Metabolic regulation and gene expression of root phosphoenolpyruvate carboxylase by different nitrogen sources

Alfalfa (Medicago sativa L.) N-sufficient plants were fed 1·5 mM N in the form of NO3 -, NH4 + or NO3 - in conjunction with NH4 +, or were N-deprived for 2 weeks. The specific activity of phosphoenolpyruvate carboxylase (PEPC) from the non-nodulated roots of N-sufficient plants was increased in comparison with that of N-deprived plants. The PEPC value was highest with NO3 - nutrition, lowest with NH4 + and intermediate in plants that were fed mixed salts. The protein was more abundant in NO3 --fed plants than in either NH4 +- or N mixed-fed plants. Nitrogen starvation decreased the level of PEPC mRNA, and nitrate was the N form that most stimulated PEPC gene expression. The malate content was significantly lower in NO3 -- deprived than in NO3 --sufficient plants. Root malate accumulation was high in NO3 --fed plants, but decreased significantly in plants that were fed with NH4 +. The effect of malate on the desalted enzyme was also investigated. Root PEPC was not very sensitive to malate and PEPC activity was inhibited only by very high concentrations of malate. Asparagine and glutamine enhanced PEPC activity markedly in NO3 --fed plants, but failed to affect plants that were either treated with other N types or N starved. Glutamate and citrate inhibited PEPC activity only at optimal pH. N-nutrition also influenced root nitrate and ammonium accumulation. Nitrate accumulated in the roots of NO3 -- and (NO3 - + NH4 +)-fed plants, but was undetectable in those administered NH4 +. Both the nitrate and the ammonium contents were significantly reduced in NO3 -- and (NO3 - + NH4 +)-starved plants. Root accumulation of free amino acids was strongly influenced by the type of N administered. It was highest in NH4 +-fed plants and the most abundant amides were asparagine and glutamine. It was concluded that root PEPC from alfalfa plants is N regulated and that nitrate exerts a strong influence on the PEPC enzyme by enhancing both PEPC gene expression and activity.