Paddy rice (Oryza sativa) is able to accumulate high concentrations of Mn without showing toxicity; however, the molecular mechanisms underlying Mn uptake are unknown. Here, we report that a member of the Nramp (for the Natural ResistanceAssociated Macrophage Protein) family, Nramp5, is involved in Mn uptake and subsequently the accumulation of high concentrations of Mn in rice. Nramp5 was constitutively expressed in the roots and encodes a plasma membrane-localized protein. Nramp5 was polarly localized at the distal side of both exodermis and endodermis cells. Knockout of Nramp5 resulted in a significant reduction in growth and grain yield, especially when grown at low Mn concentrations. This growth reduction could be partially rescued by supplying high concentrations of Mn but not by the addition of Fe. Mineral analysis showed that the concentration of Mn and Cd in both the roots and shoots was lower in the knockout line than in wild-type rice. A short-term uptake experiment revealed that the knockout line lost the ability to take up Mn and Cd. Taken together, Nramp5 is a major transporter of Mn and Cd and is responsible for the transport of Mn and Cd from the external solution to root cells.

Multidrug and toxic compound extrusion (MATE) transporters represent a large family in plants, but their functions are poorly understood. Here, we report the function of a rice (Oryza sativa) MATE gene (Os03g0216700, OsFRDL1), the closest homolog of barley (Hordeum vulgare) HvAACT1 (aluminum [Al]-activated citrate transporter 1), in terms of metal stress (iron [Fe] deficiency and Al toxicity). This gene was mainly expressed in the roots and the expression level was not affected by either Fe deficiency or Al toxicity. Knockout of this gene resulted in leaf chlorosis, lower leaf Fe concentration, higher accumulation of zinc and manganese concentration in the leaves, and precipitation of Fe in the root's stele. The concentration of citrate and ferric iron in the xylem sap was lower in the knockout line compared to the wild-type rice. Heterologous expression of OsFRDL1 in Xenopus oocytes showed transport activity for citrate. Immunostaining showed that OsFRDL1 was localized at the pericycle cells of the roots. On the other hand, there was no difference in the Al-induced secretion of citrate from the roots between the knockout line and the wild-type rice. Taken together, our results indicate that OsFRDL1 is a citrate transporter localized at the pericycle cells, which is necessary for efficient translocation of Fe to the shoot as a Fe-citrate complex.

Developing tissues such as meristems and reproductive organs require high zinc, but the molecular mechanisms of how zinc taken up by the roots is preferentially delivered to these tissues with low transpiration are unknown. Here, we report that rice (Oryza sativa) heavy metal ATPase2 (OsHMA2), a member of P-type ATPases, is involved in preferential delivery of zinc to the developing tissues in rice. OsHMA2 was mainly expressed in the mature zone of the roots at the vegetative stage, but higher expression was also found in the nodes at the reproductive stage. The expression was unaffected by either zinc deficiency or zinc excess. OsHMA2 was localized at the pericycle of the roots and at the phloem of enlarged and diffuse vascular bundles in the nodes. Heterologous expression of OsHMA2 in yeast (Saccharomyces cerevisiae) showed influx transport activity for zinc as well as cadmium. Two independent Tos17 insertion lines showed decreased zinc concentration in the crown root tips, decreased concentration of zinc and cadmium in the upper nodes and reproductive organs compared with wild-type rice. Furthermore, a short-term labeling experiment with 67 Zn showed that the distribution of zinc to the panicle and uppermost node I was decreased, but that, to the lower nodes, was increased in the two mutants. Taken together, OsHMA2 in the nodes plays an important role in preferential distribution of zinc as well as cadmium through the phloem to the developing tissues.

SUMMARYA number of plant species, including rice, secretes citrate from roots in response to Al stress. Here we characterized the functions of a gene, OsFRDL4 (Os01g0919100) that belongs to the multidrug and toxic compound extrusion (MATE) family in rice (Oryza sativa). Heterologous expression in Xenopus oocyte showed that the OsFRDL4 protein was able to transport citrate and was activated by Al. The expression level of the OsFRDL4 gene in roots was very low in the absence of Al, but was greatly enhanced by Al after short exposure. Furthermore, the OsFRDL4 expression was regulated by ART1, a C2H2-type zinc finger transcription factor for Al tolerance. Transient expression of OsFRDL4 in onion epidermal cells showed that it localized to the plasma membrane. Immunostaining showed that OsFRDL4 was localized in all cells in the root tip. These expression patterns and cell specificity of localization of OsFRDL4 are different from other MATE members identified previously. Knockout of OsFRDL4 resulted in decreased Al tolerance and decreased citrate secretion compared with the wild-type rice, but did not affect citrate concentration in the xylem sap. Furthermore, there is a positive correlation between OsFRDL4 expression level and the amount of citrate secretion in rice cultivars that are differing in Al tolerance. Taken together, our results show that OsFRDL4 is an Al-induced citrate transporter localized at the plasma membrane of rice root cells and is one of the components of high Al tolerance in rice.

Cadmium (Cd) is a highly toxic heavy metal for plants, but several unique Cd-hyperaccumulating plant species are able to accumulate this metal to extraordinary concentrations in the aboveground tissues without showing any toxic symptoms. However, the molecular mechanisms underlying this hypertolerance to Cd are poorly understood. Here we have isolated and functionally characterized an allelic gene, TcHMA3 (heavy metal ATPase 3) from two ecotypes (Ganges and Prayon) of Thlaspi caerulescens contrasting in Cd accumulation and tolerance. The TcHMA3 alleles from the higher (Ganges) and lower Cd-accumulating ecotype (Prayon) share 97.8% identity, and encode a P(1B)-type ATPase. There were no differences in the expression pattern, cell-specificity of protein localization and transport substrate-specificity of TcHMA3 between the two ecotypes. Both alleles were characterized by constitutive expression in the shoot and root, a tonoplast localization of the protein in all leaf cells and specific transport activity for Cd. The only difference between the two ecotypes was the expression level of TcHMA3: Ganges showed a sevenfold higher expression than Prayon, partly caused by a higher copy number. Furthermore, the expression level and localization of TcHMA3 were different from AtHMA3 expression in Arabidopsis. Overexpression of TcHMA3 in Arabidopsis significantly enhanced tolerance to Cd and slightly increased tolerance to Zn, but did not change Co or Pb tolerance. These results indicate that TcHMA3 is a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens.

Mineral nutrients, such as manganese, are required for the development of plants and their reproductive organs, but these can be toxic if accumulated at high concentrations. Therefore, plants must have a system for preferentially delivering an adequate amount of minerals to these organs for active growth and development, while preventing mineral overaccumulation in the face of changing environments. Here we show that a member of the Nramp transporter family, OsNramp3, functions as a switch in response to environmental Mn changes. OsNramp3 is constitutively expressed in the node, a junction of vasculatures connecting leaves, stems and panicles. At low Mn concentration, OsNramp3 preferentially transports Mn to young leaves and panicles. However, at high Mn concentration, the OsNramp3 protein is rapidly degraded within a few hours, resulting in the distribution of Mn to old tissues. Our results reveal the OsNramp3-mediated strategy of rice for adapting to a wide change of Mn in the environment.

Originating from the Fertile Crescent in the Middle East, barley has now been cultivated widely on different soil types including acid soils, where aluminium toxicity is a major limiting factor. Here we show that the adaptation of barley to acid soils is achieved by the modification of a single gene (HvAACT1) encoding a citrate transporter. We find that the primary function of this protein is to release citrate from the root pericycle cells to the xylem to facilitate the translocation of iron from roots to shoots. However, a 1-kb insertion in the upstream of the HvAACT1 coding region occurring only in the Al-tolerant accessions, enhances its expression and alters the location of expression to the root tips. The altered HvAACT1 has an important role in detoxifying aluminium by secreting citrate to the rhizosphere. Thus, the insertion of a 1-kb sequence in the HvAACT1 upstream enables barley to adapt to acidic soils.

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 if these effects were due to selective pressure on a single gene or multiple genes. Here, resequencing data from over 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 reduction in 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 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.