Role Of Citrate Transporter And Pectin Methylesterase In Plant Aluminum Resistance

Aluminum (Al) toxicity is one of the most important deleterious factors limiting crop production on acid soils. Al resistance mechanisms in plant have been classified into external exclusion and internal tolerance. Among them, secretion of organic acid anions is an important external exclusion mechanism. The transporter on the plasma membrane mediates the secretion of organic acid anion, which is the crucial step of the secretion. However, only a few organic acid anion transporters have been cloned by now, and transporter induced completely by Al stress hasn't been isolated although the secretion of organic acid anion has the obvious Al-induced characteristics. Our previous study showed that citrate secretion could be induced with a lag phase after the initiation of Al stress in rice bean. Therefore, the aim of this study was to clone the citrate transporter responsible for citrate efflux from rice bean. The function of the transporter was also investigated. On the other hand, the secretion of organic acid anion cannot explain the Al resistance mechanism in some plants. Thus, another focus of our research was to investigate the relationship between cell wall and Al resistance in rice as it had been demonstrated that Al-induced organic acid anions secretion was not involved in high Al resistance in rice. We tried to associate some specific pectin methylesterase genes with Al-induced root elongation inhibition in rice. The results were summarized as following:1. A de novo synthesis citrate transporter VuMATE confers aluminum resistance in rice bean(Vigna umbellata)Al specifically induces citrate efflux in rice bean, and the efflux is delayed for several hours. Here we conducted homologous cloning and RACE technology to isolate a gene encoding a de novo synthesis (newly synthesized) citrate transporter from the root tip of rice bean, which belongs to MATE gene family and was named as VuMATE(Vigna umbellata multidrug and toxic compound extrusion). The deduced protein of VuMATE comprised565amino acids.VuMATE showed high protein sequence homolog to the known citrate transporters. They all are twelve-span transmembrane proteins and share a common highly conserved amino acid sequences in intracellular loop between the2nd and3rd transmembrane domain. The intracellular loop is cytoplasmic and unique to the known citrate transporters of MATE family, so it is possible that the conserved sequences in the loop play an important role in citrate transport. Phylogenetic relationship analysis showed that VuMATE clustered most nearly with LaMATE from white lupin.In the absence of Al, the VuMATE mRNA transcription was not detected neither in root tip nor leaf tissue. Al treatment induced the expression only in the root segment0-1cm from the root tip, while still no expression could be detected in the1-2cm region and nor in leaf tissue. Al-induction of VuMATE expression was both Al-concentration and time dependent, increasing at higher Al levels and durations of Al exposure, which was consistent with citrate secretion pattern. To determine the Al-specificity of VuMATE induction of expression, the effect of the other metals was tested. While cadmium (Cd) and copper (Cu) alone didn't induce VuMATE expression, lanthanum (La) induced VuMATE expression in an additive manner, in that VuMATE expression in response to La+Al was higher than in response to La alone.The transient expression assays with a VuMATE::GFP translational fusion in onion epidermal cell showed that VuMATE localized at plasma membrane.Electrophysiological analysis of Xenopus oocytes expressing VuMATE indicated this transporter could mediate significant anion efflux across the plasma membrane. VuMATE-mediated inward currents were pH dependent (and possibily Na+dependent). After injecting14C-labeled citrate, VuMATE-expressing cells showed enhanced14C efflux, indicating that VuMATE could mediate citrate efflux directly. Moreover,14C efflux from cells expressing VuMATE was also pH dependent.Overexpression of VuMATE in transgenic tomato conferred citrate efflux in root and consequently Al resistance increase.All these indicated that VuMATE was the citrate transporter responsible for citrate efflux in the root apex of rice bean. By comparing the citrate transporter in rice bean with the other known citrate transporters, we summarized some specific characteristics possessed by the citrate transporter in rice bean. 2. Association of specific pectin methylesterase genes with Al-induced root elongation inhibition in riceRice (Oryza sativa L.) is the most Al resistance species among small-grain cereal crops. The secretion of organic acid anion cannot explain its Al resistance. It has been reported that the negative charges of cell wall pectin molecules determined by pectin methylesterase (PME, EC3.1.1.11) contribute to Al binding. Therefore, this study focused on the relationship between PME gene family with Al-induced root elongation inhibition in rice.Thirty-five putative Oryza sativa L. PME gene sequences were retrieved from DFCI (http://compbio.dfci.harvard.edu/tgi/plant.html. The GC%of PME genes in rice is very high. Each member has different gene structure. Their deduced proteins were classified as group1(only having a PME domain) or group2[having a PMEI (PME Inhibitor) domain preceding the PME domain]. The phylogenetic tree analysis showed that there were five distinct clades (Clade I to V) in rice.After treatment Al-sensitive rice sp. indica'Zhefu802'with25μM Al for3h, root elongation was inhibited by40%, and CW PME activity was increased by20%. Meanwhile, the expression pattern of PME could be divided into4classes. Nine of PME genes had no expression signals (Class A), eight genes were up-regulated by Al (Class B), two genes were down-regulated (Class C), and the remainder exhibited constitutive expression pattern which was not affected by Al treatment (Class D). As the result of the consistency between8up-regulated genes with PME activity increase, it could be speculated that the eight up-regulated PMEs might be associated with Al-induced root elongation inhibition in rice.We further investigated the eight genes'expression pattern in an Al-resistant rice cultivar sp. japonica'Nipponbare'. While root elongation of rice'Nipponbare'was not significantly affected by25μM Al treatment for3h, the expression of these eight genes was not affected. However, when Al concentration was increased to50μM, by which the root elongation was also inhibited by40%, the expression of the eight PME genes was up-regulated except two no signal genes. These results suggested that the eight PME genes were likely related to the Al-induced inhibition of root elongation both in Al-sensitive and Al-resistant rice cultivars.The eight PME genes behaved differently under CdCl2and LaCl3treatment, implying the specificity of different PME genes in response to different metal toxicities. Altogether, we chose the eight members from PME gene family to be associated with Al-induced inhibition of root elongation in rice. Our results provided a breakthrough point to study the effect of Al stress on cell wall.