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

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 (⌬cit123) showed lower levels of citrate accumulation and reduced Al tolerance, whereas ⌬aco1-and ⌬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 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.Aluminum toxicity is one of the major factors limiting crop productivity in acid soils. The root apex is considered the primary site of Al-induced injury, and inhibition of root elongation is one of the most visible symptoms of Al toxicity. Although most plants are sensitive to Al, several crop species exhibit genetic variation in their ability to tolerate Al. One possible mechanism of Al tolerance is the chelation of Al by organic anions within root cells or in the rhizosphere (Taylor, 1991). Exudation of a variety of low M r organic anions such as citrate, malate, or oxalate has been reported in several crop species upon exposure to Al (Miyasaka et al., 1991; Basu et al., 1994;Pellet et al., 1995;Larsen et al., 1998). Although a rapid release of organic anions (malate 2Ϫ ) was observed in near isogenic, Al-tolerant wheat (Triticum aestivum) lines (Delhaize et al., 1993), several lines of evidence suggest that a lag phase may also exist between exposure of roots to Al and excretion of organic anions. Citrate exudation from roots of rye was observed only after 120 h of exposure (Li et al., 2000). Similarly, an Alinduced, de novo synthesis of malate leading to enhanced malate efflux 24 h after exposure was demonstrated in Al-tolerant wheat cv Katepwa (Basu et al., 1994). Delayed exudation of organic anions could possibly ...

Engineering high-level aluminum tolerance in barley with the ALMT1 gene

“…Al toxicity is manifest by inhibition of root growth resulting in poor uptake of water and nutrients (2). Plant production on acid soils can be maintained by neutralizing the acidity with lime (CaCO 3 ) and through the use of Al-tolerant plant species. Lime can take decades to correct acidity at depth, and many important crop and pasture species lack sufficient Al tolerance within their germplasm to allow effective breeding for this character.…”

“…Genetic engineering provides an opportunity to enhance the Al tolerance of sensitive species through the overexpression of endogenous genes or by the expression of foreign genes. Toward this end, the Al tolerance of canola (Brassica napus) (3), Arabidopsis thaliana (4), tobacco (Nicotiana tabacum) (5), and alfalfa (Medicago sativum) (6) have been reported to be enhanced by increasing organic acid biosynthesis through overexpression of citrate synthase or malate dehydrogenase genes derived from plants or bacteria. Other strategies have sought to increase Al tolerance by overexpression of genes associated with stress responses (7)(8)(9).…”

“…Soil experiments used an acid subsoil (10-to 40-cm layer, CaCl 2 extracted pH of 3.9, water-extracted pH of 5.2, and 40 g͞g of CaCl 2 -extractable Al) derived from Chiltern in Australia (24) and a nonallophanic andosol obtained from the Field Science Center at Tohoku University (Sendai, Japan). In the first experiment, pregerminated seeds, where the longest root was 8-35 mm, were planted in 300 g of either unamended Chiltern soil or Chiltern soil mixed with 0.75 g of CaCO 3 per kg of soil (water-extracted pH of 6.0). Each pot contained a single plant with each combination of genotype and treatment consisting of eight replicates.…”

A Formate Dehydrogenase Confers Tolerance to Aluminum and Low pH

“…For example, genes coding for citrate synthase have been introduced into tobacco (Nicotiana tabacum; de la Fuente et al, 1997), Arabidopsis (Koyama et al, 2000), canola (Brassica napus; Anoop et al, 2003), and alfalfa (Medicago sativa; Barone et al, 2008), and genes coding for malate dehydrogenase have been introduced into tobacco and alfalfa (Tesfaye et al, 2001). Similarly, genes related to protection from oxidative stress including manganese superoxide dismutase, dehydroascorbate reductase, peroxidase, and glutathione S-transferase have also been introduced into plants (Ezaki et al, 2000;Basu et al, 2001;Yin et al, 2010).…”

Molecular Aspect of Al Tolerance in Crop Plants: Novel Al-Activated Malate Transporter Gene in Wheat Roots

“…The increase of antioxidants like ascorbate, glutathione as well as their synthetic enzymes, ROS scavenging enzyme like MnSOD, peroxidase and glutathione reductase may be useful candidates for the repression of oxidative stress caused by Al. Expression of the genes encoding enzyme proteins mentioned above have been reported to confer resistance to A1 in some plants (Koyama et al 2000;Basu et al 2001;Tesfaye et al 2001;Anoop et al 2003). Some of the proposed mechanisms mentioned above will be recognized as a real internal tolerance mechanism, but comparative study showing this fact should be done with plant species of a similar genetic background.…”