Molecular and whole-plant responses to selection for enzyme activity in alfalfa root nodules: evidence for molecular compensation of aspartate aminotransferase expression

Abstract

The enzyme aspartate aminotransferase (AAT) plays a key role in the assimilation of fixed-N in alfalfa (Medicago sativa L.) root nodules. AAT activity in alfalfa nodules is due to the activity of two dimeric isozymes, AAT-1 and AAT-2, that are products of two distinct genes. Three forms of AAT-2 (AAT-2a, -2b, and-2c) have been identified. It was hypothesized that two alleles occur at the AAT-2 locus, giving rise to the three AAT-2 enzymes. In a prior study bidirectional selection for root nodule AAT and asparagine synthetase (AS) activities on a nodule fresh weight basis in two diverse alfalfa germ plasms resulted in high nodule enzyme activity subpopulations with about 20% more nodule AAT activity than low enzyme activity subpopulations. The objectives of the study presented here were to determine the inheritance of nodule AAT-2 production and to evaluate the effect of bidirectional selection for AAT and AS on AAT-2 allelic frequencies, the relative contributions of AAT-1 and AAT-2 to total nodule activity, nodule enzyme concentration, and correlated traits. Two alleles at the AAT-2 locus were verified by evaluating segregation of isozyme phenotypes among F1 and S1 progeny of crosses or selfs. Characterization of subpopulations for responses associated with selection was conducted using immunoprecipitation of in vitro nodule AAT activity, quantification of AAT enzyme protein by ELISA, and AAT activity staining of native isozymes on PAGE. Results indicate that selection for total AAT activity specifically altered the expression of the nodule AAT-2 isozyme. AAT-2 activity was significantly greater in high compared to low activity subpopulations, and high AAT subpopulations from both germ plasms had about 18% more AAT-2 enzyme (on a nodule fresh weight basis). No significant or consistent changes in AAT-2 genotypic frequencies in subpopulations were caused by selection for AAT activity. Since changes in AAT activity were not associated with changes in AAT-2 genotype, selection must have affected a change(s) at another locus (or loci), which indirectly effects the expression of nodule AAT.