ABSTRACT
In acidic soils, aluminum (Al) toxicity is a significant limitation to crop production worldwide. Given its Al-binding capacity, malate allows internal as well as external detoxification strategies to cope with Al stress, but little is known about the metabolic processes involved in this response. Here, we analyzed the relevance of NADP-dependent malic enzyme (NADP-ME), which catalyzes the oxidative decarboxylation of malate, in Al tolerance. Plants lacking NADP-ME1 (nadp-me1) display reduced inhibition of root elongation along Al treatment compared with the wild type (wt). Moreover, wt roots exposed to Al show a drastic decrease in NADP-ME1 transcript levels. Although malate levels in seedlings and root exudates are similar in nadp-me1 and wt, a significant increase in intracellular malate is observed in roots of nadp-me1 after long exposure to Al. The nadp-me1 plants also show a lower H2 O2 content in root apices treated with Al and no inhibition of root elongation when exposed to glutamate, an amino acid implicated in Al signaling. Proteomic studies showed several differentially expressed proteins involved in signal transduction, primary metabolism and protection against biotic and other abiotic stimuli and redox processes in nadp-me1, which may participate directly or indirectly in Al tolerance. The results indicate that NADP-ME1 is involved in adjusting the malate levels in the root apex, and its loss results in an increased content of this organic acid. Furthermore, the results suggest that NADP-ME1 affects signaling processes, such as the generation of reactive oxygen species and those that involve glutamate, which could lead to inhibition of root growth.
Subject(s)
Aluminum/toxicity , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Malate Dehydrogenase (NADP+)/metabolism , Malates/metabolism , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Loss of Function Mutation , Malate Dehydrogenase (NADP+)/genetics , Plant Roots/enzymology , Plant Roots/genetics , Plant Roots/physiology , Proteomics , Stress, PhysiologicalABSTRACT
NAD(P)-malic enzyme (NAD(P)-ME) catalyzes the reversible oxidative decarboxylation of malate to pyruvate, CO2 , and NAD(P)H and is present as a multigene family in Arabidopsis thaliana. The carboxylation reaction catalyzed by purified recombinant Arabidopsis NADP-ME proteins is faster than those reported for other animal or plant isoforms. In contrast, no carboxylation activity could be detected in vitro for the NAD-dependent counterparts. In order to further investigate their putative carboxylating role in vivo, Arabidopsis NAD(P)-ME isoforms, as well as the NADP-ME2del2 (with a decreased ability to carboxylate pyruvate) and NADP-ME2R115A (lacking fumarate activation) versions, were functionally expressed in the cytosol of pyruvate carboxylase-negative (Pyc- ) Saccharomyces cerevisiae strains. The heterologous expression of NADP-ME1, NADP-ME2 (and its mutant proteins), and NADP-ME3 restored the growth of Pyc- S. cerevisiae on glucose, and this capacity was dependent on the availability of CO2 . On the other hand, NADP-ME4, NAD-ME1, and NAD-ME2 could not rescue the Pyc- strains from C4 auxotrophy. NADP-ME carboxylation activity could be measured in leaf crude extracts of knockout and overexpressing Arabidopsis lines with modified levels of NADP-ME, where this activity was correlated with the amount of NADP-ME2 transcript. These results indicate that specific A. thaliana NADP-ME isoforms are able to play an anaplerotic role in vivo and provide a basis for the study on the carboxylating activity of NADP-ME, which may contribute to the synthesis of C4 compounds and redox shuttling in plant cells.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/enzymology , Malate Dehydrogenase (NADP+)/genetics , Malates/metabolism , NADP/metabolism , NAD/metabolism , Pyruvic Acid/metabolism , Saccharomyces cerevisiae/enzymology , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Carbon Dioxide/metabolism , Cloning, Molecular , Gene Expression , Genetic Complementation Test , Genetic Engineering , Glucose/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Malate Dehydrogenase (NADP+)/metabolism , Plant Leaves/enzymology , Plant Leaves/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Transformation, Genetic , TransgenesABSTRACT
Malic enzymes (ME) catalyze the decarboxylation of malate generating pyruvate, CO2 and NADH or NADPH. In some organisms it has been established that ME is involved in lipids biosynthesis supplying carbon skeletons and reducing power. In this work we studied the MEs of soybean and castor, metabolically different oilseeds. The comparison of enzymatic activities, transcript profiles and organic acid contents suggest different metabolic strategies operating in soybean embryo and castor endosperm in order to generate precursors for lipid biosynthesis. In castor, the malate accumulation pattern agrees with a central role of this metabolite in the provision of carbon to plastids, where the biosynthesis of fatty acids occurs. In this regard, the genome of castor possesses a single gene encoding a putative plastidic NADP-ME, whose expression level is high when lipid deposition is active. On the other hand, NAD-ME showed an important contribution to the maturation of soybean embryos, perhaps driving the carbon relocation from mitochondria to plastids to support the fatty acids synthesis in the last stages of seed filling. These findings provide new insights into intermediary metabolism in oilseeds and provide new biotechnological targets to improve oil yields.
