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Plant Physiology 132:2205-2217 (2003)
© 2003 American Society of Plant Biologists

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ENVIRONMENTAL STRESS AND ADAPTATION

Modulation of Citrate Metabolism Alters Aluminum Tolerance in Yeast and Transgenic Canola Overexpressing a Mitochondrial Citrate Synthase1

Valar M. Anoop*, Urmila Basu, Mark T. McCammon, Lee McAlister-Henn and Gregory J. Taylor

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 (V.M.A., U.B., G.J.T.); and Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229–3900 (M.T.M., L.M.-H.)

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 ({Delta}cit123) showed lower levels of citrate accumulation and reduced Al tolerance, whereas {Delta}aco1- and {Delta}idh12-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 µM 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.

1 This work was supported by the Natural Sciences and Engineering Research Council of Canada, by the Department of Biological Sciences, University of Alberta (Edmonton, Alberta, Canada), and by the National Institutes of Health (grant no. AG17477 to L.M.-H.).

* Corresponding author; e-mail mv{at}ualberta.ca; fax 780–492–9234.

Received March 19, 2003; returned for revision April 21, 2003; accepted May 12, 2003.




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