A tonoplast-localized magnesium transporter is crucial for stomatal opening in Arabidopsis under high Mg2+ conditions.

Plant stomata play an important role in CO2 uptake for photosynthesis and transpiration, but the mechanisms underlying stomatal opening and closing under changing environmental conditions are still not completely understood. Through large-scale genetic screening, we isolated an Arabidopsis mutant (closed stomata2 (cst2)) that is defective in stomatal opening. We cloned the causal gene (MGR1/CST2) and functionally characterized this gene. The mutant phenotype was caused by a mutation in a gene encoding an unknown protein with similarities to the human magnesium (Mg2+ ) efflux transporter ACDP/CNNM. MGR1/CST2 was localized to the tonoplast and showed transport activity for Mg2+ . This protein was constitutively and highly expressed in guard cells. Knockout of this gene resulted in stomatal closing, decreased photosynthesis and growth retardation, especially under high Mg2+ conditions, while overexpression of this gene increased stomatal opening and tolerance to high Mg2+ concentrations. Furthermore, guard cell-specific expression of MGR1/CST2 in the mutant partially restored its stomatal opening. Our results indicate that MGR1/CST2 expression in the leaf guard cells plays an important role in maintaining cytosolic Mg2+ concentrations through sequestering Mg2+ into vacuoles, which is required for stomatal opening, especially under high Mg2+ conditions.

A crucial role for a node-localized transporter, HvSPDT, in loading phosphorus into barley grains.

Grains are the major sink of phosphorus (P) in cereal crops, accounting for 60-85% of total plant P, but the mechanisms underlying P loading into the grains are poorly understood. We functionally characterized a transporter gene required for the distribution of P to the grains in barley (Hordeum vulgare), HvSPDT (SULTR-like phosphorus distribution transporter). HvSPDT encoded a plasma membrane-localized Pi/H+ cotransporter. It was mainly expressed in the nodes at both the vegetative and reproductive stages. Furthermore, its expression was induced by inorganic phosphate (Pi) deficiency. In the nodes, HvSPDT was expressed in both the xylem and phloem region of enlarged and diffuse vascular bundles. Knockout of HvSPDT decreased the distribution of P to new leaves, but increased the distribution to old leaves at the vegetative growth stage under low P supply. However, knockout of HvSPDT did not alter the redistribution of P from old to young organs. At the reproductive stage, knockout of HvSPDT significantly decreased P allocation to the grains, resulting in a considerable reduction in grain yield, especially under P-limited conditions. Our results indicate that node-based HvSPDT plays a crucial role in loading P into barley grains through preferentially distributing P from the xylem and further to the phloem.

Buckwheat FeNramp5 Mediates High Manganese Uptake in Roots.

Manganese (Mn) is an essential element for plant growth and development, but transporters required for Mn uptake have only been identified in a few plant species. Here, we functionally characterized a member of the natural resistance-associated macrophage proteins (Nramps) family, FeNramp5 in buckwheat (Fagopyrum esculentum Moench), which is known as a species well adapted to acidic soils. FeNramp5 was mainly expressed in the roots, and its expression was upregulated by the deficiency of Mn and Fe. Furthermore, spatial and tissue-specific expression analysis showed that FeNramp5 was expressed in all tissues of the basal root regions. FeNramp5-GFP protein was localized to the plasma membrane when transiently expressed in buckwheat leaf protoplast. FeNramp5 showed the transport activity for Mn2+ and Cd2+ but not for Fe2+ when expressed in yeast. Furthermore, the transport activity for Mn2+ was higher in yeast expressing FeNramp5 than in yeast expressing AtNramp1. FeNramp5 was also able to complement the phenotype of Arabidopsis atnramp1 mutant in terms of the growth and accumulation of Mn and Cd. The absolute expression level of AtNramp1 was comparable to that of FeNramp5 in the roots, but buckwheat accumulated higher Mn than Arabidopsis when grown under the same condition. Further analysis showed that at least motif B in FeNramp5 seems important for its high transport activity for Mn. These results indicate that FeNramp5 is a transporter for the uptake of Mn and Cd and its higher transport activity for Mn is probably associated with higher Mn accumulation in buckwheat.

Diel magnesium fluctuations in chloroplasts contribute to photosynthesis in rice.

Photosynthesis provides food, fibre and fuel that support our society; understanding the mechanisms controlling dynamic changes in this process helps identify new options to improve photosynthesis. Photosynthesis shows diel changes, which have been largely attributed to external light/dark conditions, as well as internal gene expression and the post-translational modification of critical enzymes. Here we report diel fluctuations of magnesium (Mg) in rice (Oryza sativa) chloroplasts, which may function as a rhythm regulator contributing to the post-translational regulation of photosynthetic CO2 assimilation in rice. We found that a chloroplast-localized Mg2+ transporter gene, OsMGT3, which is rhythmically expressed in leaf mesophyll cells, partly modulates Mg fluctuations in rice chloroplasts. Knockout of OsMGT3 substantially reduced Mg2+ uptake, as well as the amplitude of free Mg2+ fluctuations in chloroplasts, which was closely associated with a decrease in ribulose 1,5-bisphosphate carboxylase activity in vivo and a consequent decline in the photosynthetic rate. In addition, the mesophyll-specific overexpression of OsMGT3 remarkably improved photosynthetic efficiency and growth performance in rice. Taken together, these observations demonstrate that OsMGT3-dependent diel Mg fluctuations in chloroplasts may contribute to Mg-dependent enzyme activities for photosynthesis over the daily cycle. Enhancing Mg2+ input to chloroplasts could be a potential approach to improving photosynthetic efficiency in plants.

Simultaneous changes in seed size, oil content and protein content driven by selection of SWEET homologues during soybean domestication.

Soybean accounts for more than half of the global production of oilseed and more than a quarter of the protein used globally for human food and animal feed. Soybean domestication involved parallel increases in seed size and oil content, and a concomitant decrease in protein content. However, science has not yet discovered whether these effects were due to selective pressure on a single gene or multiple genes. Here, re-sequencing data from >800 genotypes revealed a strong selection during soybean domestication on GmSWEET10a. The selection of GmSWEET10a conferred simultaneous increases in soybean-seed size and oil content as well as a reduction in the protein content. The result was validated using both near-isogenic lines carrying substitution of haplotype chromosomal segments and transgenic soybeans. Moreover, GmSWEET10b was found to be functionally redundant with its homologue GmSWEET10a and to be undergoing selection in current breeding, leading the the elite allele GmSWEET10b, a potential target for present-day soybean breeding. Both GmSWEET10a and GmSWEET10b were shown to transport sucrose and hexose, contributing to sugar allocation from seed coat to embryo, which consequently determines oil and protein contents and seed size in soybean. We conclude that past selection of optimal GmSWEET10a alleles drove the initial domestication of multiple soybean-seed traits and that targeted selection of the elite allele GmSWEET10b may further improve the yield and seed quality of modern soybean cultivars.

Vascular Cambium-Localized AtSPDT Mediates Xylem-to-Phloem Transfer of Phosphorus for Its Preferential Distribution in Arabidopsis.

During plant growth and development mineral elements are preferentially delivered to different organs and tissues to meet the differential demand. It has been shown that the preferential distribution of mineral nutrients in gramineous plants is mediated by node-based transporters, but the mechanisms of preferential distribution in dicots are poorly understood. Here, we report a distinct mechanism for the preferential distribution of phosphorus (P) in Arabidopsis plants, revealed by detailed functional analysis of AtSPDT/AtSULTR3;4 (SULTR-like P Distribution Transporter), a homolog of rice OsSPDT. Like OsSPDT, AtSPDT is localized at the plasma membrane and showed proton-dependent transport activity for P. Interestingly, we found that AtSPDT is mainly expressed in the rosette basal region and leaf petiole, and its expression is up-regulated by P deficiency. Tissue-specific analysis showed that AtSPDT is mainly located in the vascular cambium of different organs, as well as in the parenchyma tissues of both xylem and phloem regions. Knockout of AtSPDT inhibited the growth of new leaves under low P due to decreased P distribution to those organs. The seed yields of the wild-type and atspdt mutant plants are similar, but the seeds of mutant plants contain - less P. These results indicate that AtSPDT localized in the vascular cambium is involved in preferential distribution of P to the developing tissues, through xylem-to-phloem transfer mainly at the rosette basal region and leaf petiole.

A Vacuolar Phytosiderophore Transporter Alters Iron and Zinc Accumulation in Polished Rice Grains.

Essential metals, such as iron (Fe) and zinc (Zn), in grains are important sources for seed germination and nutritional requirements, but the molecular mechanisms underlying their loading into grains are poorly understood. Recently, nodes in rice (Oryza sativa) were reported to play an important role in the preferential distribution of mineral elements to the grains. In this study, we functionally characterized a rice gene highly expressed in nodes, OsVMT (VACUOLAR MUGINEIC ACID TRANSPORTER), belonging to a major facilitator superfamily. OsVMT is highly expressed in the parenchyma cell bridges of node I, where Fe and Zn are highly deposited. The expression of OsVMT was induced by Fe deficiency in the roots but not in the shoot basal region and uppermost node. OsVMT localized to the tonoplast and showed efflux transport activity for 2'-deoxymugineic acid (DMA). At the vegetative stage, knockout of OsVMT resulted in decreased DMA but increased ferric Fe in the root cell sap. As a result, the concentration of DMA in the xylem sap increased but that of ferric Fe decreased in the xylem sap in the mutants. In the polished rice grain, the mutants accumulated 1.8- to 2.1-fold, 1.5- to 1.6-fold, and 1.4- to 1.5-fold higher Fe, Zn, and DMA, respectively, than the wild type. Taken together, our results indicate that OsVMT is involved in sequestering DMA into the vacuoles and that knockout of this gene enhances the accumulation of Fe and Zn in polished rice grains through DMA-increased solubilization of Fe and Zn deposited in the node.

The Soybean Sugar Transporter GmSWEET15 Mediates Sucrose Export from Endosperm to Early Embryo.

Soybean (Glycine max) seed is primarily composed of a mature embryo that provides a major source of protein and oil for humans and other animals. Early in development, the tiny embryos grow rapidly and acquire large quantities of sugars from the liquid endosperm of developing seeds. An insufficient supply of nutrients from the endosperm to the embryo results in severe seed abortion and yield reduction. Hence, an understanding of the molecular basis and regulation of assimilate partitioning involved in early embryo development is important for improving soybean seed yield and quality. Here, we used expression profiling analysis to show that two paralogous sugar transporter genes from the SWEET (Sugars Will Eventually be Exported Transporter) family, GmSWEET15a and GmSWEET15b, were highly expressed in developing soybean seeds. In situ hybridization and quantitative real-time PCR showed that both genes were mainly expressed in the endosperm at the cotyledon stage. GmSWEET15b showed both efflux and influx activities for sucrose in Xenopus oocytes. In Arabidopsis (Arabidopsis thaliana), knockout of three AtSWEET alleles is required to see a defective, but not lethal, embryo phenotype, whereas knockout of both GmSWEET15 genes in soybean caused retarded embryo development and endosperm persistence, resulting in severe seed abortion. In addition, the embryo sugar content of the soybean knockout mutants was greatly reduced. These results demonstrate that the plasma membrane sugar transporter, GmSWEET15, is essential for embryo development in soybean by mediating Suc export from the endosperm to the embryo early in seed development.

Two Genes Encoding a Bacterial-Type ATP-Binding Cassette Transporter are Implicated in Aluminum Tolerance in Buckwheat.

Buckwheat (Fagopyrum esculentum Moench) shows high tolerance to aluminum (Al) toxicity, but the molecular mechanisms underlying its high Al tolerance are poorly understood. Here, we functionally characterized two genes (FeSTAR1 and FeSTAR2), which encode a nucleotide-binding domain and a membrane domain, respectively, of a bacterial-type ATP-binding cassette (ABC) transporter. The expression of FeSTAR1 and FeSTAR2 was induced by Al in both roots and leaves with higher expression in the roots. Spatial and tissue-specific expression analysis showed that the Al-induced expression of these two genes was found in both the root tips and basal root regions with higher expression in the root outer cell layers. The expression was neither induced by other metals including Cd and La nor by low pH and phosphorus-deficiency. FeSTAR1 and FeSTAR2 were present in a single copy in the genome, but the Al-induced transcript copy number of FeSTAR1 and FeSTAR2 was much higher than their homologous genes in rice and Arabidopsis. FeSTAR1 and FeSTAR2 form a complex when co-expressed in onion epidermal cells. Introduction of FeSTAR1 and FeSTAR2 into Arabidopsis mutants atstar1 and als3/atstar2, respectively, rescued the sensitivity of the mutants to Al. Taken together, our results indicate that FeSTAR1 and FeSTAR2 are involved in Al tolerance and that their high expression level may contribute to high Al tolerance in buckwheat.

Retrotransposon Insertion and DNA Methylation Regulate Aluminum Tolerance in European Barley Accessions.

Aluminum (Al) toxicity is a major stress factor limiting crop productivity in acid soil. Although there is great genotypic variation in tolerance to Al toxicity, the underlying molecular mechanisms are poorly understood. Here, we report that, in barley (Hordeum vulgare), the fourth largest cereal crop produced in the world, both retrotransposon insertion and DNA methylation are involved in regulating differential Al tolerance. HvAACT1 is a major gene responsible for citrate secretion from the roots for external detoxification of Al. A multiretrotransposon-like (MRL) sequence insertion at least 15.3 kb in length was detected in the upstream genomic region of HvAACT1 that displayed promoter activity and significantly enhanced HvAACT1 expression, especially in the root tips of Al-tolerant accessions. Furthermore, in a number of accessions with low levels of HvAACT1 expression, this MRL insertion was present but highly methylated. Geographical analysis showed that accessions with this MRL insertion are distributed mainly in European areas with acid soils. Two wild barley accessions were found to possess this MRL insertion, but with a high degree of methylation. These results indicate that the MRL insertion and its degree of DNA methylation influence HvAACT1 expression and that demethylation of this MRL insertion, which facilitates adaptation to acid soils, occurred following barley domestication. Moreover, our results indicate that barley accessions in East Asia and Europe have developed independent but equivalent strategies to withstand Al toxicity in acid soils.

Functional characterization of an aluminum (Al)-inducible transcription factor, ART2, revealed a different pathway for Al tolerance in rice.

High aluminum (Al) tolerance in rice (Oryza sativa) is controlled by a Cys2His2-type zinc finger transcription factor ART1 (Al resistance transcription factor 1). There are five close homologs of ART1 in the rice genome, but the role of these homologs is unknown. We functionally characterized one of the ART1 homologs, ART2, in terms of tissue and spatial expression, subcellular localization, transcriptional activation activity, and phenotypic analysis of the knockout lines. ART2 was localized to the nucleus and showed a transcriptional activation potential in yeast. ART2 was mainly expressed in the roots, but the expression level was much lower than that of ART1. The ART2 expression was rapidly induced by Al in the roots of the wild-type rice, but not in art1 mutant. Knockout of ART2 resulted in increased sensitivity to Al toxicity, but did not alter sensitivity to different pH values. Expression profile analysis by RNA-sequencing showed that ART2 was not involved in activation of genes regulated by ART1; however, four genes seems to be regulated by ART2, which are implicated in Al tolerance. These results indicate that ART1 and ART2 regulate different pathways leading to Al tolerance, and ATR2 plays a supplementary role in Al tolerance in rice.

Transcription factor WRKY22 promotes aluminum tolerance via activation of OsFRDL4 expression and enhancement of citrate secretion in rice (Oryza sativa).

Whilst WRKY transcription factors are known to be involved in diverse plant responses to biotic stresses, their involvement in abiotic stress tolerance is poorly understood. OsFRDL4, encoding a citrate transporter, has been reported to be regulated by ALUMINUM (Al) RESISTANCE TRANSCRIPTION FACTOR 1 (ART1) in rice, but whether it is also regulated by other transcription factors is unknown. We define the role of OsWRKY22 in response to Al stress in rice by using mutation and transgenic complementation assays, and characterize the regulation of OsFRDL4 by OsWRKY22 via yeas one-hybrid, electrophoretic mobility shift assay and ChIP-quantitative PCR. We demonstrate that loss of OsWRKY22 function conferred by the oswrky22 T-DNA insertion allele causes enhanced sensitivity to Al stress, and a reduction in Al-induced citrate secretion. We next show that OsWRKY22 is localized in the nucleus, functions as a transcriptional activator and is able to bind to the promoter of OsFRDL4 via W-box elements. Finally, we find that both OsFRDL4 expression and Al-induced citrate secretion are significantly lower in art1 oswrky22 double mutants than in the respective single mutants. We conclude that OsWRKY22 promotes Al-induced increases in OsFRDL4 expression, thus enhancing Al-induced citrate secretion and Al tolerance in rice.

Preferential Distribution of Boron to Developing Tissues Is Mediated by the Intrinsic Protein OsNIP3.

Boron is especially required for the growth of meristem and reproductive organs, but the molecular mechanisms underlying the preferential distribution of B to these developing tissues are poorly understood. Here, we show evidence that a member of nodulin 26-like intrinsic protein (NIP), OsNIP3;1, is involved in this preferential distribution in rice (Oryza sativa). OsNIP3;1 was highly expressed in the nodes and its expression was up-regulated by B deficiency, but down-regulated by high B. OsNIP3;1 was polarly localized at the xylem parenchyma cells of enlarged vascular bundles of nodes facing toward the xylem vessels. Furthermore, this protein was rapidly degraded within a few hours in response to high B. Knockout of this gene hardly affected the uptake and root-to-shoot translocation of B, but altered B distribution in different organs in the above-ground parts, decreased distribution of B to the new leaves, and increased distribution to the old leaves. These results indicate that OsNIP3;1 located in the nodes is involved in the preferential distribution of B to the developing tissues by unloading B from the xylem in rice and that it is regulated at both transcriptional and protein level in response to external B level.

Two MATE Transporters with Different Subcellular Localization are Involved in Al Tolerance in Buckwheat.

Buckwheat (Fagopyrum esculentum) shows high tolerance to aluminum (Al) toxicity, but the molecular mechanisms responsible for this high Al tolerance are still poorly understood. Here, we investigated the involvement of two MATE (multi-drug and toxic compound extrusion) genes in Al tolerance. Both FeMATE1 and FeMATE2 showed efflux transport activity for citrate, but not for oxalate when expressed in Xenopus oocytes. A transient assay with buckwheat leaf protoplasts using green fluorescent protein (GFP) fusion showed that FeMATE1 was mainly localized to the plasma membrane, whereas FeMATE2 was localized to the trans-Golgi and Golgi. The expression of FeMATE1 was induced by Al only in the roots, but that of FeMATE2 was up-regulated in both the roots and leaves. Furthermore, the expression of both genes only responded to Al toxicity, but not to other stresses including low pH, cadmium (Cd) and lanthanum (La). Heterologous expression of FeMATE1 or FeMATE2 in the Arabidopsis mutant atmate partially rescued its Al tolerance. Expression of FeMATE1 also partially recovered the Al-induced secretion of citrate in the transgenic lines, whereas expression of FeMATE2 did not complement the citrate secretion. Further physiological analysis showed that buckwheat roots also secreted citrate in addition to oxalate in response to Al in a dose-responsive manner. Taken together, our results indicate that FeMATE1 is involved in the Al-activated citrate secretion in the roots, while FeMATE2 is probably responsible for transporting citrate into the Golgi system for the internal detoxification of Al in the roots and leaves of buckwheat.

Functional characterization of two half-size ABC transporter genes in aluminium-accumulating buckwheat.

Buckwheat (Fagopyrum esculentum Moench) is able to detoxify high aluminium (Al) internally by sequestering it to the vacuoles in the leaves; however, the molecular mechanisms underlying this sequestration are unknown. We performed proteomic analysis with the leaf tonoplast-rich fraction and identified two half-size ABC transporters; FeASL1.1 and FeALS1.2. We investigated the gene expression patterns and subcellular localization. To demonstrate their physiological role, we expressed FeALS1.1 or FeALS1.2 in the Arabidopsis atals1 mutant under the control of AtALS1 promoter. FeALS1.1 expression was upregulated by Al in both the leaves and the roots, and its expression level in the roots was six times higher than its homologous gene (AtALS1) of Arabidopsis. FeALS1.2 expression, however, was not affected by Al but showed a 39 times higher expression level than AtALS1 in the leaves. When FeALS1.1 or FeALS1.2 was expressed in atals1, both of them recovered their Al tolerance through altering the subcellular localization of Al in root cells. Taken together, our results indicate that FeALS1.1 and FeALS1.2 are involved in the internal detoxification of Al in the roots and leaves, respectively, by sequestering Al into the vacuoles. Their high expression is probably required for high Al tolerance in buckwheat.

Retrotransposon-Mediated Aluminum Tolerance through Enhanced Expression of the Citrate Transporter OsFRDL4.

High aluminum (Al) tolerance of rice (Oryza sativa) is controlled by multiple tolerance genes, but the regulatory mechanisms underlying the differential expression of these genes are poorly understood. Here, we investigated the factors regulating the expression of OsFRDL4, a gene encoding a citrate efflux transporter involved in Al-induced citrate secretion from the roots. Analysis with chromosome segment substitution lines derived from cv Nipponbare (high OsFRDL4 expression) and cv Kasalath (low OsFRDL4 expression) revealed that the differential expression of OsFRDL4 is responsible for the quantitative trait locus for Al tolerance detected previously on chromosome 1. Comparison of the OsFRDL4 gene structure in cv Nipponbare and cv Kasalath showed that there was no difference in the position of the transcriptional start site, but a 1.2-kb insertion showing high similarity to the solo long terminal repeat of the retrotransposon was found in the promoter region of OsFRDL4 in cv Nipponbare. This insertion showed higher promoter activity and contained nine cis-acting elements for ALUMINUM RESISTANCE TRANSCRIPTION FACTOR1 (ART1). However, this insertion did not alter the spatial expression or cellular localization of OsFRDL4. Furthermore, this insertion was found in most japonica varieties but was largely absent from indica varieties or wild rice species. These results indicate that the 1.2-kb insertion in the OsFRDL4 promoter region in japonica subspecies is responsible for their higher expression level of OsFRDL4 due to the increased number of cis-acting elements of ART1. Our results also suggest that this insertion event happened at the initial stage of domestication of japonica subspecies.

OsFRDL1 expressed in nodes is required for distribution of iron to grains in rice.

Iron (Fe) is essential for plant growth and development, but the molecular mechanisms underlying its distribution to different organs are poorly understood. We found that OsFRDL1 (FERRIC REDUCTASE DEFECTIVE LIKE 1), a plasma membrane-localized transporter for citrate, was highly expressed in the upper nodes of rice at the reproductive growth stage. OsFRDL1 was expressed in most cells of enlarged vascular bundles, diffuse vascular bundles, and the interjacent parenchyma cell bridges of uppermost node I, as well as vascular tissues of the leaf blade, leaf sheath, peduncle, rachis, husk, and stamen. Knockout of OsFRDL1 decreased pollen viability and grain fertility when grown in a paddy field. Iron was deposited in the parenchyma cell bridges, a few of the cell layers of the parenchyma tissues outside of the bundle sheath of enlarged vascular bundles in node I in both the wild-type rice and osfrdl1 mutant, but the mutant accumulated more Fe than the wild-type rice in this area. A stem-fed experiment with stable isotope 57Fe showed that the distribution of Fe in the anther and panicle decreased in the knockout line, but that in the flag leaf it increased compared with the wild-type rice. Taken together, our results show that OsFRDL1 expressed in the upper nodes is required for the distribution of Fe in the panicles through solubilizing Fe deposited in the apoplastic part of nodes in rice.

An Aluminum-Inducible IREG Gene is Required for Internal Detoxification of Aluminum in Buckwheat.

Buckwheat (Fagopyrum esculentum Moench) is able to detoxify aluminum (Al) both externally and internally, but the molecular mechanisms underlying its high Al tolerance are not understood. We functionally characterized a gene (FeIREG1) belonging to IRON REGULATED/ferroportin in buckwheat, which showed high expression in our previous genome-wide transcriptome analysis. FeIREG1 was mainly expressed in the roots, and its expression was up-regulated by Al, but not by other metals and low pH. Furthermore, in contrast to AtIREG1 and AtIREG2 in Arabidopsis, the expression of FeIREG1 was not induced by Fe deficiency. Spatial expression analysis showed that the Al-induced expression of FeIREG1 was found in the root tips and higher expression was detected in the outer layers of this part. Immunostaining also showed that FeIREG1 was localized at the outer cell layers in the root tip. A FeIREG1-green fluorescent protein (GFP) fusion protein was localized to the tonoplast when transiently expressed in onion epidermal cells. Overexpression of FeIREG1 in Arabidopsis resulted in increased Al tolerance, but did not alter the tolerance to Cd, Co and Fe. The tolerance to Ni was slightly enhanced in the overexpression lines. Mineral analysis showed that the accumulation of total root Al and other essential mineral elements was hardly altered in the overexpression lines. Taken together, our results suggest that FeIREG1 localized at the tonoplast plays an important role in internal Al detoxification by sequestering Al into the root vacuoles in buckwheat.

Functional Analysis of a MATE Gene OsFRDL2 Revealed its Involvement in Al-Induced Secretion of Citrate, but a Lower Contribution to Al Tolerance in Rice.

The multidrug and toxic compound extrusion (MATE) transporters represent a large transporter family in plants, but the role of most genes in this family has not been examined. We functionally characterized a MATE family member, OsFRDL2, in rice (Oryza sativa). OsFRDL2 showed an efflux transport activity for citrate when it was expressed in both Xenopus oocytes and cultured tobacco cells. OsFRDL2 was mainly expressed in the roots and its expression was not induced by iron (Fe) deficiency, but it was rapidly up-regulated by aluminum (Al). Furthermore, the expression of OsFRDL2 was regulated by ART1, a C2H2-type zinc-finger transcription factor for Al tolerance. OsFRDL2 protein was localized at unidentified vesicles in the cytosol, but not co-localized with either mitochondria or peroxisomes when expressed in both onion epidermal cells and cultured tobacco cells. Knockout of OsFRDL2 decreased Al-induced secretion of citrate from the roots, but did not affect the internal citrate concentration. The Al-induced inhibition of root elongation was similar between the OsFRDL2 knockout line and its wild-type rice. Knockout of OsFRDL2 did not affect the translocation of Fe from the roots to the shoots. A double mutant between osfrdl2 and osfrdl4 or osfrdl1 did not further decrease the Al-induced citrate secretion and Fe translocation compared with the single mutant. Collectively, our results indicate that although OsFRDL2 is involved in the Al-induced secretion of citrate, its contribution to high Al tolerance is relatively small in rice.

AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis.

Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl(-)-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.