The regulation of Phosphoenolpyruvate (PEP) metabolism via Phosphoenolpyruvate Carboxylase (PEPC) in P-deficient roots and nodules of Virgilia divaricata

[摘要] ENGLISH ABSTRACT: Plants exhibit a flexible array of morphological, physiological and biochemical adaptations duringphosphorous limitation. Legumes are vulnerable to P deficiency, because it affects their ability tofix atmospheric nitrogen (N2). In particular, legumes from nutrient-poor ecosystems, such as theFynbos in the Cape Floristic Region (CFR) evolved on P deficient soils and may therefore displayunique adaptations to soil P stress. In general, very few studies on legumes have focussed on thebelowground structures of nodules as a plant organ. Moreover, even less is known about the Pstressed responses in nodules from legumes in nutrient-poor ecosystems. The aim of this researchwas to investigate the metabolic flexibility of organic acid and amino acid metabolism in thenodulated root system of the Fynbos legume Virgilia divaricata, during low P stress.Virgilia divaricata, which grows in the Cape Floristic Region, was used in this study to enhanceour knowledge regarding the vital role that the cytosolic enzyme, phosphoenol pyruvatecarboxylase (PEPC) plays in phosphoenol pyruvate (PEP) metabolism, in roots and nodules of thislegume during phosphate stress. V. divaricata was grown under glasshouse conditions (20 - 25°C)in sterilized quartz sand for 2-3 months whilst being inoculated with the nitrogen fixation bacteria,Burkholderia phytofirmans, which was isolated from V. divaricata nodules grown in fynbos soil.Two phosphate treatments, 5 μM and 500 μM, were applied simulating low-phosphate and highphosphate conditions respectively using a modified Long Ashton Nutrient Solution to simulate alow nutrient ecosystem such as the Cape Floristic Region. Roots and nodules were then analysedfor growth kinetics, nutrient acquisition and distribution, enzyme activity and genetic responses. Itwas shown that during phosphate deficiency, V. divaricata nodules experienced less Pi stress thanroots, due to increased metabolic phosphate conservation reactions during organic acid synthesisvia an increased PEPC activity. The increased PEPC activity resulted in an increase in downstreammetabolic products such as organic acids, (malic acid and citric acid), and amino acids (glutamate,aspartate and asparagine). Although the biological nitrogen fixation (BNF) declined, the highefficiency of BNF may be underpinned by these altered phosphate conservation pathways andenhanced resource allocation during growth particularly under low phosphate (LP) conditions. Therefore, it can be concluded that the efficiency of the nodules via an increased allocation ofresources and P acquiring mechanisms in V. divaricata may be the key to the plant's ability toadapt to poor P environments and thus sustaining its reliance on BNF. From the data obtained aswell as previous findings, it has been established that the phosphate conservation mechanisms in roots and nodules, involve the non-adenylate requiring PEPC-bypass route. 13C Nuclear magneticresonance (NMR) gave us a better understanding regarding the incorporation rates of the PEPCderivedC into malate, α-ketoglutarate and asparagine. It therefore is suggested that V. divaricatanodules may use their large PEPC-derived malate pool to prevent large declines in BNF under lowphosphate conditions. The nodules of V. divaricata were able to offset an excessive drop in BNF,despite a decline in inorganic phophosphate (Pi) levels. It therefore appears that nodules haveevolved to acquire different mechanisms than roots to adapt to phosphate deficiency in order tomaintain their function. This was achieved via increased regulation of nodule PEPC and itsdownstream products. This implies that compared to roots under low P, nodules alter themetabolism of PEPC derived C, in order to maintain nodule respiration and amino acid synthesis.This trait could be observed in the synthesis of larger 13C malate pools of nodules compared toroots, from PEPC, which was underpinned by their different regulation mechanisms of enzymeactivity, of the same protein isoform. Since malate is a potent inhibitor of PEPC activity, rootsappear to have invested in more PEPC protein compared to nodules. In contrast, nodules withlower PEPC protein, achieved greater enzyme activity than roots, possibly due to higherphosphorylation in order to reduce the malate effect. The subsequent metabolism of this PEPCderivedmalate, caused roots and nodules to synthesise asparagine via different pathways. Thesefindings imply that roots and nodules under P stress, synthesise their major export amino acid,asparagine, via different routes. This research has generated new knowledge regarding thephysiological impact of the organic and amino acid metabolism, derived from PEPC-C in the rootsand nodules of legumes growing in nutrient poor ecosystems. It has demonstrated for the first timethat the nodules of legume from a nutrient-poor ecosystem rely on improved resource allocation,Pi distribution, and PEPC-derived organic acids to maintain the efficient functioning of Nassimilation under P stress. This may be a consequence of having evolved in a nutrient-poorecosystem, so that nodule-bacteroid respiration and N metabolism can be maintained in P-poorsoils such as the Fynbos.

[发布机构] Stellenbosch University