The functional responses of phosphate-deficient lupin nodules as mediated by phosphoenolpyruvate carboxylase and alteredcarbon and nitrogen metabolism

[摘要] ENGLISH ABSTRACT: In soils, the concentration of available phosphate (P) for plants is normally very low(ca. 1 µM in the soil solution), because most of the P combines with iron, aluminiumand calcium to form relatively insoluble compounds. Inorganic P (Pi)-deficiency isthought to be one of the limiting factors of nitrogen fixation due to the high energyrequirement for nitrogenase function of plants taking part in nitrogen fixation. Pideficiencyhas important implications for the metabolic Pi and adenylate pools ofplants, which influence respiration and nitrogen fixation. An alternative route ofpyruvate supply during Pi stress has been proposed involving the combined activitiesof phosphoenolpyruvate carboxylase (PEPc), malate dehydrogenase (MDH) andNAD-malic enzyme (ME) supplying pyruvate to the mitochondrion during Pi stress.Previously, three isoforms of PEPc were isolated from lupin nodules and roots, withtwo forms being nodule specific. The aim of this project was to determine the effect ofPi stress on these PEPc isoforms in Lupinus luteus at transcript and proteinexpression level with a view to produce genetically modified crops for nutrient-poorsoils.Cytosolic P levels were measured over a time course to give an indication oftemporal development of P stress in nodules. The changes in enzyme activities ofPEPc, MDH and PK (pyruvate kinase) under P stress were measured and thedownstream effect on amino and organic acid pools were analysed. Two novel PEPcisoforms, LUP1 (AM235211) and LUP2 (AM237200) were isolated from nodules,followed by transcriptional and protein expression analyses.Nodules under P stress had lower amounts of metabolically available Pi and as Pstressed developed, the amount of Pi decreased. This decline in Pi levels wasassociated with lower growth, but higher biological nitrogen fixation (BNF). A greaterproportion of root-nodule respiration was devoted to nutrient acquisition than to newgrowth. A typical P-stress response is higher anaplerotic carbon fixation via PEPc.However, in this study, no significant differences were found for PEPc, MDH or PK inP-stressed plants compared to P-sufficient plants which would lead to an increase inorganic acids. An increase in key amino acids was reported along with unchangedlevels of organic acids. These levels of organic and amino acid are in congruencewith the increases in BNF under P-starvation.No significant differences were found in expression of PEPC1 or PEPC2 at 12 and20 days for both P-sufficient and P-stressed plants which further supported the lackof engagement of the PEPc-MDH-ME bypass. PEPc activity appeared not to beregulated by gene expression or phosphorylation indicating that other posttranslationalmodifications such as a decrease in protein degradation may be ofimportance.

[发布机构] Stellenbosch University