enhanced MDH is important because it explains how such large quantities of malate are produced to provide carbon for N2 fixation. Moreover, the discovery of this MDH gene provides a unique tool to use with biotechnological approaches to improve carbon metabolism and N2 fixation in legumes.

Technical Abstract: Malate dehydrogenase (MDH) catalyzes the readily reversible reaction converting oxaloacetate to malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C4 metabolism, pH balance, stomatal and pulvinal movement, respiration, B-oxidation of fatty acids, and legume root nodule functioning. Due to its sdiverse roles, the enzyme occurs as numerous isozymes in various organelles. Although antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Consistent with this finding, polypeptides corresponding to neMDH occur in both ureide- and amide-transporting legume species. Four of the five forms could effectively complement an E. coli Mdh mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.

Last Modified: 4/22/2015
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