Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase.

Valar M Anoop,Lee Mcalister-Henn,Gregory J Taylor,Mark T Mccammon,Urmila Basu

doi:10.1104/pp.103.023903

Valar M Anoop, Lee Mcalister-Henn + Show 3 more

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https://doi.org/10.1104/pp.103.023903

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Abstract

Aluminum (Al) toxicity is a major constraint for crop production in acid soils, although crop cultivars vary in their tolerance to Al. We have investigated the potential role of citrate in mediating Al tolerance in Al-sensitive yeast (Saccharomyces cerevisiae; MMYO11) and canola (Brassica napus cv Westar). Yeast disruption mutants defective in genes encoding tricarboxylic acid cycle enzymes, both upstream (citrate synthase [CS]) and downstream (aconitase [ACO] and isocitrate dehydrogenase [IDH]) of citrate, showed altered levels of Al tolerance. A triple mutant of CS (Deltacit123) showed lower levels of citrate accumulation and reduced Al tolerance, whereas Deltaaco1- and Deltaidh12-deficient mutants showed higher accumulation of citrate and increased levels of Al tolerance. Overexpression of a mitochondrial CS (CIT1) in MMYO11 resulted in a 2- to 3-fold increase in citrate levels, and the transformants showed enhanced Al tolerance. A gene for Arabidopsis mitochondrial CS was overexpressed in canola using an Agrobacterium tumefaciens-mediated system. Increased levels of CS gene expression and enhanced CS activity were observed in transgenic lines compared with the wild type. Root growth experiments revealed that transgenic lines have enhanced levels of Al tolerance. The transgenic lines showed enhanced levels of cellular shoot citrate and a 2-fold increase in citrate exudation when exposed to 150 micro M Al. Our work with yeast and transgenic canola clearly suggest that modulation of different enzymes involved in citrate synthesis and turnover (malate dehydrogenase, CS, ACO, and IDH) could be considered as potential targets of gene manipulation to understand the role of citrate metabolism in mediating Al tolerance.

Highlights

Aluminum (Al) toxicity is a major constraint for crop production in acid soils, crop cultivars vary in their tolerance to Al
Northern analysis of WT yeast exposed to Al (0–400 ␮m) showed that the transcript level of CIT1 increased by 40% to 50% from 100 to 300 ␮m Al, and no significant increase in transcript abundance was observed at 400 ␮m Al
Transcript abundance for ACO1 increased by 430% to 477% of control at 100 to 400 ␮m Al, whereas the IDH1 transcript level was downregulated to 60% of control by increasing concentrations of Al (Fig. 2, B and C)

Summary

Introduction

Aluminum (Al) toxicity is a major constraint for crop production in acid soils, crop cultivars vary in their tolerance to Al. Yeast disruption mutants defective in genes encoding tricarboxylic acid cycle enzymes, both upstream (citrate synthase [CS]) and downstream (aconitase [ACO] and isocitrate dehydrogenase [IDH]) of citrate, showed altered levels of Al tolerance. The transgenic lines showed enhanced levels of cellular shoot citrate and a 2-fold increase in citrate exudation when exposed to 150 ␮m Al. Our work with yeast and transgenic canola clearly suggest that modulation of different enzymes involved in citrate synthesis and turnover (malate dehydrogenase, CS, ACO, and IDH) could be considered as potential targets of gene manipulation to understand the role of citrate metabolism in mediating Al tolerance. CS is a key enzyme involved in condensation of oxaloacetate (OAA) and acetyl CoA to produce citrate This biochemical reaction plays an important role in the Krebs cycle, in ␤-oxidation of fatty acids, and in photo-respiratory glycolate pathways. It is important to undertake a holistic approach that considers enzymes and metabolites both upstream and downstream of citrate, while attempting to engineer citrate metabolism

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