Uptake and Accumulation of Cobalt Is Mediated by OsNramp5 in Rice.

Cobalt (Co) contamination in soils potentially affects human health through the food chain. Although rice (Oryza sativa) as a staple food is a major dietary source of human Co intake, it is poorly understood how Co is taken up by the roots and accumulated in rice grain. In this study, we physiologically characterized Co accumulation and identified the transporter for Co2+ uptake in rice. A dose-dependent experiment showed that Co mainly accumulated in rice roots. Further analysis with LA-ICP-MS showed Co deposited in most tissue of the roots, including exodermis, endodermis and stele region. Co accumulation analysis using mutants defective in divalent cation uptake showed that Co2+ uptake in rice is mediated by the Mn2+/Cd2+/Pb2+ transporter OsNramp5, rather than OsIRT1 for Fe2+ and OsZIP9 for Zn2+. Knockout of OsNramp5 enhanced tolerance to Co toxicity. Heterologous expression of OsNramp5 showed transport activity for Co2+ in Saccharomyces cerevisiae. Co2+ uptake was inhibited by either Mn2+ or Cd2+ supply. At the reproductive stage, the Co concentration in the straw and grains of the OsNramp5 knockout lines was decreased by 41%-48% and 28%-36%, respectively, compared with that of the wild-type rice. The expression level of OsNramp5 in the roots was not affected by Co2+. Taken together, our results indicate that OsNramp5 is a major transporter for Co2+ uptake in rice, which ultimately mediates Co accumulation in the grains.

OsHAK4 functions in retrieving sodium from the phloem at the reproductive stage of rice.

Soil salinity significantly limits rice productivity, but it is poorly understood how excess sodium (Na+) is delivered to the grains at the reproductive stage. Here, we functionally characterized OsHAK4, a member of the clade IV HAK/KUP/KT transporter subfamily in rice. OsHAK4 was localized to the plasma membrane and exhibited influx transport activity for Na+, but not for K+. Analysis of organ- and growth stage-dependent expression patterns showed that very low expression levels of OsHAK4 were detected at the vegetative growth stage, but its high expression in uppermost node I, peduncle, and rachis was found at the reproductive stage. Immunostaining indicated OsHAK4 localization in the phloem region of node I, peduncle, and rachis. Knockout of OsHAK4 did not affect the growth and Na+ accumulation at the vegetative stage. However, at the reproductive stage, the hak4 mutants accumulated higher Na+ in the peduncle, rachis, husk, and brown rice compared to the wild-type rice. Element imaging revealed higher Na+ accumulation at the phloem region of the peduncle in the mutants. These results indicate that OsHAK4 plays a crucial role in retrieving Na+ from the phloem in the upper nodes, peduncle, and rachis, thereby preventing Na+ distribution to the grains at the reproductive stage of rice.

Tissue-specific deposition, speciation and transport of antimony in rice.

Rice (Oryza sativa) as a staple food is a potential intake source of antimony (Sb), a toxic metalloid. However, how rice accumulates this element is still poorly understood. Here, we investigated tissue-specific deposition, speciation, and transport of Sb in rice. We found that Sb(III) is the preferential form of Sb uptake in rice, but most Sb accumulates in the roots, resulting in a very low root-to-shoot translocation (less than 2%). Analysis of Sb deposition with laser ablation-inductively coupled plasma-mass spectrometry showed that most Sb deposits at the root exodermis. Furthermore, we found that Sb is mainly present as Sb(III) in the root cell sap after uptake. Further characterization showed that Sb(III) uptake is mediated by Low silicon rice 1 (Lsi1), a Si permeable transporter. Lsi1 showed transport activity for Sb(III) rather than Sb(V) in yeast (Saccharomyces cerevisiae). Knockout of Lsi1 resulted in a significant decrease in Sb accumulation in both roots and shoots. Sb concentration in the root cell sap of two independent lsi1 mutants decreased to less than 3% of that in wild-type rice, indicating that Lsi1 is a major transporter for Sb(III) uptake. Knockout of Lsi1 also enhanced rice tolerance to Sb toxicity. However, knockout of Si efflux transporter genes, including Lsi2 and Lsi3, did not affect Sb accumulation. Taken together, our results showed that Sb(III) is taken up by Lsi1 localized at the root exodermis and is deposited at this cell layer due to lack of Sb efflux transporters in rice.

An oligo peptide transporter family member, OsOPT7, mediates xylem unloading of Fe for its preferential distribution in rice.

Iron (Fe) needs to be delivered to different organs and tissues of above-ground parts for playing its multiple physiological functions once it is taken up by the roots. However, the mechanisms underlying Fe distribution are poorly understood. We functionally characterized OsOPT7, a member of oligo peptide transporter family in terms of expression patterns, localization, transport activity and phenotypic analysis of knockdown lines. OsOPT7 was highly expressed in the nodes, especially in the uppermost node I, and its expression was upregulated by Fe-deficiency. OsOPT7 transports ferrous iron into the cells coupled with proton. Immunostaining revealed that OsOPT7 is mainly localized in the xylem parenchyma cells of the enlarged vascular bundles in the nodes and vascular tissues in the leaves. Knockdown of OsOPT7 did not affect the Fe uptake, but altered Fe distribution; less Fe was distributed to the new leaf, upper nodes and developing panicle, but more Fe was distributed to the old leaves. Furthermore, knockdown of OsOPT7 also resulted in less Fe distribution to the leaf sheath, but more Fe to the leaf blade. Taken together, OsOPT7 is involved in the xylem unloading of Fe for both long-distance distribution to the developing organs and local distribution within the leaf in rice.

Metal Transport Systems in Plants.

Plants take up metals, including essential micronutrients [iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn)] and the toxic heavy metal cadmium (Cd), from soil and accumulate these metals in their edible parts, which are direct and indirect intake sources for humans. Multiple transporters belonging to different families are required to transport a metal from the soil to different organs and tissues, but only a few of them have been fully functionally characterized. The transport systems (the transporters required for uptake, translocation, distribution, redistribution, and their regulation) differ with metals and plant species, depending on the physiological roles, requirements of each metal, and anatomies of different organs and tissues. To maintain metal homeostasis in response to spatiotemporal fluctuations of metals in soil, plants have developed sophisticated and tightly regulated mechanisms through the regulation of transporters at the transcriptional and/or posttranscriptional levels. The manipulation of some transporters has succeeded in generating crops rich in essential metals but low in Cd accumulation. A better understanding of metal transport systems will contribute to better and safer crop production.

Local distribution of manganese to leaf sheath is mediated by OsNramp5 in rice.

To play essential roles of manganese (Mn) in plant growth and development, it needs to be transported to different organs and tissues after uptake. However, the molecular mechanisms underlying Mn distribution between different tissues are poorly understood. We functionally characterized a member of rice natural resistance-associated macrophage protein (NRAMP) family, OsNramp5 in terms of its tissue specificity of gene expression, cell-specificity of protein localization, phenotypic analysis of leaf growth and response to Mn fluctuations. OsNramp5 is highly expressed in the leaf sheath. Immunostaining revealed that OsNramp5 is polarly localized at the proximal side of xylem parenchyma cells of the leaf sheath. Both the gene expression and protein abundance of OsNramp5 are unaffected by different Mn concentrations. Knockout of OsNramp5 decreased the distribution of Mn to the leaf sheath, but increased the distribution to the leaf blade at both low and high Mn supplies, resulting in reduced growth of leaf sheath. Furthermore, expression of OsNramp5 under the control of root-specific promoter in osnramp5 mutant complemented Mn uptake, but could not complement Mn distribution to the leaf sheath. These results indicate that OsNramp5 expressed in the leaf sheath plays an important role in unloading Mn from the xylem for the local distribution in rice.

Knockout of a rice K5.2 gene increases Ca accumulation in the grain.

Rice is a staple food for half of the world's population, but it is a poor dietary source of calcium (Ca) due to the low concentration. It is an important issue to boost Ca concentration in this grain to improve Ca deficiency risk, but the mechanisms underlying Ca accumulation are poorly understood. Here, we obtained a rice (Oryza sativa) mutant with high shoot Ca accumulation. The mutant exhibited 26%-53% higher Ca in shoots than did wild-type rice (WT) at different Ca supplies. Ca concentration in the xylem sap was 36% higher in the mutant than in the WT. There was no difference in agronomic traits between the WT and mutant, but the mutant showed 25% higher Ca in the polished grain compared with the WT. Map-based cloning combined with a complementation test revealed that the mutant phenotype was caused by an 18-bp deletion of a gene, OsK5.2, belonging to the Shaker-like K+ channel family. OsK5.2 was highly expressed in the mature region of the roots and its expression in the roots was not affected by Ca levels, but upregulated by low K. Immunostaining showed that OsK5.2 was mainly expressed in the pericycle of the roots. Taken together, our results revealed a novel role for OsK5.2 in Ca translocation in rice, and will be a good target for Ca biofortification in rice.

A silicon transporter gene required for healthy growth of rice on land.

Silicon (Si) is the most abundant mineral element in the earth's crust. Some plants actively accumulate Si as amorphous silica (phytoliths), which can protect plants from stresses. Here, we report a gene (SIET4) that is required for the proper accumulation and cell-specific deposition of Si in rice and show that it is essential for normal growth. SIET4 is constitutively expressed in leaves and encodes a Si transporter. SlET4 polarly localizes at the distal side of epidermal cells and cells surrounding the bulliform cells (motor cells) of the leaf blade, where Si is deposited. Knockout of SIET4 leads to the death of rice in the presence but not absence of Si. Further analysis shows that SIET4 knockout induces abnormal Si deposition in mesophyll cells and the induction of hundreds of genes related to various stress responses. These results indicate that SIET4 is required for the proper export of Si from leaf cells to the leaf surface and for the healthy growth of rice on land.

A vacuolar transporter plays important roles in zinc and cadmium accumulation in rice grain.

Rice grain is a poor dietary source of zinc (Zn) but the primary source of cadmium (Cd) for humans; however, the molecular mechanisms for their accumulation in rice grain remain incompletely understood. This study functionally characterized a tonoplast-localized transporter, OsMTP1. OsMTP1 was preferentially expressed in the roots, aleurone layer, and embryo of seeds. OsMTP1 knockout decreased Zn concentration in the root cell sap, roots, aleurone layer and embryo, and subsequently increased Zn concentration in shoots and polished rice (endosperm) without yield penalty. OsMTP1 haplotype analysis revealed elite alleles associated with increased Zn level in polished rice, mostly because of the decreased OsMTP1 transcripts. OsMTP1 expression in yeast enhanced Zn tolerance but did not affect that of Cd. While OsMTP1 knockout resulted in decreased uptake, translocation and accumulation of Cd in plant and rice grain, which could be attributed to the indirect effects of altered Zn accumulation. Our results suggest that rice OsMTP1 primarily functions as a tonoplast-localized transporter for sequestrating Zn into vacuole. OsMTP1 knockout elevated Zn concentration but prevented Cd deposition in polished rice without yield penalty. Thus, OsMTP1 is a candidate gene for enhancing Zn level and reducing Cd level in rice grains.

Polar localization of a rice silicon transporter requires isoleucine at both C- and N-termini as well as positively charged residues.

Silicon (Si) is important for stable and high yields in rice (Oryza sativa), a typical Si hyperaccumulator. The high Si accumulation is achieved by the cooperation of 2 Si transporters, LOW SILICON 1 (OsLsi1) and OsLsi2, which are polarly localized in cells of the root exodermis and endodermis. However, the mechanism underlying their polar localization is unknown. Here, we identified amino acid residues critical for the polar localization of OsLsi1. Deletion of both N- and C-terminal regions resulted in the loss of its polar localization. Furthermore, the deletion of the C-terminus inhibited its trafficking from the endoplasmic reticulum to the plasma membrane. Detailed site-directed mutagenesis analysis showed that Ile18 at the N-terminal region and Ile285 at the C-terminal region were essential for the polar localization of OsLsi1. Moreover, a cluster of positively charged residues at the C-terminal region is also required for polar localization. Phosphorylation and Lys modifications of OsLsi1 are unlikely to be involved in its polar localization. Finally, we showed that the polar localization of OsLsi1 is required for the efficient uptake of Si. Our study not only identified critical residues required for the polar localization of OsLsi1, but also provided experimental evidence for the importance of transporter polarity for efficient nutrient uptake.

FE UPTAKE-INDUCING PEPTIDE1 maintains Fe translocation by controlling Fe deficiency response genes in the vascular tissue of Arabidopsis.

FE UPTAKE-INDUCING PEPTIDE1 (FEP1), also named IRON MAN3 (IMA3) is a short peptide involved in the iron deficiency response in Arabidopsis thaliana. Recent studies uncovered its molecular function, but its physiological function in the systemic Fe response is not fully understood. To explore the physiological function of FEP1 in iron homoeostasis, we performed a transcriptome analysis using the FEP1 loss-of-function mutant fep1-1 and a transgenic line with oestrogen-inducible expression of FEP1. We determined that FEP1 specifically regulates several iron deficiency-responsive genes, indicating that FEP1 participates in iron translocation rather than iron uptake in roots. The iron concentration in xylem sap under iron-deficient conditions was lower in the fep1-1 mutant and higher in FEP1-induced transgenic plants compared with the wild type (WT). Perls staining revealed a greater accumulation of iron in the cortex of fep1-1 roots than in the WT root cortex, although total iron levels in roots were comparable in the two genotypes. Moreover, the fep1-1 mutation partially suppressed the iron overaccumulation phenotype in the leaves of the oligopeptide transporter3-2 (opt3-2) mutant. These data suggest that FEP1 plays a pivotal role in iron movement and in maintaining the iron quota in vascular tissues in Arabidopsis.

A Golgi-localized glycosyltransferase, OsGT14;1, is required for growth of both roots and shoots in rice.

Glycosyltransferases (GTs) form a large family in plants and are important enzymes for the synthesis of various polysaccharides, but only a few members have been functionally characterized. Here, through mutant screening with gene mapping, we found that an Oryza sativa (rice) mutant with a short-root phenotype was caused by a frame-shift mutation of a gene (OsGT14;1) belonging to the glycosyltransferase gene family 14. Further analysis indicated that the mutant also had a brittle culm and produced lower grain yield compared with wild-type rice, but the roots showed similar root structure and function in terms of the uptake of mineral nutrients. OsGT14;1 was broadly expressed in all organs throughout the entire growth period, with a relatively high expression in the roots, stems, node I and husk. Furthermore, OsGT14;1 was expressed in all tissues of these organs. Subcellular observation revealed that OsGT14;1 encoded a Golgi-localized protein. Mutation of OsGT14;1 resulted in decreased cellulose content and increased hemicellulose, but did not alter pectin in the cell wall of roots and shoots. The knockout of OsGT14;1 did not affect the tolerance to toxic mineral elements, including Al, As, Cd and salt stress, but did increase the sensitivity to low pH. Taken together, OsGT14;1 located at the Golgi is required for growth of both roots and shoots in rice through affecting cellulose synthesis.

NRAMP6 and NRAMP1 cooperatively regulate root growth and manganese translocation under manganese deficiency in Arabidopsis.

The essential micronutrient manganese (Mn) in plants regulates multiple biological processes including photosynthesis and oxidative stress. Some Natural Resistance-Associated Macrophage Proteins (NRAMPs) have been reported to play critical roles in Mn uptake and reutilization in low Mn conditions. NRAMP6 was demonstrated to regulate cadmium tolerance and iron utilization in Arabidopsis. Nevertheless, it is unclear whether NRAMP6 plays a role in Mn nutrition. Here, we report that NRAMP6 cooperates with NRAMP1 in Mn utilization. Mutation of NRAMP6 in nramp1 but not in a wild-type background reduces root growth and Mn translocation from the roots to shoots under Mn deficient conditions. Grafting experiments revealed that NRAMP6 expression in both the roots and shoots is required for root growth and Mn translocation under Mn deficiency. We also showed that NRAMP1 could replace NRAMP6 to sustain root growth under Mn deficiency, but not vice versa. Mn deficiency does not affect the transcript level of NRAMP6, but is able to increase and decrease the protein accumulation of NRAMP6 in roots and shoots, respectively. Furthermore, NRAMP6 can be localized to both the plasma membrane and endomembranes including the endoplasmic reticulum, and Mn deficiency enhances the localization of NRAMP6 to the plasma membrane in Arabidopsis plants. NRAMP6 could rescue the defective growth of the yeast mutant Δsmf2, which is deficient in endomembrane Mn transport. Our results reveal the important role of NRAMP6 in Mn nutrition and in the long-distance signaling between the roots and shoots under Mn deficient conditions.

Effects of polygalacturonase overexpression on pectin distribution in the elongation zones of roots under aluminium stress.

The roots of many plant species contain large amounts of pectin and it contributes to the formation of the rhizosphere. In the present study, the relationship between the root-tip pectin content and aluminium (Al) tolerance in wild-type (WT) and demethylesterified pectin degradation enzyme gene overexpressor (OsPG2-FOX) rice lines was compared. OsPG2-FOX rice showed reduced pectin content in roots, even under control conditions; Al treatment reduced root elongation and the pectin content in the root elongation zone. Wild-type rice showed more pectin accumulation in the root elongation zone after Al treatment. Relative to WT rice, OsPG2-FOX rice showed more Al accumulation in the root elongation zone. These results indicate that the amount of pectin influences Al tolerance and that the distribution of pectin in the root elongation zone inhibits Al accumulation in rice roots. Pectin accumulation in cell walls in the root elongation zone may play a role in protecting rice plants from the Al-induced inhibition of root elongation by regulating pectin distribution.

Cell-Type-Dependent but CME-Independent Polar Localization of Silicon Transporters in Rice.

Silicon (Si) is an important nutrient required for sustainable and high production of rice and its uptake is mediated by a pair of influx (OsLsi1)-efflux (OsLsi2) transporters showing polar localization. However, the mechanisms underlying their polarity are unknown. Here, we revealed that the polarity of the Si transporters depends on cell types. The polar localization of both OsLsi1 and OsLsi2 was not altered by Si supply, but their protein abundance was reduced. Double immunostaining showed that localization of OsLsi1 and OsLsi2 was separated at the edge of the lateral polar domain by Casparian strips in the endodermis, whereas they were slightly overlapped at the transversal side of the exodermis. When OsLsi1 was ectopically expressed in the shoots, it showed polar localization at the xylem parenchyma cells of the basal node and leaf sheath, but not at the phloem companion cells. Ectopic expression of non-polar Si transporters, barley HvLsi2 and maize ZmLsi2 in rice, resulted in their polar localization at the proximal side. The polar localization of OsLsi1 and OsLsi2 was not altered by inhibition of clathrin-mediated endocytosis (CME) by dominant-negative induction of dynamin-related protein1A and knockout of mu subunit of adaptor protein 2 complex, although the knockout mutants of OsAP2M gene showed dwarf phenotype. These results indicate that CME is not required for the polar localization of Si transporters. Taken together, our results indicate that CME-independent machinery controls the polar localization of Si transporters in exodermis, endodermis of root cells and xylem parenchyma cells.

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.

A pericycle-localized silicon transporter for efficient xylem loading in rice.

Rice is able to accumulate high concentrations of silicon (Si) in the shoots, and this ability is required for the mitigation of abiotic and biotic stresses. Although transporters for Si uptake have been identified, a transporter for the xylem loading of Si has not been found. We functionally characterized a Si transporter, OsLsi3, in terms of tissue-specific localization, knockout line phenotype and mathematic simulation. OsLsi3 was shown to be an efflux Si transporter. OsLsi3 was mainly expressed in the mature root region, and its expression was downregulated by Si. Immunostaining with a specific antibody showed that OsLsi3 was localized to the pericycle in the roots, without polarity. However, when it was expressed under the control of the OsLsi2 promoter, OsLsi3 became polarly localized to the proximal side of both the exodermis and endodermis. Knockout of this gene resulted in decreased Si uptake and concentration in the xylem sap under low Si supply, but not under high Si supply. Mathematical modeling showed that localization of OsLsi3 to the pericycle accounts for c. 30% of the total Si loading to the xylem under low Si concentrations. In summary, OsLsi3 was involved in the xylem loading of Si in rice roots, which is required for the efficient root-to-shoot translocation of Si.

Duplication of a manganese/cadmium transporter gene reduces cadmium accumulation in rice grain.

Global contamination of soils with toxic cadmium (Cd) is a serious health threat. Here we found that a tandem duplication of a gene encoding a manganese/Cd transporter, OsNramp5, was responsible for low-Cd accumulation in Pokkali, an old rice cultivar. This duplication doubled the expression of OsNramp5 gene but did not alter its spatial expression pattern and cellular localization. Higher expression of OsNramp5 increased uptake of Cd and Mn into the root cells but decreased Cd release to the xylem. Introgression of this allele into Koshihikari, an elite rice cultivar, through backcrossing significantly reduced Cd accumulation in the grain when cultivated in soil heavily contaminated with Cd but did not affect both grain yield and eating quality. This study not only reveals the molecular mechanism underlying low-Cd accumulation but also provides a useful target for breeding rice cultivars with low-Cd accumulation.

Zinc transport in rice: how to balance optimal plant requirements and human nutrition.

Zinc (Zn) is an essential micronutrient for both plants and animals, while its deficiency in crops and humans is a global problem that affects both crop productivity and human health. Since plants and humans differ in their Zn requirements, it is crucial to balance plant nutrition and human nutrition for Zn. In this review, we focus on the transport system of Zn from soil to grain in rice (Oryza sativa), which is a major dietary source of Zn for people subsiding on rice-based diets. We describe transporters belonging to the different families that are involved in the uptake, vacuolar sequestration, root-to-shoot translocation, and distribution of Zn, and discuss their mechanisms of regulation. We give examples for enhancing Zn accumulation and bioavailability in rice grains through the manipulation of genes that are highly expressed in the nodes, where Zn is deposited at high concentrations. Finally, we provide our perspectives on breeding rice cultivars with both increased tolerance to Zn-deficiency stress and high Zn density in the grains.

Boron uptake in rice is regulated post-translationally via a clathrin-independent pathway.

Uptake of boron (B) in rice (Oryza sativa) is mediated by the Low silicon rice 1 (OsLsi1) channel, belonging to the NOD26-like intrinsic protein III subgroup, and the efflux transporter B transporter 1 (OsBOR1). However, it is unknown how these transporters cooperate for B uptake and how they are regulated in response to B fluctuations. Here, we examined the response of these two transporters to environmental B changes at the transcriptional and posttranslational level. OsBOR1 showed polar localization at the proximal side of both the exodermis and endodermis of mature root region, forming an efficient uptake system with OsLsi1 polarly localized at the distal side of the same cell layers. Expression of OsBOR1 and OsLsi1 was unaffected by B deficiency and excess. However, although OsLsi1 protein did not respond to high B at the protein level, OsBOR1 was degraded in response to high B within hours, which was accompanied with a significant decrease of total B uptake. The high B-induced degradation of OsBOR1 was inhibited in the presence of MG-132, a proteasome inhibitor, without disturbance of the polar localization. In contrast, neither the high B-induced degradation of OsBOR1 nor its polarity was affected by induced expression of dominant-negative mutated dynamin-related protein 1A (OsDRP1AK47A) or knockout of the mu subunit (AP2M) of adaptor protein-2 complex, suggesting that clathrin-mediated endocytosis is not involved in OsBOR1 degradation and polar localization. These results indicate that, in contrast to Arabidopsis thaliana, rice has a distinct regulatory mechanism for B uptake through clathrin-independent degradation of OsBOR1 in response to high B.