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      Enhancing the aluminium tolerance of barley by expressing the citrate transporter genes SbMATE and FRD3

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          Summary

          Al 3+ tolerance of barley was enhanced in transgenic plants by expression of the SbMATE gene from sorghum and the FRD3 gene from Arabidopsis, which increased citrate efflux in roots.

          Abstract

          Malate and citrate efflux from root apices is a mechanism of Al 3+ tolerance in many plant species. Citrate efflux is facilitated by members of the MATE (multidrug and toxic compound exudation) family localized to the plasma membrane of root cells. Barley ( Hordeum vulgare) is among the most Al 3+-sensitive cereal species but the small genotypic variation in tolerance that is present is correlated with citrate efflux via a MATE transporter named HvAACT1. This study used a biotechnological approach to increase the Al 3+ tolerance of barley by transforming it with two MATE genes that encode citrate transporters: SbMATE is the major Al 3+-tolerance gene from sorghum whereas FRD3 is involved with Fe nutrition in Arabidopsis. Independent transgenic and null T3 lines were generated for both transgenes. Lines expressing SbMATE showed Al 3+-activated citrate efflux from root apices and greater tolerance to Al 3+ toxicity than nulls in hydroponic and short-term soil trials. Transgenic lines expressing FRD3 exhibited similar phenotypes except citrate release from roots occurred constitutively. The Al 3+ tolerance of these lines was compared with previously generated transgenic barley lines overexpressing the endogenous HvAACT1 gene and the TaALMT1 gene from wheat. Barley lines expressing TaALMT1 showed significantly greater Al 3+ tolerance than all lines expressing MATE genes. This study highlights the relative efficacy of different organic anion transport proteins for increasing the Al 3+ tolerance of an important crop species.

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          A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum.

          Crop yields are significantly reduced by aluminum toxicity on highly acidic soils, which comprise up to 50% of the world's arable land. Candidate aluminum tolerance proteins include organic acid efflux transporters, with the organic acids forming non-toxic complexes with rhizosphere aluminum. In this study, we used positional cloning to identify the gene encoding a member of the multidrug and toxic compound extrusion (MATE) family, an aluminum-activated citrate transporter, as responsible for the major sorghum (Sorghum bicolor) aluminum tolerance locus, Alt(SB). Polymorphisms in regulatory regions of Alt(SB) are likely to contribute to large allelic effects, acting to increase Alt(SB) expression in the root apex of tolerant genotypes. Furthermore, aluminum-inducible Alt(SB) expression is associated with induction of aluminum tolerance via enhanced root citrate exudation. These findings will allow us to identify superior Alt(SB) haplotypes that can be incorporated via molecular breeding and biotechnology into acid soil breeding programs, thus helping to increase crop yields in developing countries where acidic soils predominate.
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            The FRD3-mediated efflux of citrate into the root vasculature is necessary for efficient iron translocation.

            Iron, despite being an essential micronutrient, becomes toxic if present at high levels. As a result, plants possess carefully regulated mechanisms to acquire iron from the soil. The ferric reductase defective3 (frd3) mutant of Arabidopsis (Arabidopsis thaliana) is chlorotic and exhibits constitutive expression of its iron uptake responses. Consequently, frd3 mutants overaccumulate iron; yet, paradoxically, the frd3 phenotypes are due to a reduction in the amount of iron present inside frd3 leaf cells. The FRD3 protein belongs to the multidrug and toxin efflux family, members of which are known to export low-M(r) organic molecules. We therefore hypothesized that FRD3 loads an iron chelator necessary for the correct distribution of iron throughout the plant into the xylem. One such potential chelator is citrate. Xylem exudate from frd3 plants contains significantly less citrate and iron than the exudate from wild-type plants. Additionally, supplementation of growth media with citrate rescues the frd3 phenotypes. The ectopic expression of FRD3-GFP results in enhanced tolerance to aluminum in Arabidopsis roots, a hallmark of organic acid exudation. Consistent with this result, approximately 3 times more citrate was detected in root exudate from plants ectopically expressing FRD3-GFP. Finally, heterologous studies in Xenopus laevis oocytes reveal that FRD3 mediates the transport of citrate. These results all strongly support the hypothesis that FRD3 effluxes citrate into the root vasculature, a process important for the translocation of iron to the leaves, as well as confirm previous reports suggesting that iron moves through the xylem as a ferric-citrate complex. Our results provide additional answers to long-standing questions about iron chelation in the vasculature and organic acid transport.
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              Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance.

              Aluminum-activated root malate and citrate exudation play an important role in plant Al tolerance. This paper characterizes AtMATE, a homolog of the recently discovered sorghum and barley Al-tolerance genes, shown here to encode an Al-activated citrate transporter in Arabidopsis. Together with the previously characterized Al-activated malate transporter, AtALMT1, this discovery allowed us to examine the relationship in the same species between members of the two gene families for which Al-tolerance genes have been identified. AtMATE is expressed primarily in roots and is induced by Al. An AtMATE T-DNA knockdown line exhibited very low AtMATE expression and Al-activated root citrate exudation was abolished. The AtALMT1 AtMATE double mutant lacked both Al-activated root malate and citrate exudation and showed greater Al sensitivity than the AtALMT1 mutant. Therefore, although AtALMT1 is a major contributor to Arabidopsis Al tolerance, AtMATE also makes a significant but smaller contribution. The expression patterns of AtALMT1 and AtMATE and the profiles of Al-activated root citrate and malate exudation are not affected by the presence or absence of the other gene. These results suggest that AtALMT1-mediated malate exudation and AtMATE-mediated citrate exudation evolved independently to confer Al tolerance in Arabidopsis. However, a link between regulation of expression of the two transporters in response to Al was identified through work on STOP1, a transcription factor that was previously shown to be necessary for AtALMT1 expression. Here we show that STOP1 is also required for AtMATE expression and Al-activated citrate exudation.
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                Author and article information

                Journal
                J Exp Bot
                J. Exp. Bot
                jexbot
                exbotj
                Journal of Experimental Botany
                Oxford University Press (UK )
                0022-0957
                1460-2431
                June 2014
                1 April 2014
                1 April 2014
                : 65
                : 9
                : 2381-2390
                Affiliations
                1Tasmanian Institute of Agriculture, University of Tasmania , PO Box 46, Kings Meadows, TAS 7249, Australia
                2CSIRO Plant Industry , GPO Box 1600, Canberra, ACT 2601, Australia
                3Embrapa Wheat , Rodovia BR 285 km 294, CEP 99001-970, Passo Fundo, RS, Brazil
                4Embrapa Maize and Sorghum , Rod. MG 424, Km 65, 35701-970, Sete Lagoas, Minas Gerais, Brazil
                Author notes
                * Current address: Department of Agriculture & Food WA, 3 Baron-Hay Court, South Perth, WA 6155, Australia.
                To whom correspondence should be addressed. E-mail: Peter.Ryan@ 123456csiro.au
                Article
                10.1093/jxb/eru121
                4036506
                24692647
                065f9260-8029-44e3-8eff-a35602963889
                © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Page count
                Pages: 10
                Categories
                Research Paper

                Plant science & Botany
                acid soil,cereal,citrate,hordeum vulgare,mate transporters,resistance,root exudates,transgenic.

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