Aluminum (Al) is the most abundant metal in the Earth's crust, but its trivalent ionic form is highly toxic to all organisms at low concentrations. How Al enters cells has not been elucidated in any organisms. Herein, we report a transporter, Nrat1 (Nramp aluminum transporter 1), specific for trivalent Al ion in rice. Nrat1 belongs to the Nramp (natural resistance-associated macrophage protein) family, but shares a low similarity with other Nramp members. When expressed in yeast, Nrat1 transports trivalent Al ion, but not other divalent ions, such as manganese, iron, and cadmium, or the Al–citrate complex. Nrat1 is localized at the plasma membranes of all cells of root tips except epidermal cells. Knockout of Nrat1 resulted in decreased Al uptake, increased Al binding to cell wall, and enhanced Al sensitivity, but did not affect the tolerance to other metals. Expression of Nrat1 is up-regulated by Al in the roots and regulated by a C2H2 zinc finger transcription factor (ART1). We therefore concluded that Nrat1 is a plasma membrane-localized transporter for trivalent Al, which is required for a prior step of final Al detoxification through sequestration of Al into vacuoles.

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 ⁶⁷Zn 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.

Cadmium (Cd) is a highly toxic heavy metal in nature, which causes severe damage to plant growth. The molecular mechanisms for Cd detoxification are poorly understood. Here, we report that a G-type ATP-binding cassette transporter, OsABCG36, is involved in Cd tolerance in rice. OsABCG36 was expressed in both roots and shoots at a low level, but expression in the roots rather than the shoots was greatly up-regulated by a short exposure to Cd. A spatial expression analysis showed that Cd-induced expression of OsABCG36 was found in both the root tip and the mature root region. Transient expression of OsABCG36 in rice protoplast cells showed that it was localized to the plasma membrane. Immunostaining showed that OsABCG36 was localized in all root cells except the epidermal cells. Knockout of OsABCG36 resulted in increased Cd accumulation in root cell sap and enhanced Cd sensitivity, but did not affect tolerance to other metals including Al, Zn, Cu, and Pb. The concentration of Cd in the shoots was similar between the knockout lines and wild-type rice. Heterologous expression of OsABCG36 in yeast showed an efflux activity for Cd, but not for Zn. Taken together, our results indicate that OsABCG36 is not involved in Cd accumulation in the shoots, but is required for Cd tolerance by exporting Cd or Cd conjugates from the root cells in rice.

Sterility is common in hybrids between divergent populations, such as the indica and japonica subspecies of Asian cultivated rice ( Oryza sativa ). Although multiple loci for plant hybrid sterility have been identified, it remains unknown how alleles of the loci interact at the molecular level. Here we show that a locus for indica-japonica hybrid male sterility, Sa , comprises two adjacent genes, SaM and SaF , encoding a small ubiquitin-like modifier E3 ligase-like protein and an F-box protein, respectively. Most indica cultivars contain a haplotype SaM ⁺ SaF ⁺ , whereas all japonica cultivars have SaM ⁻ SaF ⁻ that diverged by nucleotide variations in wild rice. Male semi-sterility in this heterozygous complex locus is caused by abortion of pollen carrying SaM ⁻ . This allele-specific gamete elimination results from a selective interaction of SaF ⁺ with SaM ⁻ , a truncated protein, but not with SaM ⁺ because of the presence of an inhibitory domain, although SaM ⁺ is required for this male sterility. Lack of any one of the three alleles in recombinant plants does not produce male sterility. We propose a two-gene/three-component interaction model for this hybrid male sterility system. The findings have implications for overcoming male sterility in inter-subspecific hybrid rice breeding.

Heavy metal-transporting P-type ATPase (HMA) has been implicated in the transport of heavy metals in plants. Here, we report the function and role of an uncharacterized member of HMA, OsHMA5 in rice (Oryza sativa). Knockout of OsHMA5 resulted in a decreased copper (Cu) concentration in the shoots but an increased Cu concentration in the roots at the vegetative stage. At the reproductive stage, the concentration of Cu in the brown rice was significantly lower in the mutants than in the wild-type rice; however, there was no difference in the concentrations of iron, manganese, and zinc between two independent mutants and the wild type. The Cu concentration of xylem sap was lower in the mutants than in the wild-type rice. OsHMA5 was mainly expressed in the roots at the vegetative stage but also in nodes, peduncle, rachis, and husk at the reproductive stage. The expression was up-regulated by excess Cu but not by the deficiency of Cu and other metals, including zinc, iron, and manganese, at the vegetative stage. Analysis of the transgenic rice carrying the OsHMA5 promoter fused with green fluorescent protein revealed that it was localized at the root pericycle cells and xylem region of diffuse vascular bundles in node I, vascular tissues of peduncle, rachis, and husk. Furthermore, immunostaining with an antibody against OsHMA5 revealed that it was localized to the plasma membrane. Expression of OsHMA5 in a Cu transport-defective mutant yeast (Saccharomyces cerevisiae) strain restored the growth. Taken together, OsHMA5 is involved in loading Cu to the xylem of the roots and other organs.

Yellow Stripe-Like (YSL) proteins belong to the oligopeptide transporter family and have been implicated in metal transport and homeostasis in different plant species. Here, we functionally characterized a rice (Oryza sativa) YSL member, OsYSL6. Knockout of OsYSL6 resulted in decreased growth of both roots and shoots only in the high-manganese (Mn) condition. There was no difference in the concentration of total Mn and other essential metals between the wild-type rice and the knockout line, but the knockout line showed a higher Mn concentration in the leaf apoplastic solution and a lower Mn concentration in the symplastic solution than wild-type rice. OsYSL6 was constitutively expressed in both the shoots and roots, and the expression level was not affected by either deficiency or toxicity of various metals. Furthermore, the expression level increased with leaf age. Analysis with OsYSL6 promoter-green fluorescent protein transgenic rice revealed that OsYSL6 was expressed in all cells of both the roots and shoots. Heterogolous expression of OsYSL6 in yeast showed transport activity for the Mn-nicotianamine complex but not for the Mn-mugineic acid complex. Taken together, our results suggest that OsYSL6 is a Mn-nicotianamine transporter that is required for the detoxification of excess Mn in rice.