The development of clustered tertiary lateral roots (proteoid roots) and the expression of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in roots were studied in white lupin (Lupinus albus L.) grown with either 1 mM P (+P-treated) or without P (-P-treated). The +P-treated plants initiated fewer clustered tertiary meristems and the emergence of these meristems was delayed compared with -P-treated plants. Proteoid root zones could be identified 9 d after emergence in both P treatments. Amounts of PEPC mRNA, PEPC specific activity, and enzyme protein were greater in proteoid roots than in normal roots beginning at 10, 12, and 14 d after emergence, respectively. The increases in PEPC mRNA, PEPC enzyme, and PEPC specific activity suggest that this enzyme is in part under transcriptional regulation. Recovery of organic acids from root exudates coincided with the increases in PEPC specific activity. The -P-treated plants exuded 40-, 20-, and 5-fold more citrate, malate, and succinate, respectively, than did +P-treated plants. Data presented support the hypothesis that white lupin has concerted regulation of proteoid root development, transcriptional regulation of PEPC, and biosynthesis of organic acids for exudation in response to P deficiency.

Proteoid roots develop in Lupinus albus L. in response to nutrient stress, especially P. Proteoid roots excrete citrate and thus increase the availability of P, Fe, and Mn in the rhizosphere. In an effort to understand citrate synthesis and organic acid metabolism in proteoid roots of lupin, we have evaluated in vitro enzyme activities of citrate synthase (CS), malate dehydrogenase (MDH), and phosphoenolpyruvate carboxylase (PEPC) in proteoid and normal roots of plants grown with or without P. Organic acid concentrations, respiration rates, and dark 14CO2-labeling patterns were also determined. The in vitro specific activities of CS, MDH, and PEPC and in vivo dark 14CO2 fixation were higher in proteoid roots compared to normal roots, particularly under P stress. Western blot analysis showed that PEPC enzyme protein was more highly expressed in -P proteoid roots compared to other tissues. The majority of the fixed 14C was found in organic acids, predominantly malate and citrate. A larger fraction of citrate was labeled in P- stressed proteoid roots compared to other root tissue. Respiration rates of proteoid roots were 31% less than those of normal roots. The data provide evidence for increased synthesis of citrate in proteoid roots compared to normal roots, particularly under P stress. A portion of the carbon for citrate synthesis is derived from nonautotrophic CO2 fixation via PEPC in proteoid roots.