Massively parallel single-cell genomics of microbiomes in rice paddies.

Plant growth-promoting microbes (PGPMs) have attracted increasing attention because they may be useful in increasing crop yield in a low-input and sustainable manner to ensure food security. Previous studies have attempted to understand the principles underlying the rhizosphere ecology and interactions between plants and PGPMs using ribosomal RNA sequencing, metagenomic sequencing, and genome-resolved metagenomics; however, these approaches do not provide comprehensive genomic information for individual species and do not facilitate detailed analyses of plant-microbe interactions. In the present study, we developed a pipeline to analyze the genomic diversity of the rice rhizosphere microbiome at single-cell resolution. We isolated microbial cells from paddy soil and determined their genomic sequences by using massively parallel whole-genome amplification in microfluidic-generated gel capsules. We successfully obtained 3,237 single-amplified genomes in a single experiment, and these genomic sequences provided insights into microbial functions in the paddy ecosystem. Our approach offers a promising platform for gaining novel insights into the roles of microbes in the rice rhizomicrobiome and to develop microbial technologies for improved and sustainable rice production.

Effects of improved sodium uptake ability on grain yields of rice plants under low potassium supply.

Sodium uptake is a factor that determines potassium use efficiency in plants as sodium can partially replace potassium in plant cells. Rice (Oryza sativa) roots usually exclude sodium but actively take it up when the plant is deficient in potassium. In rice roots, a sodium transporter OsHKT2;1 mediates active sodium uptake. We previously revealed that variation in the expression of OsHKT2;1 underlies the variation in sodium accumulation between a low-sodium-accumulating indica cultivar, IR64, and a high-sodium-accumulating japonica cultivar, Koshihikari. In the present study, we evaluated IR64 and its near-isogenic line IR64-K carrying OsHKT2;1 and neighboring genes inherited from Koshihikari for grain yield. IR64-K had a greater average grain yield and harvest index than IR64 in a pot culture experiment with three levels of potassium fertilizer. The differences were most significant under treatment without the potassium fertilizer. IR64-K also showed a slightly higher grain yield than IR64 when grown in a paddy field without applying the potassium fertilizer. These results suggest that enhanced sodium uptake ability improves the grain yield of rice plants under low-potassium-input conditions.

Overexpression of exogenous biuret hydrolase in rice plants confers tolerance to biuret toxicity.

Biuret, a common impurity in urea fertilizers, is toxic to plants, but little is known about the physiological mechanisms underlying its toxicity. Here, we analyzed biuret toxicity in rice (Oryza sativa) plants. We carried out uptake experiments using 15N-labelled biuret and demonstrated that biuret could reach sub millimolar concentrations in rice plants. We also demonstrated that the hydrolysis of biuret in plant cells could confer biuret tolerance to rice plants. This occurred because transgenic rice plants that overexpressed an exogenous biuret hydrolase cloned from a soil bacterium gained improved tolerance to biuret toxicity. Our results indicate that biuret toxicity is not an indirect toxicity caused by the presence of biuret outside the roots, and that biuret is not quickly metabolized in wild-type rice plants. Additionally, it was suggested that biuret was used as an additional nitrogen source in transgenic rice plants, because biuret hydrolase-overexpressing rice plants accumulated more biuret-derived N, as compared to wild-type rice.

Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl-glutathione synthetase.

Glutathione is a ubiquitous thiol tripeptide in land plants, and glutathione-like tripeptides can also be found in some plant species. Rice (Oryza sativa) plants synthesize hydroxymethyl-glutathione, in which the terminal glycine residue of glutathione is replaced by a serine residue; however, the biosynthetic pathway of hydroxymethyl-glutathione has not been identified. We isolated three rice glutathione synthetase homologs, designated OsGS1, OsGS2, and OsGS3, and found that knockdown of OsGS2 via RNA interference markedly decreased hydroxymethyl-glutathione concentration in rice plants. The in vitro enzyme assay, using purified recombinant protein, demonstrated that OsGS2 catalyzed the synthesis of hydroxymethyl-glutathione from γ-glutamylcysteine (γEC) and L-serine in an ATP-dependent manner. OsGS2 could also utilize glycine as a cosubstrate with γEC, but the enzyme-substrate affinity for L-serine was tenfold higher than that for glycine. These results indicate that OsGS2 codes for hydroxymethyl-glutathione synthetase.

A new monoclonal antibody against rhamnogalacturonan II and its application to immunocytochemical detection of rhamnogalacturonan II in Arabidopsis roots.

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. RG-II can be solubilized from cell walls as a borate-RG-II complex (B-RG-II), where two RG-II fragments are cross-linked via a borate diester linkage. Here, a rabbit monoclonal antibody against B-RG-II was prepared, which recognized both B-RG-II and RG-II monomers without borate ester-crosslinking. A pectic fragment with unknown structure was also recognized by the antibody, but neither homogalacturonan nor rhamnogalacturonan I was recognized. Immunoelectron microscopic analyses of Arabidopsis root tip cells were performed using this antibody. The signal was detected in developing cell plates and cell walls, which were denser in longitudinal walls than in transverse walls. These results coincide with our previous results obtained in suspension cultured tobacco cells, confirming that RG-II is present in cell plates at an early stage of their assembly. ABBREVIATIONS: B: boron; B-RG-II: borate-RG-II complex; ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; mBSA: methylated bovine serum albumin; PGA: polygalacturonic acid; PLL: poly-l-lysine; RG-I: rhamnogalacturonan I; RG-II: rhamnogalacturonan II.

Rice phytochelatin synthases OsPCS1 and OsPCS2 make different contributions to cadmium and arsenic tolerance.

Cadmium (Cd) and arsenic (As) pollution in paddy soil and their accumulation in rice (Oryza sativa) pose serious threats to human health. Rice internally detoxifies these toxic metal and metalloid to some extent, resulting in their accumulation within the edible parts. However, the mechanisms of Cd and As detoxification in rice have been poorly elucidated. Plants synthesize thiol-rich metal-chelating peptides, termed phytochelatins (PCs). We characterized rice PC synthase (PCS) and investigated its contribution to Cd and As tolerance in rice. We identified two PCS homolog genes, OsPCS1 and OsPCS2, in the rice genome. The expression of OsPCS1 was upregulated by As(III) stress in the roots but that of OsPCS2 was not significantly affected. The expression level of OsPCS2 was higher than that of OsPCS1 in the shoots and roots. Recombinant OsPCS1 and OsPCS2 proteins differed in their metal activation. OsPCS1 was more strongly activated by As(III) than by Cd; however, OsPCS2 was more strongly activated by Cd than by As(III). Genetically engineered plants having their OsPCS2 expression silenced via RNA interference (OsPCS2 RNAi) contained less PCs and more glutathione (GSH), a substrate of PC synthesis, than wild-type plants, although there was no significant difference in OsPCS1 RNAi plants. OsPCS2 RNAi plants were sensitive to As(III) stress, but Cd tolerance was little affected. On the other hand, treatment with buthionine sulfoximine, an inhibitor of GSH biosynthesis, significantly decreased Cd and As tolerance of rice seedlings. These findings indicate that OsPCS2 is a major isozyme controlling PC synthesis, and that PCs are important for As tolerance in rice. However, PC synthesis may make a smaller contribution to Cd tolerance in rice, and GSH plays crucial roles, not only as a substrate of PC synthesis.

Immunocytochemical detection of rhamnogalacturonan II on forming cell plates in cultured tobacco cells.

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. The RG-II region serves as the site of borate cross-linking within pectin, via which pectin macromolecules link together to form a gel. In this study, we examined whether RG-II is present in the cell plate, the precursor of primary cell walls that forms during cytokinesis. A structure inside dividing cells was labeled with a rabbit polyclonal anti-RG-II antibody and detected by immunofluorescence microscopy. An antibody against callose, a marker polysaccharide for the cell plate, also labeled the structure. In immunoelectron microscopy analyses using the anti-RG-II antibody, gold particles were distributed in electron-lucent vesicular structures that appeared to correspond to the forming cell plates in late anaphase cells. Together, these results suggest that RG-II is present in cell plates from the early phase of their assembly.

Genetic diversity of the wx flanking region in rice landraces in northern Laos.

A glutinous texture of endosperm is one of the important traits of rice (Oyza sativa L.). Northern Laos is known as a center of glutinous rice diversity. We genotyped INDEL, SSR and SNP markers in a sample of 297 rice landraces collected in northern Laos. These glutinous varieties were confirmed to share a loss-of-function mutation in Granule bound starch synthase I (Wx). INDEL markers revealed a high frequency of recombinant genotypes between indica and japonica. Principal component analysis using SSR genotypes of Wx flanking region revealed that glutinous indica landraces were scattered between non-glutinous indica and glutinous-japonica types. High ratios of heterozygosity were found especially in glutinous indica. Haplotype analysis using SNP markers around Wx locus revealed that glutinous indica landraces would have a few chromosome segments of glutinous japonica. Frequent recombinations were confirmed outside of this region in glutinous indica. This intricate genetic structure of landraces suggested that glutinous indica landraces in Laos were generated through repeated natural crossing with glutinous-japonica landraces and severe selection by local farmers.

Proton-dependent coniferin transport, a common major transport event in differentiating xylem tissue of woody plants.

Lignin biosynthesis is an essential physiological activity of vascular plants if they are to survive under various environmental stresses on land. The biosynthesis of lignin proceeds in the cell wall by polymerization of precursors; the initial step of lignin polymerization is the transportation of lignin monomers from the cytosol to the cell wall, which is critical for lignin formation. There has been much debate on the transported form of the lignin precursor, either as free monolignols or their glucosides. In this study, we performed biochemical analyses to characterize the membrane transport mechanism of lignin precursors using angiosperms, hybrid poplar (Populus sieboldii × Populus grandidentata) and poplar (Populus sieboldii), as well gymnosperms, Japanese cypress (Chamaecyparis obtusa) and pine (Pinus densiflora). Membrane vesicles prepared from differentiating xylem tissues showed clear ATP-dependent transport activity of coniferin, whereas less than 4% of the coniferin transport activity was seen for coniferyl alcohol. Bafilomycin A1 and proton gradient erasers markedly inhibited coniferin transport in hybrid poplar membrane vesicles; in contrast, vanadate had no effect. Cis-inhibition experiments suggested that this transport activity was specific for coniferin. Membrane fractionation of hybrid poplar microsomes demonstrated that transport activity was localized to the tonoplast- and endomembrane-rich fraction. Differentiating xylem of Japanese cypress exhibited almost identical transport properties, suggesting the involvement of a common endomembrane-associated proton/coniferin antiport mechanism in the lignifying tissues of woody plants, both angiosperms and gymnosperms.

Pathogenic diversity of Phytophthora sojae and breeding strategies to develop Phytophthora-resistant soybeans.

Phytophthora stem and root rot, caused by Phytophthora sojae, is one of the most destructive diseases of soybean [Glycine max (L.) Merr.], and the incidence of this disease has been increasing in several soybean-producing areas around the world. This presents serious limitations for soybean production, with yield losses from 4 to 100%. The most effective method to reduce damage would be to grow Phytophthora-resistant soybean cultivars, and two types of host resistance have been described. Race-specific resistance conditioned by single dominant Rps ("resistance to Phytophthora sojae") genes and quantitatively inherited partial resistance conferred by multiple genes could both provide protection from the pathogen. Molecular markers linked to Rps genes or quantitative trait loci (QTLs) underlying partial resistance have been identified on several molecular linkage groups corresponding to chromosomes. These markers can be used to screen for Phytophthora-resistant plants rapidly and efficiently, and to combine multiple resistance genes in the same background. This paper reviews what is currently known about pathogenic races of P. sojae in the USA and Japan, selection of sources of Rps genes or minor genes providing partial resistance, and the current state and future scope of breeding Phytophthora-resistant soybean cultivars.

Characterization of Arabidopsis CTP:3-deoxy-D-manno-2-octulosonate cytidylyltransferase (CMP-KDO synthetase), the enzyme that activates KDO during rhamnogalacturonan II biosynthesis.

In plant cells, boron (B) occurs predominantly as a borate ester associated with rhamnogalacturonan II (RG-II), but the function of this B-RG-II complex has yet to be investigated. 3-Deoxy-D-manno-2-octulosonic acid (KDO) is a specific component monosaccharide of RG-II. Mutant plants defective in KDO biosynthesis are expected to have altered RG-II structure, and would be useful for studying the physiological function of the B-RG-II complex. Here, we characterized Arabidopsis CTP:KDO cytidylyltransferase (CMP-KDO synthetase; CKS), the enzyme activating KDO as a nucleotide sugar prior to its incorporation into RG-II. Our analyses localized the Arabidopsis CKS protein to mitochondria. The Arabidopsis CKS gene occurs as a single-copy gene in the genome, and we could not obtain cks null mutants from T-DNA insertion lines. Analysis using +/cks heterozygotes in the quartet1 background demonstrated that the cks mutation rendered pollen infertile through the inhibition of pollen tube elongation. These results suggest that KDO is an indispensable component of RG-II, and that the complete B-RG-II complex is essential for the cell wall integrity of rapidly growing tissues.

Suppression of a NAC-like transcription factor gene improves boron-toxicity tolerance in rice.

We identified a gene responsible for tolerance to boron (B) toxicity in rice (Oryza sativa), named BORON EXCESS TOLERANT1. Using recombinant inbred lines derived from the B-toxicity-sensitive indica-ecotype cultivar IR36 and the tolerant japonica-ecotype cultivar Nekken 1, the region responsible for tolerance to B toxicity was narrowed to 49 kb on chromosome 4. Eight genes are annotated in this region. The DNA sequence in this region was compared between the B-toxicity-sensitive japonica cultivar Wataribune and the B-toxicity-tolerant japonica cultivar Nipponbare by eco-TILLING analysis and revealed a one-base insertion mutation in the open reading frame sequence of the gene Os04g0477300. The gene encodes a NAC (NAM, ATAF, and CUC)-like transcription factor and the function of the transcript is abolished in B-toxicity-tolerant cultivars. Transgenic plants in which the expression of Os04g0477300 is abolished by RNA interference gain tolerance to B toxicity.

Boron nutrition of cultured tobacco BY-2 cells. VI. Calcium is involved in early responses to boron deprivation.

Boron (B) deprivation induces various responses in plant cells, some of which can be observed very early. However, it has been unknown what kind of signal is generated by the stress. We found that B deprivation induced the expression of stress-responsive genes within 1 h in suspension-cultured tobacco BY-2 cells. The induction was largely suppressed by withholding medium Ca(2+) or by adding a Ca(2+) channel blocker. Analysis using aequorin-expressing cells showed that B-deprived cells took up more Ca(2+) than control cells. These results suggest that Ca(2+) influx plays a role in B deprivation stress signaling.

Boron nutrition of tobacco BY-2 cells. V. oxidative damage is the major cause of cell death induced by boron deprivation.

Boron (B) is an essential micronutrient for vascular plants. However, it remains unclear how B deficiency leads to various metabolic disorders and cell death. To understand this mechanism, we analyzed the physiological changes in suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells upon B deprivation. When 3-day-old cells were transferred to B-free medium, cell death was detectable as early as 12 h after treatment. The B-deprived cells accumulated more reactive oxygen species and lipid peroxides than control cells, and showed a slight but significant decrease in the cellular ascorbate pool. Supplementing the media with lipophilic antioxidants effectively suppressed the death of B-deprived cells, suggesting that the oxidative damage is the immediate and major cause of cell death under B deficiency. Dead cells in B-free culture exhibited a characteristic morphology with a shrunken cytoplasm, which is often seen in cells undergoing programmed cell death (PCD). However, they did not display other hallmarks of PCD such as internucleosomal DNA fragmentation, decreased ascorbate peroxidase expression and protection from death by cycloheximide. These results suggest that the death of tobacco cells induced by B deprivation is not likely to be a typical PCD.

Boron nutrition of cultured tobacco BY-2 cells. IV. Genes induced under low boron supply.

Genes whose expression was up-regulated in low boron (B)-acclimated tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) cells, which had been selected under a low supply of B, were screened by the cDNA differential subtraction method. Thirteen genes were identified, including early salicylate-inducible glucosyltransferase, glutamine synthetase, glutathione S-transferase, and a pathogenesis-related protein, which might constitute a rescue system for oxidative damage. This indicates that B deficiency might impose cellular redox imbalance on the cells. Two of the 13 genes were induced within 30 min of B removal in the parent cells, indicating fast signal transfer from the cell walls to the cytoplasm.

Arabidopsis 3-deoxy-D-manno-oct-2-ulosonate-8-phosphate synthase: cDNA cloning and expression analyses.

The molecular characterization of two isoforms of 3-deoxy-d-manno-oct-2-ulosonate (KDO) -8-phosphate synthase (AtkdsA1 and AtkdsA2) from Arabidopsis is reported here. First, by isolating a full-length cDNA for AtkdsA1, it was confirmed that the deduced primary structures of AtkdsA1 and AtkdsA2 proteins were 93% identical. Functional expression and purification studies demonstrated the efficient catalytic activity of the AtkdsA1 enzyme to produce KDO-8-phosphate from phosphoenolpyruvate and d-arabinose-5-phosphate. RT-PCR and RNA-gel blot analysis revealed different expression profiles for both genes; the AtkdsA1 gene was predominantly expressed in the shoots, while the AtkdsA2 transcript accumulated to a higher level in the roots, implicating differential roles of these isoforms in planta.

Purification and cDNA cloning of UDP-D-glucuronate carboxy-lyase (UDP-D-xylose synthase) from pea seedlings.

Uridine diphospho-D-glucuronate carboxy-lyase (UDP-D-xylose synthase; EC 4.1.1.35), which catalyzes the conversion of UDP-D-glucuronate to UDP-D-xylose, was purified to apparent homogenity from pea (Pisum sativum L.) seedlings. The pH optimum for enzyme activity was around 5-6, and the activity was not affected by exogeneously supplied NAD+ and NADH. The purified enzyme had a molecular weight of 250 kDa and consisted of 42 kDa polypeptides. Based on the amino acid sequence, a probe (400 bp) was prepared with degenerate primers by a reverse transcriptase-PCR. Using this probe, a clone encoding 346 amino acid residues was screened from a pea cDNA library. The recombinant protein expressed in Escherichia coli catalyzed conversion of UDP-D-glucuronate to UDP-D-xylose, confirming that the isolated clone encoded UDP-D-glucuronate carboxy-lyase.

Short-term boron deprivation inhibits endocytosis of cell wall pectins in meristematic cells of maize and wheat root apices.

By using immunofluorescence microscopy, we observed rapidly altered distribution patterns of cell wall pectins in meristematic cells of maize (Zea mays) and wheat (Triticum aestivum) root apices. This response was shown for homogalacturonan pectins characterized by a low level (up to 40%) of methylesterification and for rhamnogalacturonan II pectins cross-linked by a borate diol diester. Under boron deprivation, abundance of these pectins rapidly increased in cell walls, whereas their internalization was inhibited, as evidenced by a reduced and even blocked accumulation of these cell wall pectins within brefeldin A-induced compartments. In contrast, root cells of species sensitive to the boron deprivation, like zucchini (Cucurbita pepo) and alfalfa (Medicago sativa), do not internalize cell wall pectins into brefeldin A compartments and do not show accumulation of pectins in their cell walls under boron deprivation. For maize and wheat root apices, we favor an apoplastic target for the primary action of boron deprivation, which signals deeper into the cell via endocytosis-mediated pectin signaling along putative cell wall-plasma membrane-cytoskeleton continuum.