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 (LA-ICP-MS) 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.

Diverse roles of the metal binding domains and transport mechanism of copper transporting P-type ATPases.

Copper transporting P-type (P1B-1-) ATPases are essential for cellular homeostasis. Nonetheless, the E1-E1P-E2P-E2 states mechanism of P1B-1-ATPases remains poorly understood. In particular, the role of the intrinsic metal binding domains (MBDs) is enigmatic. Here, four cryo-EM structures and molecular dynamics simulations of a P1B-1-ATPase are combined to reveal that in many eukaryotes the MBD immediately prior to the ATPase core, MBD-1, serves a structural role, remodeling the ion-uptake region. In contrast, the MBD prior to MBD-1, MBD-2, likely assists in copper delivery to the ATPase core. Invariant Tyr, Asn and Ser residues in the transmembrane domain assist in positioning sulfur-providing copper-binding amino acids, allowing for copper uptake, binding and release. As such, our findings unify previously conflicting data on the transport and regulation of P1B-1-ATPases. The results are critical for a fundamental understanding of cellular copper homeostasis and for comprehension of the molecular bases of P1B-1-disorders and ongoing clinical trials.

Enhancing fuel characteristics and combustion performance of cellulose-rich straws through CO2-assisted torrefaction.

Cellulose-rich straws of corn and rice were torrefied under carbon dioxide, and the fuel characteristics and combustion performance of the obtained biochar were investigated. A high severity resulted in surface collapse, greater pore volume, elimination of oxygen, elevated calorific value, and improved hydrophobicity in biochar. Following carbon dioxide torrefaction, the cellulose content in solid biochar experienced a slight decrease when the temperature was raised to 220 °C for longer residence durations. At 300 °C, the cellulose content in the biochar was nearly eliminated, while the relative proportion of non-sugar organic matter in corn stover and rice straw increased to 87.40 % and 77.27 %, respectively. The maximum calorific values for biochar from corn and rice straws were 22.38 ± 0.03 MJ/kg and 18.72 ± 0.05 MJ/kg. The comprehensive combustion indexes of rice and corn straw samples decreased to 1.06 × 10-7 and 1.31 × 10-7 after torrefaction at 300 °C, respectively. In addition, the initial decomposition temperatures increased by 38 °C and 45 °C, while the ultimate combustion temperatures rose by 13 °C and 16 °C for corn and rice straws, respectively. These results imply an extended combustion timeframe for the torrefied samples.

Metal Transport Systems in Plants.

Plants take up metals, including the 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. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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.

Dissecting the promotional effect of zinc on cadmium translocation from roots to shoots in rice.

It is often expected that Zn decreases Cd accumulation in plants due to competition for the same transporters. Here, we found that increasing Zn supply markedly increased the root-to-shoot translocation of Cd in rice. RNA sequencing showed that high Zn up-regulated expression of genes involved in glutathione biosynthesis and metabolism and the Zn/Cd transporter gene OsHMA2, but down-regulated expression of genes related to Zn uptake. Knockout of the iron or Zn transporter genes OsIRT1, OsIRT2, or OsZIP9 did not affect the Zn promotional effect on Cd translocation. Knockout of the manganese/Cd transporter gene OsNRAMP5 greatly reduced Cd uptake but did not affect the Zn promotional effect. Variation in the tonoplast transporter gene OsHMA3 affected Cd translocation but did not change the Zn promotional effect. Knockout of the Zn/Cd transporter gene OsHMA2 not only decreased Cd and Zn translocation, but also abolished the Zn promotional effect. Increased expression of OsHMA2 under high Zn conditions supports the hypothesis that this transporter participates in the promotional effect of Zn on Cd translocation. The results also show that OsIRT1, OsIRT2, and OsZIP9 made only small contributions to Cd uptake under low Zn conditions but not under high Zn conditions, whereas the dominant role of OsNRAMP5 in Cd uptake diminished under low Zn conditions.

Knockout of a gene encoding a Gγ protein boosts alkaline tolerance in cereal crops.

Sorghum is highly tolerant to alkaline stress, but the underlying mechanisms are not well understood. Here, based on genotypic difference in alkaline tolerance of sorghum, it was found that AT1 (Alkaline tolerance 1) encoding a G protein is involved in alkaline tolerance through negatively modulating the phosphorylation level of PIP2, an aquaporin with transport activity for H2O2. Knockout of AT1 releases its inhibition of PIP2, thereby resulting in an increased transport of H2O2 from the cytosol into the apoplast, subsequently boosting alkaline tolerance.

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.

ART1 and putrescine contribute to rice aluminum resistance via OsMYB30 in cell wall modification.

Cell wall is the first physical barrier to aluminum (Al) toxicity. Modification of cell wall properties to change its binding capacity to Al is one of the major strategies for plant Al resistance; nevertheless, how it is regulated in rice remains largely unknown. In this study, we show that exogenous application of putrescines (Put) could significantly restore the Al resistance of art1, a rice mutant lacking the central regulator Al RESISTANCE TRANSCRIPTION FACTOR 1 (ART1), and reduce its Al accumulation particularly in the cell wall of root tips. Based on RNA-sequencing, yeast-one-hybrid and electrophoresis mobility shift assays, we identified an R2R3 MYB transcription factor OsMYB30 as the novel target in both ART1-dependent and Put-promoted Al resistance. Furthermore, transient dual-luciferase assay showed that ART1 directly inhibited the expression of OsMYB30, and in turn repressed Os4CL5-dependent 4-coumaric acid accumulation, hence reducing the Al-binding capacity of cell wall and enhancing Al resistance. Additionally, Put repressed OsMYB30 expression by eliminating Al-induced H2 O2 accumulation, while exogenous H2 O2 promoted OsMYB30 expression. We concluded that ART1 confers Put-promoted Al resistance via repression of OsMYB30-regulated modification of cell wall properties in rice.

The ferroxidases LPR1 and LPR2 control iron translocation in the xylem of Arabidopsis plants.

Iron (Fe) deficiency is common in agricultural crops and affects millions of people worldwide. Translocation of Fe in the xylem is a key step for Fe distribution in plants. The mechanism controlling this process remains largely unknown. Here, we report that two Arabidopsis ferroxidases, LPR1 and LPR2, play a crucial and redundant role in controlling Fe translocation in the xylem. LPR1 and LPR2 are mainly localized in the cell walls of xylem vessels and the surrounding cells in roots, leaves, and stems. Knockout of both LPR1 and LPR2 increased the proportion of Fe(II) in the xylem sap, and caused Fe deposition along the vascular bundles especially in the petioles and main veins of leaves, which was alleviated by blocking blue light. The lpr1 lpr2 double mutant displayed constitutive expression of Fe deficiency response genes and overaccumulation of Fe in the roots and mature leaves under Fe-sufficient supply, but Fe deficiency chlorosis in the new leaves and inflorescences under low Fe supply. Moreover, the lpr1 lpr2 double mutant showed lower Fe concentrations in the xylem and phloem saps, and impaired 57Fe translocation along the xylem. In vitro assays showed that Fe(III)-citrate, the main form of Fe in xylem sap, is easily photoreduced to Fe(II)-citrate, which is unstable and prone to adsorption by cell walls. Taken together, these results indicate that LPR1 and LPR2 are required to oxidize Fe(II) and maintain Fe(III)-citrate stability and mobility during xylem translocation against photoreduction. Our study not only uncovers an essential physiological role of LPR1 and LPR2 but also reveals a new mechanism by which plants maintain Fe mobility during long-distance translocation in the xylem.

Plant Hormone and Inorganic Ion Concentrations in the Xylem Exudate of Grafted Plants Depend on the Scion-Rootstock Combination.

In grafted plants, inorganic ions and plant hormones in the xylem exudate transported from the rootstock to the scion directly or indirectly affect the scion, thereby improving the traits. Therefore, the concentration of these components in the xylem exudate of grafted plants may be an indicator for rootstock selection. On the other hand, few reports have presented a comprehensive analysis of substances transferred from the rootstock to the scion in plants grafted onto different rootstocks, primarily commercial cultivars. In this study, we measured inorganic ions and plant hormones in the xylem exudate from the rootstock to the scion in various grafted plants of tomato and eggplant. The results revealed that the concentrations of inorganic ions and plant hormones in the xylem exudate significantly differed depending on the type of rootstock. In addition, we confirmed the concentration of the inorganic ions and plant hormones in the xylem exudate of plants grafted onto the same tomato rootstock cultivars as rootstock with tomato or eggplant as the scions. As a result, the concentrations of inorganic ions and plant hormones in the xylem exudate were significantly different in the grafted plants with eggplant compared with tomato as the scion. These results suggest that signals from the scion (shoot) control the inorganic ions and plant hormones transported from the rootstock (root).

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 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.

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.