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1.  The role of VuMATE1 expression in aluminium-inducible citrate secretion in rice bean (Vigna umbellata) roots 
Journal of Experimental Botany  2013;64(7):1795-1804.
Aluminium (Al)-activated citrate secretion plays an important role in Al resistance in a number of plant species, such as rice bean (Vigna umbellata). This study further characterized the regulation of VuMATE1, an aluminium-activated citrate transporter. Al stress induced VuMATE1 expression, followed by the secretion of citrate. Citrate secretion was specific to Al stress, whereas VuMATE1 expression was not, which could be explained by a combined regulation of VuMATE1 expression and Al-specific activation of VuMATE1 protein. Pre-treatment with a protein translation inhibitor suppressed VuMATE1 expression, indicating that de novo biosynthesis of proteins is required for gene expression. Furthermore, post-treatment with a protein translation inhibitor inhibited citrate secretion, indicating that post-transcriptional regulation of VuMATE1 is critical for citrate secretion. Protein kinase and phosphatase inhibitor studies showed that reversible phosphorylation was important not only for transcriptional regulation of VuMATE1 expression but also for post-translational regulation of VuMATE1 protein activity. These results suggest that citrate secretion is dependent on both transcriptional and post-transcriptional regulation of VuMATE1. Additionally, VuMATE1 promoter–β-glucuronidase fusion lines revealed that VuMATE1 expression was restricted to the root apex and was entirely Al induced, indicating the presence of cis-acting elements regulating root tip-specific and Al-inducible gene expression, which will be an important resource for genetic improvement of plant Al resistance.
PMCID: PMC3638816  PMID: 23408830
aluminium toxicity; cis-acting element; promoter; reversible phosphorylation; signalling transduction; transcription factor.
2.  Role of IKK/NF-κB Signaling in Extinction of Conditioned Place Aversion Memory in Rats 
PLoS ONE  2012;7(6):e39696.
The inhibitor κB protein kinase/nuclear factor κB (IKK/NF-κB) signaling pathway is critical for synaptic plasticity. However, the role of IKK/NF-κB in drug withdrawal-associated conditioned place aversion (CPA) memory is unknown. Here, we showed that inhibition of IKK/NF-κB by sulphasalazine (SSZ; 10 mM, i.c.v.) selectively blocked the extinction but not acquisition or expression of morphine-induced CPA in rats. The blockade of CPA extinction induced by SSZ was abolished by sodium butyrate, an inhibitor of histone deacetylase. Thus, the IKK/NF-κB signaling pathway might play a critical role in the extinction of morphine-induced CPA in rats and might be a potential pharmacotherapy target for opiate addiction.
PMCID: PMC3383688  PMID: 22761874
3.  TcOPT3, a Member of Oligopeptide Transporters from the Hyperaccumulator Thlaspi caerulescens, Is a Novel Fe/Zn/Cd/Cu Transporter 
PLoS ONE  2012;7(6):e38535.
Thlaspi caerulescens is a natural selected heavy metal hyperaccumulator that can not only tolerate but also accumulate extremely high levels of heavy metals in the shoots. Thus, to identify the transportors involved in metal long-distance transportation is very important for understanding the mechanism of heavy metal accumulation in this hyperaccumulator.
Methodology/Principal Findings
We cloned and characterized a novel gene TcOPT3 of OPT family from T. caerulescens. TcOPT3 was pronouncedly expressed in aerial parts, including stem and leaf. Moreover, in situ hybridization analyses showed that TcOPT3 expressed in the plant vascular systems, especially in the pericycle cells that may be involved in the long-distance transportation. The expression of TcOPT3 was highly induced by iron (Fe) and zinc (Zn) deficiency, especially in the stem and leaf. Sub-cellular localization showed that TcOPT3 was a plasma membrane-localized protein. Furthermore, heterogonous expression of TcOPT3 by mutant yeast (Saccharomyces cerevisiae) complementation experiments demonstrated that TcOPT3 could transport Fe2+ and Zn2+. Moreover, expression of TcOPT3 in yeast increased metal (Fe, Zn, Cu and Cd) accumulation and resulted in an increased sensitivity to cadmium (Cd) and copper (Cu).
Our data demonstrated that TcOPT3 might encode an Fe/Zn/Cd/Cu influx transporter with broad-substrate. This is the first report showing that TcOPT3 may be involved in metal long-distance transportation and contribute to the heavy metal hyperaccumulation.
PMCID: PMC3382247  PMID: 22761683
4.  Genotypic differences in Al resistance and the role of cell-wall pectin in Al exclusion from the root apex in Fagopyrum tataricum 
Annals of Botany  2010;107(3):371-378.
Background and Aims
Aluminium (Al) toxicity is one of the factors limiting crop production on acid soils. However, genotypic differences exist among plant species or cultivars in response to Al toxicity. This study aims to investigate genotypic differences among eight cultivars of tatary buckwheat (Fagopyrum tataricum) for Al resistance and explore the possible mechanisms of Al resistance.
Al resistance was evaluated based on relative root elongation (root elongation with Al/root elongation without Al). Root apex Al content, pectin content and exudation of root organic acids were determined and compared.
Key Results
Genotypic differences among the eight cultivars were correlated with exclusion of Al from the root apex. However, there was a lack of correlation between Al exclusion and Al-induced oxalate secretion. Interestingly, cell-wall pectin content of the root apex was generally lower in Al-resistant cultivars than in Al-sensitive cultivars. Although we were unable to establish a significant correlation between Al exclusion and pectin content among the eight cultivars, a strong correlation could be established among six cultivars, in which the pectin content in the most Al-resistant cultivar ‘Chuan’ was significantly lower than that in the most Al-sensitive cultivar ‘Liuku2’. Furthermore, root apex cell-wall pectin methylesterase activity (PME) was similar in ‘Chuan’ and ‘Liuku2’ in the absence of Al, but Al treatment resulted in increased PME activity in ‘Liuku2’ compared with ‘Chuan’. Immunolocalization of pectins also showed that the two cultivars had similar amounts of either low-methyl-ester pectins or high-methyl-ester pectins in the absence of Al, but Al treatment resulted in a more significant increase of low-methyl-ester pectins and decrease of high-methyl-ester pectins in ‘Liuku2’.
Cell-wall pectin content may contribute, at least in part, to differential Al resistance among tatary buckwheat cultivars.
PMCID: PMC3043930  PMID: 21183454
Aluminium resistance; cell wall; exclusion mechanism; Fagopyrum tataricum; pectin; pectin methylesterase; oxalate; toxicity
5.  Disorganized distribution of homogalacturonan epitopes in cell walls as one possible mechanism for aluminium-induced root growth inhibition in maize 
Annals of Botany  2009;104(2):235-241.
Background and Aims
Aluminium (Al) toxicity is one of the most severe limitations to crop production in acid soils. Inhibition of root elongation is the primary symptom of Al toxicity. However, the underlying basis of the process is unclear. Considering the multiple physiological and biochemical functions of pectin in plants, possible involvement of homogalacturonan (HG), one of the pectic polysaccharide domains, was examined in connection with root growth inhibition induced by Al.
An immunolabelling technique with antibodies specific to HG epitopes (JIM5, unesterified residues flanked by methylesterifed residues; JIM7, methyl-esterified residues flanked by unesterified residues) was used to visualize the distribution of different types of HG in cell walls of root apices of two maize cultivars differing in Al resistance.
Key Results
In the absence of Al, the JIM5 epitope was present around the cell wall with higher fluorescence intensity at cell corners lining the intercellular spaces, and the JIM7 epitope was present throughout the cell wall. However, treatment with 50 µm Al for 3 h produced 10 % root growth inhibition in both cultivars and caused the disappearance of fluorescence in the middle lamella of both epitopes. Prolonged Al treatment (24 h) with 50 % root growth inhibition in ‘B73’, an Al-sensitive cultivar, resulted in faint and irregular distribution of both epitopes. In ‘Nongda3138’, an Al-resistant cultivar, the distribution of HG epitopes was also restricted to the lining of intercellular spaces when a 50 % inhibition to root growth was induced by Al (100 µm Al, 9 h). Altered distribution of both epitopes was also observed when of roots were exposed to 50 µm LaCl3 for 24 h, resulting in 40 % inhibition of root growth.
Changes in HG distribution and root growth inhibition were highly correlated, indicating that Al-induced perturbed distribution of HG epitopes is possibly involved in Al-induced inhibition of root growth in maize.
PMCID: PMC2710910  PMID: 19483201
Al toxicity; cell wall; homogalacturnonan; immunofluorescence; methylesterification; pectin
6.  Citrate Transporters Play a Critical Role in Aluminium-stimulated Citrate Efflux in Rice Bean (Vigna umbellata) Roots 
Annals of Botany  2006;97(4):579-584.
• Background and Aims Aluminium (Al) stimulates the efflux of citrate from apices of rice bean (Vigna umbellata) roots. This response is delayed at least 3 h when roots are exposed to 50 µm Al, indicating that some inducible processes leading to citrate efflux are involved. The physiological bases responsible for the delayed response were examined here.
• Methods The effects of several antagonists of anion channels and citrate carriers, and of the protein synthesis inhibitor, cycloheximide (CHM) on Al-stimulated citrate efflux and/or citrate content were examined by high-pressure liquid chromatography (HPLC) or an enzymatic method.
• Key Results Both anion channel inhibitors and citrate carrier inhibitors can inhibit Al-stimulated citrate efflux, with anthracene-9-carboxylic acid (A-9-C, an anion channel inhibitor) and phenylisothiocyanate (PI, a citrate carrier inhibitor) the most effective inhibitors. A 6 h pulse of 50 µm Al induced a significant increase of citrate content in root apices and release of citrate. However, the increase in citrate content preceded the efflux. Furthermore, the release of citrate stimulated by the pulse treatment was inhibited by both A-9-C and PI, indicating the importance of the citrate carrier on the mitochondrial membrane and the anion channel on the plasma membrane for the Al-stimulated citrate efflux. CHM (20 µm) also significantly inhibited Al-stimulated citrate efflux, confirming that de novo protein synthesis is required for Al-stimulated citrate efflux.
• Conclusions These results indicate that the activation of genes possibly encoding citrate transporters plays a critical role in Al-stimulated citrate efflux.
PMCID: PMC2803670  PMID: 16446286
Aluminium resistance; anion channel; citrate carrier; inhibitor; organic acid anions; protein synthesis; rice bean; toxicity; transporter; Vigna umbellata

Results 1-6 (6)