Phosphorus deficiency-induced modifications in citrate catabolism and in cytosolic pH as related to citrate exudation in cluster roots of white lupin

A possible contribution of alterations in metabolic sequences involved in citrate catabolism, to intracellular accumulation and subsequent release of citrate was investigated in cluster roots of phosphorus (P)-deficient white lupin (Lupinus albus L.). Citrate accumulation during maturation of root clusters was associated with decreased levels of intracellular soluble P-i and ATP, and with reduced rates of respiration. Inhibitor studies with KCN and salicylhydroxamic acid (SHAM) suggest a reduced capacity of both the cytochrome pathway and of the alternative respiration with a concomitant decrease of immunochemically detectable protein levels of the alternative oxidase. Reduced respiration seems to be related to a general impairment of the respiratory system, rather than to limitation of respiratory substrates such as P-i and adenylates, as indicated by the absence of stimulatory effects of the uncoupler CCCP. The citrate/malate ratio in juvenile root clusters with high rates of respiration and low inherent levels of citrate accumulation was increased by short-term application (4-8 h) of azide and SHAM as respiration inhibitors. During maturation of root clusters, a shift from intracellular malic acid to citric acid accumulation was associated also with down-regulation of ATP citrate lyase (ACL), which catalyzes cleavage of citrate into acetylCoA and oxaloacetate with a putative function as anapleurotic source for the production of acetyl-CoA under P-deficient conditions. Inhibition of nitrate uptake and assimilation is a general response to P limitation in many plant species including white lupin. Reduced consumption of the amino acceptor 2-oxoglutaric acid as a product of citrate turnover may therefore contribute to increased citrate accumulation. Accordingly, artificial inhibition of nitrate reduction by localized application of tungstate significantly increased the citrate/malate ratio in juvenile root clusters. Lowering the cytosolic pH by external application of propionate stimulated citrate and malate exudation in non-cluster lateral roots and in developing root clusters. This effect was reverted by preincubation with phosphonate to buffer the cytosol. The results suggest that acidification of the cytosol may be an important factor, triggering the transient release of citrate and protons from mature root clusters in P-deficient white lupin.