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.

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.

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.

DNA hypomethylation in tetraploid rice potentiates stress-responsive gene expression for salt tolerance.

Polyploidy is a prominent feature for genome evolution in many animals and all flowering plants. Plant polyploids often show enhanced fitness in diverse and extreme environments, but the molecular basis for this remains elusive. Soil salinity presents challenges for many plants including agricultural crops. Here we report that salt tolerance is enhanced in tetraploid rice through lower sodium uptake and correlates with epigenetic regulation of jasmonic acid (JA)-related genes. Polyploidy induces DNA hypomethylation and potentiates genomic loci coexistent with many stress-responsive genes, which are generally associated with proximal transposable elements (TEs). Under salt stress, the stress-responsive genes including those in the JA pathway are more rapidly induced and expressed at higher levels in tetraploid than in diploid rice, which is concurrent with increased jasmonoyl isoleucine (JA-Ile) content and JA signaling to confer stress tolerance. After stress, elevated expression of stress-responsive genes in tetraploid rice can induce hypermethylation and suppression of the TEs adjacent to stress-responsive genes. These induced responses are reproducible in a recurring round of salt stress and shared between two japonica tetraploid rice lines. The data collectively suggest a feedback relationship between polyploidy-induced hypomethylation in rapid and strong stress response and stress-induced hypermethylation to repress proximal TEs and/or TE-associated stress-responsive genes. This feedback regulation may provide a molecular basis for selection to enhance adaptation of polyploid plants and crops during evolution and domestication.

Plant Nutrition for Human Nutrition: Hints from Rice Research and Future Perspectives.

Both plants and humans require mineral elements for their healthy growth and development. Mineral elements in the soil are taken up by the plant roots and transported to the edible parts for human consumption through various different transporters. An ideal future crop for human health should be rich in essential mineral elements but with less toxic elements in the edible parts. However, due to the great difference in the numbers and amounts of mineral elements required between plants and humans, it is a challenge to balance plant growth and nutrient requirement for humans. In this article, we mainly focus on the transport system of mineral elements from soil to grain in rice, a staple food for half of the world's population, and discuss recent progress on the underlying genetic and physiological mechanisms. Examples are given for silicon, zinc, and iron essential/beneficial for both plants and humans, selenium and iodine only essential for humans, and toxic cadmium and arsenic for all organisms. Manipulation of some transporters for these elements, especially those localized in the node for allocation of mineral elements to the grain, has been successful in generating rice with higher density and bioavailability of essential elements but with less accumulation of toxic elements. We provide our perspectives toward breeding future crops for human health.

Plastic transport systems of rice for mineral elements in response to diverse soil environmental changes.

Climate change will increase frequency of drought and flooding, which threaten global crop productivity and food security. Rice (Oryza sativa) is unique in that it is able to grow in both flooded and upland conditions, which have large differences in the concentrations and chemical forms of mineral elements available to plants. To comprehensively understand the mechanisms of rice for coping with different water status, we performed ionomics and transcriptomics analysis of the roots, nodes and leaves of rice grown in flooded and upland conditions. Focusing the analysis on genes encoding proteins involved in transport functions for mineral elements, it was found that, although rice plants maintained similar levels of each element in the shoots for optimal growth, different transporters for mineral elements were utilised for nitrogen, iron, copper and zinc to deal with different soil water conditions. For example, under flooded conditions, rice roots take up nitrogen using transporters for both ammonium (OsAMT1/2) and nitrate (OsNPF2.4, OsNRT1.1A and OsNRT2.3), whereas under upland conditions, nitrogen uptake is mediated by different nitrate transporters (OsNRT1.1B and OsNRT1.5A). This study shows that rice possesses plastic transport systems for mineral elements in response to different water conditions (upland and flooding).

ARSENATE INDUCED CHLOROSIS 1/ TRANSLOCON AT THE OUTER ENVOLOPE MEMBRANE OF CHLOROPLASTS 132 Protects Chloroplasts from Arsenic Toxicity.

Arsenic (As) is highly toxic to plants and detoxified primarily through complexation with phytochelatins (PCs) and other thiol compounds. To understand the mechanisms of As toxicity and detoxification beyond PCs, we isolated an arsenate-sensitive mutant of Arabidopsis (Arabidopsis thaliana), arsenate induced chlorosis1 (aic1), in the background of the PC synthase-defective mutant cadmium-sensitive1-3 (cad1-3). Under arsenate stress, aic1 cad1-3 showed larger decreases in chlorophyll content and the number and size of chloroplasts than cad1-3 and a severely distorted chloroplast structure. The aic1 single mutant also was more sensitive to arsenate than the wild type (Columbia-0). As concentrations in the roots, shoots, and chloroplasts were similar between aic1 cad1-3 and cad1-3 Using genome resequencing and complementation, TRANSLOCON AT THE OUTER ENVOLOPE MEMBRANE OF CHLOROPLAST132 (TOC132) was identified as the mutant gene, which encodes a translocon protein involved in the import of preproteins from the cytoplasm into the chloroplasts. Proteomic analysis showed that the proteome of aic1 cad1-3 chloroplasts was more affected by arsenate stress than that of cad1-3 A number of proteins related to chloroplast ribosomes, photosynthesis, compound synthesis, and thioredoxin systems were less abundant in aic1 cad1-3 than in cad1-3 under arsenate stress. Our results indicate that chloroplasts are a sensitive target of As toxicity and that AIC1/Toc132 plays an important role in protecting chloroplasts from As toxicity.

OsWRKY28 Regulates Phosphate and Arsenate Accumulation, Root System Architecture and Fertility in Rice.

WRKYs are transcriptional factors involved in stress tolerance and development of plants. In the present study, we characterized OsWRKY28, a group IIa WRKY gene, in rice, because its expression was found to be upregulated by arsenate exposure in previous transcriptomic studies. Subcellular localization using YFP-OsWRKY28 fusion protein showed that the protein was localized in the nuclei. Transgenic rice plants expressing pOsWRKY28::GUS suggested that the gene was expressed in various tissues in the whole plant, with a strong expression in the root tips, lateral roots and reproductive organs. The expression of OsWRKY28 was markedly induced by arsenate and other oxidative stresses. In a hydroponic experiment, loss-of-function mutation in OsWRKY28 resulted in lower accumulation of arsenate and phosphate concentration in the shoots. The mutants showed altered root system architecture, with fewer lateral roots and shorter total root length than wild-type plants. In a soil pot experiment, the mutants produced lower grain yield than wild-type because of reduced fertility and smaller effective tiller numbers. Transcriptomic profiling using RNA-seq showed altered expression in the mutant of genes involved in the biosynthesis of phytohormones, especially jasmonic acid (JA). Exogenous JA treatments mimicked the phenotypes of the oswrky28 mutants with inhibited root elongation and decreased arsenate/phosphate translocation. Our results suggested that OsWRKY28 affected arsenate/phosphate accumulation, root development at the seedling stage and fertility at the reproductive stage possibly by influencing homeostasis of JA or other phytohormones.

The role of OsPT8 in arsenate uptake and varietal difference in arsenate tolerance in rice.

Arsenic (As) contamination in paddy soil can cause phytotoxicity and elevated As accumulation in rice grain. Rice varieties vary in As uptake and tolerance, but the underlying mechanisms remain unclear. In this study, the aus variety Kasalath was found to be more tolerant to arsenate [As(V)] than the japonica variety Nipponbare, but the two varieties showed similar arsenite [As(III)] tolerance. Nipponbare took up more phosphate (Pi) and As(V) than Kasalath. The expression of genes for Pi transporters or Pi homeostasis regulation was quantified. Nipponbare showed 2- to 3-fold higher expression of the Pi transporter genes OsPT2 and OsPT8 than Kasalath. Two ospt8 mutants were isolated from the Kasalath background and compared with an ospt8 mutant in the Nipponbare background. Mutation in OsPT8 in both backgrounds decreased As(V) uptake by 33-57%, increased As(V) tolerance assayed by root elongation by >100-fold, and abolished the varietal differences in As(V) uptake and tolerance. The results show that OsPT8 plays a key role in As(V) uptake and that As(V) uptake mediated by OsPT8 exerts a profound toxic effect on root elongation. The results also suggest that differential OsPT8 expression explains the varietal differences in As(V) uptake and tolerance between Kasalath and Nipponbare.

A loss-of-function allele of OsHMA3 associated with high cadmium accumulation in shoots and grain of Japonica rice cultivars.

Excessive cadmium (Cd) accumulation in rice poses a risk to food safety. OsHMA3 plays an important role in restricting Cd translocation from roots to shoots. A non-functional allele of OsHMA3 has been reported in some Indica rice cultivars with high Cd accumulation, but it is not known if OsHMA3 allelic variation is associated with Cd accumulation in Japonica cultivars. In this study, we identified a Japonica cultivar with consistently high Cd accumulation in shoots and grain in both field and greenhouse experiments. The cultivar possesses an OsHMA3 allele with a predicted amino acid mutation at the 380(th) position from Ser to Arg. The haplotype had no Cd transport activity when the gene was expressed in yeast, and the allele did not complement a known nonfunctional allele of OsHMA3 in F1 test. The allele is present only in temperate Japonica cultivars among diversity panels of 1483 rice cultivars. Different cultivars possessing this allele showed greatly increased root-to-shoot Cd translocation and a shift in root Cd speciation from Cd-S to Cd-O bonding determined by synchrotron X-ray absorption spectroscopy. Our study has identified a new loss-of-function allele of OsHMA3 in Japonica rice cultivars leading to high Cd accumulation in shoots and grain.