Exploring the complexity of malate and citrate metabolism and their roles in aluminum tolerance in yeast and transgenic canola overexpressing a glyoxysomal malate synthase and a mitochondrial citrate synthase.

摘要

Aluminum (Al) toxicity is a major constraint for crop production in acid soils. The potential role of malate and citrate in mediating Al resistance, and various genes involved in malate and citrate metabolism were investigated in Al-sensitive Saccharomyces cerevisiae (MMYO11) and Brassica napus (cv. Westar). No correlation between malate levels and Al sensitivity was observed in yeast mutants defective in various genes involved in malate metabolism (malate synthase, MLS1; isocitrate lyase, ICL1; and malate dehydrogenases, MDH1, MDH2 and MDH12). In addition, transgenic canola lines overexpressing a glyoxysomal malate synthase (MS) gene showed enhanced MS transcript levels, but no increase in MS activity and malate content. This suggested that the complexity of malate metabolism and gene redundancy should be considered if gene manipulations are attempted in an effort to generate Al-resistant yeast or canola.; Yeast disruption mutants defective in genes encoding TCA cycle enzymes, both upstream (citrate synthase, CS) and downstream (aconitase, ACO and isocitrate dehydrogenase, IDH) of citrate, showed altered levels of Al resistance. A triple mutant of CS (Δcit123) showed lower levels of citrate accumulation and reduced Al resistance, while Δ aco1 and Δidh12 mutants showed higher accumulation of citrate and increased levels of A1 resistance. Overexpression of a mitochondria) CS (CIT1) in MMYO11 yeast resulted in a 2 to 3 fold increase in citrate levels, and the transformants showed enhanced Al resistance. A gene for Arabidopsis thaliana mitochondrial CS was overexpressed in canola using an Agrobacterium-mediated system. Increased levels of CS gene expression and enhanced CS activity were observed in transgenic lines compared to the wild type (WT). The transgenic lines showed enhanced levels of cellular shoot citrate and a two-fold increase in citrate exudation when exposed to 150 μM Al. Root growth experiments revealed that transgenic lines had enhanced levels of Al resistance. This research suggests that modulation of different enzymes involved in citrate synthesis and turnover ( MDH, CS, ACO, IDH) could be considered as potential targets-of-gene manipulation to understand the role of citrate metabolism in mediating Al resistance.