Introduction

In plants, malate is a key product of metabolism. It is thought by many (see Lance, Rustin 1984) to be the ultimate product of glycolysis, rather than pyruvate. As such it plays important roles in photosynthesis (both C3 and C4), energy generation, fatty acid oxidation, ion balance, energy generation, pulvinal and stomatal function, amino acid synthesis, and nitrogen (N2) fixation (Gietl 1992; Martinoia, Rentsch 1994).

In N2-fixing nodules malate is the predominant source of energy for bacteroid respiration (Driscoll, Finan 1993) and provides a significant portion of the carbon skeletons for assimilation of fixed N2 (Rosendahl et al. 1990). Malate may also be involved in regulation of the nodule oxygen diffusion barrier through an osmoelectrical mechanism (Vance, Heichel 1991; Denison 1998; Galvez et al. 2000). The critical role that malate plays in root nodules is evidenced by the fact that ineffective nodules, whether induced by changes in either the bacterial or plant genotype, have strikingly reduced levels of malate as compared to effective nodules. Moreover, mutations in rhizobia that block organic acid use result in ineffective nodules while those that block amino acid and carbohydrate use generally have no effect on N2 fixation (Ronson et al. 1981; Driscoll, Finan 1993).

The enzyme malate dehydrogenase (MDH; EC 1.1.1.82) catalyzes the reversible reduction of oxaloacetate to malate. Because malate is important in many metabolic pathways in higher plants, it occurs in multiple forms that differ in co-enzyme specificity and subcellular localization (Gietl 1992). We have identified five different forms of MDH in alfalfa (Miller et al. 1998): glyoxysomal, gMDH; mitochondrial, mMDH; chloroplast, chMDH; cytosolic, cMDH; and nodule enhanced, neMDH. Kinetic analysis along with mRNA and protein expression data indicated that neMDH and cMDH may have important functions in effective root nodules. To further understand the role of malate in root nodule function we thought it important to: (1) isolate and characterize the plant genes encoding ne- and cMDH; (2) identify cellular expression patterns for ne- and cMDH protein and mRNA in alfalfa root nodules; and (3) assess whether constitutive overexpression of neMDH had an effect on symbiotic N2 fixation.

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