Characterization of Citrate Transport through the Plasma Membrane in a Carrot Mutant Cell Line with Enhanced Citrate Excretion

Abstract

;The superior ability of citrate excretion in a carrot (Daucus carota L.) mutant cell line, namely IPG (insoluble phosphate grower) [Takita et al. (1999a) Plant Cell Physiol. 40: 489] cells has been characterized in terms of citrate transport at the plasma membrane. IPG cells released about a 20-fold increase in citrate in comparison with malate, while the concentration of malate was only 35% lower than that of citrate in the cell sap. Citrate excretion was sensitive to anion channel blockers, such as niflumic acid and anthracene-9-carboxylic acid. These results indicate that IPG cells release citrate through the plasma membrane using citrate specific anion channels. The rate of citrate release from IPG cells was not affected by the concentration of aluminum (0 and 50 mM), soluble P i (0 or 2 mM) and the pH (4.5-5.6) of the medium, suggesting that anion channels would not be regulated by such external conditions. Citrate excretion correlated with the H + efflux, possibly from the action of H + -ATPase on the plasma membrane. The activity of plasma membrane H + -ATPase was about three times higher in IPG cells than in wild-type cells, and might be involved in the high citrate excretion ability.Keywords: Aluminum phosphate -Anion channel -Citrate excretion -Daucus carota -H + -ATPase.Abbreviations: A-9-C, anthracene-9-carboxylic acid; DIDS, 4,4¢-diisothiocyanostilbene-2,2¢-disulfonic acid; EA, ethacrynic acid; IPG, insoluble phosphate grower; NA, niflumic acid; WT, wild-type. IntroductionOrganic acid excretion from roots, which plays various roles in efficient nutrient uptake (e.g. phosphate, Schactman et al. 1998; iron, Hirsch and Sussman 1999) and the alleviation of metal toxicities (e.g. aluminum, Miyasaka et al. 1991, Ma 2000, is thought to be one of the most important plant traits for adaptation to problem soils. For example, certain plant species showing an aluminum tolerance or a high P i -acquisition ability, such as Cassia tora L. (Ma et al. 1997) and lupin (Gardner et al. 1981), release large amounts of organic acids from their roots. Genetic manipulation may be one approach to introduce such traits into crop plants. In fact, transgenic tobacco (de la Fuente et al. 1997) and Arabidopsis (Koyama et al. 2000), with overexpression of the citrate synthase gene from bacteria and plants, increased citrate excretion from the roots. On the other hand, overexpression of a malate dehydrogenase in alfalfa (Tesfaye et al. 2001) causes enhanced citrate excretion from the roots, and in turn improves growth in either the presence of Al in the growing medium or with an Al-phosphate as the sole phosphate source. These results indicate that the modification of organic acid metabolisms is a target for improving organic acid excretion from the roots of crop plants. Additionally, several studies have suggested that organic acid transport through plasma membranes is another important factor for attaining high organic acid excretion abilities from roots (Ryan et al. 1995).There are two distinct patterns of organic acid excret...

Quantitative trait loci controlling aluminium tolerance in two accessions of Arabidopsis thaliana (Landsberg erecta and Cape Verde Islands)

“…Plant culture and malate excretion were carried out under aseptic conditions. Malate concentrations in the medium were measured by an enzymatic cycling method according to Hampp, Goller & Füllgraf (1984) with minor modifications as described previously (Ohno, Koyama & Hara 2003).…”

Fusicoccin‐induced catalase inhibitor is produced independently of H+‐ATPase activation and behaves as an organic acid

“…At this point some speculation was made as to the possible weak acid identity but, as well as for SA and GABA, none of the other main organic acids known to be secreted to the apoplast under stress conditions seemed to fit with the inhibitor's characteristics. In fact, the finding that in all experimental conditions so far tested no link between activity state of the PM H + ‐pump and apoplastic inhibitor concentration was detected suggested that the inhibitor could not apparently match with either AA or citrate, whose excretion was proved to be closely linked to the PM H + ‐ATPase activity (Ohno et al 2003, Ryan et al 2001, Takahama 1996, Tomasi et al 2009). Also, the possibility that the FC‐induced increase in apoplastic H 2 O 2 content might result from an inhibiting effect of malate on catalase activity seemed quite unlikely, given that its production too is known to be strictly correlated to the activity state of the PM H + ‐ATPase (Beffagna and Romani 1994, Marrè et al 1987).…”

“…An important role for citrate excretion into the apoplast in response to environmental stresses such as P i deficiency and exposure to high Al 3+ levels was also established. Furthermore, as well as for AA, the activity state of the PM H + ‐ATPase was proved to be correlated with the release of this organic acid from cells, an increased activity of this enzyme being accompanied by an enhanced citrate excretion (Ohno et al 2003, Ryan et al 2001, Tomasi et al 2009). And else, an increase in salicylic acid (SA) synthesis was proved to be induced by FC (Schaller et al 2000, Singh and Roberts 2004) and this molecule, proposed as the putative endogenous signal for systemic‐acquired resistance (Malamy et al 1990, Mètraux et al 1990), was suggested to act by binding to and inhibiting a catalase (Chen et al 1993, Dempsey and Klessig 1994).…”

Effect of ultrasonic exposure on Ca2+-ATPase activity in plasma membrane from Aloe arborescens callus cells

“…PM fractions were isolated and purified by the procedures described by Ohno et al with some modification [6]. Briefly, aloe callus were chopped with a scalpel and homogenized in cold (4°C) grinding buffer (pH 7.8) (3 ml/g fresh weight) containing 25 mM potassium phosphate buffer (pH 7.8), 0.25 M mannitol, 1.5% (w/v) polyvinylpyrrolidone, 0.1% (w/v) bovine serum albumin (BSA, fatty acid free, Sigma), 1 mM dithiothreitol (DTT), 2 mM EGTA and 2 mM EDTA-2Na, using a mortar and pestle.…”