Management of fertilizer application is essential for maintaining adequate food production while preventing the environmental impact caused by excessive fertilizer application. Therefore, it is crucial to understand how crops respond to early nitrogen deficiency. In the present study, we analyzed the responses to early nitrogen deficiency in komatsuna (Brassica rapa var. perviridis) plants, a popular leafy green vegetable in Japan. Komatsuna plants were grown in pot cultures under seven nitrogen levels (0, 30, 60, 90, 150, 210, and 270 mg N per pot), with nitrogen as ammonium nitrate. Shoots were harvested every 5 days from 14 to 39 days after sowing to measure dry weight, total nitrogen, and nitrate concentrations. The dry weight of komatsuna shoots increased significantly even after soil inorganic nitrogen was nearly exhausted. Under the 30 mg N treatment, nitrogen accumulation at 24 days accounted for 81% of the final accumulation at 39 days, whereas dry weight accounted for only 35% of the final value. This indicates about 65% of the final dry weight accumulated after nitrogen accumulation plateaued. To elucidate the molecular mechanisms enabling growth sustained by internal nitrogen, gene expression profiles in leaves were analyzed using RNA-seq in plants grown under marginal or ample nitrogen supply. The results indicated that transcriptional regulation of purine metabolism plays an important role in adaptation to early nitrogen deficiency. These findings contribute to the optimization of nitrogen fertilization by providing a foundation for developing markers to assess the need of top-dressing and for breeding crop varieties with high nitrogen-use efficiency.

3-Deoxy-d-manno-oct-2-ulosonic acid (KDO) is an essential component of rhamnogalacturonan II (RG-II), a complex pectic polysaccharide required for plant growth and development. While most steps of the KDO biosynthetic pathway have been characterized in plants, KDO-8-phosphatase (KDO8Pase), the phosphatase responsible for converting KDO 8-phosphate (KDO8P) to KDO, remained unidentified. To identify this missing component, we performed gene co-expression analysis and identified At5g57440 (GPP2) as the primary candidate in Arabidopsis (Arabidopsis thaliana L.). Recombinant GPP2 protein exhibited KDO8P-specific phosphohydrolase activity in vitro. A green-fluorescent protein-tagged GPP2 protein was predominantly localized to mitochondria, consistent with the compartmentation of the subsequent step in KDO biosynthesis. Null mutants of GPP2 exhibited significant growth retardation under boron-limited conditions, in which expression of GPP2 and other KDO biosynthetic genes was up-regulated. The growth retardation was also observed in liquid culture in normal media, a condition that induces rapid growth and thus likely increases metabolic demand for KDO. Despite this growth defect, the KDO content per unit cell wall in gpp2 remained equivalent to that in wild-type plants. These results are consistent with the identification of GPP2 as the elusive plant KDO8Pase and suggest that KDO availability may become limiting for RG-II biosynthesis and cell wall production. Our findings complete the plant KDO biosynthetic pathway and provide new insights into the physiological significance of RG-II in cell wall biosynthesis.

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.

Excess biuret, a common impurity in urea fertilizers, is toxic to plants. Little is known about the mechanisms of biuret toxicity in plants. This study aimed to investigate the accumulation of biuret and the changes in metabolites in rice (Oryza sativa) plants under biuret toxicity. A previous study had shown that transgenic rice plants overexpressing bacterial biuret hydrolase had improved biuret tolerance. Here, we grew wild-type and bacterial biuret hydrolase-overexpressing rice plants in hydroponics at different biuret levels. Concentrations of biuret and allantoin, a nitrogenous intermediate in the purine degradation pathway, in the plants were determined. The expression levels of genes related to purine degradation and ureide metabolisms were analyzed using wild-type plants. Additionally, we performed a metabolome analysis using rice suspension cells. The biuret hydrolase-overexpressing plants did not contain biuret, whereas wild-type plants accumulated biuret in shoots in the order of mmol L−1 tissue water. The concentration of allantoin in shoots of wild-type plants under biuret toxicity was higher than those in control conditions. Inhibition of allantoinase activity by biuret was not detected, and allantoin accumulation appeared to be associated with changes in the expression of allantoinase, allantoate amidohydrolase and putative allantoin transporter genes. Furthermore, another nitrogenous compound citrulline, which is a non-protein amino acid, accumulated in rice suspension cells under biuret toxicity. The accumulation of these compounds suggests that rice plants subjected to biuret toxicity need to reduce the concentration of surplus ammonium ions via synthesizing nitrogen-rich compounds.

We aimed to clarify whether low cadmium (Cd) accumulation in varieties of rice also lower human nutrient uptakes. This trial was setup to explore the correlation between grain-Cd and iron (Fe) and zinc (Zn) among four promising rice cultivars and the prevailing KDML105 and RD6. Their grain Cd (0.10–0.60 mg kg-1 ) and Zn (17.36–22.61 mg kg-1 ) contents were significantly different, but not significant for grain Fe (11.17–13.91 mg kg-1 ). The results revealed significant positive correlations between grain Cd and Zn of the prevailing cultivar (r=0.471). But no correlations between grain Cd and Fe and Zn were observed. The RD15 and KNU2 were identified as the low grain Cd, high grain Fe and Zn compared with the prevailing cultivar. Thus the RD15 and KNU2 led to reduced Cd in rice grain without changing the human essential elements.

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.

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.

Transferring hydroponically grown Arabidopsis plants (Arabidopsis thaliana L.) to boron (B)-free medium kills the cells in root tips within an hour. To understand the mechanism underlying the induced rapid cell death, the Arabidopsis response to B deprivation was characterized using inhibitors of presumably involved cellular processes. The results suggest that stretching of the plasma membrane and the influx of calcium ions through mechanosensitive channels triggered the responses and that reactive oxygen species were overproduced under B-deprived condition. In addition, nitric oxide was observed to be involved in cell death, suggesting that Arabidopsis root cells undergo programmed cell death upon B deprivation. RNA sequencing analysis revealed that B deprivation induced transcriptome changes that resembled pathogen-induced responses. In summary, we speculate that B deprivation induces hypersensitive responses in Arabidopsis roots as a response to defective cell wall structures.

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.

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.

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.

The anaerobic digestion of livestock manure is an environmentally compatible technology used for the production of renewable energy. Anaerobically digested residual slurry has been used worldwide as a liquid fertilizer in both upland and paddy fields. However, a controversial question remains as to whether the application of slurry to rice paddy fields increases methane emissions; although methane is one of the most prevalent greenhouse gases, little is known about the effects of the long-term application of residual slurry on methane emission. In this study, we repeatedly applied slurry to a paddy field for six years at different application rates (10, 15, and 20 g N m(-2) based on ammonium-nitrogen content). At the fifth and sixth years of application, we evaluated the effect in terms of methane flux and soil total carbon content. The effect of the long-term application of the slurry (10 g N m(-2)) on grain yield was equivalent to that of chemical fertilizer (10 g N m(-2)). The application of the residual slurry was likely to increase the cumulative methane emissions during rice growing season in both 2006 and 2007. On the other hand, we observed that soil total carbon did not accumulate significantly in the soil. Thus, we cannot rule out the potential risk of additional methane emissions caused by the application of the residuary slurry to paddy fields.

Aluminum (Al) minerals form a strong complex with organic matter (OM) in soil, affecting the stability and degradability of OM and carbon (C) dynamics in soil. However, plant effects on the accumulation of Al minerals (particularly Al hydroxide minerals) that are not crystallized and actually or potentially associated with OM (active Al minerals) in soil have received little attention. Al also interacts with silicon (Si) in soil to form aluminosilicate minerals, and plants remove Si from soil through Si uptake. Plant Si uptake may thus lead to an increase in active Al minerals in soil because of a reduced influence of Si in soil and the resulting reduced formation of aluminosilicate minerals. To test whether plant Si uptake changes active Al minerals in root-zone soil, we performed two cultivation experiments using plants varying in Si uptake and compared changes in soil mineral indices during cultivation, including soil extractable Al concentrations in root-zone soils. In the first experiment, using five plant species varying in Si uptake, plant species with greater Si uptake, in particular grass species, showed higher pH (NaF) and CuCl2-extractable Al in their root-zone soils, suggesting that plant Si uptake increases the amount of active Al in soil. This suggestion was supported by the second experiment, using two types of rice (Oryza sativa), a wild type and a mutant defective for a Si transporter. The results showed that pH (NaF) and extractable Al in root-zone soil decreased under the low-Si-uptake mutant, especially in the vicinity of their roots. These observations suggest that Si uptake by plant roots leads to an increase in active Al minerals, possibly enhancing OM retention and C accumulation in root-zone soil and may partly explain why grassland soils are often rich in C.

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.

Under potassium (K)-deficient conditions, rice (Oryza sativa L.) actively takes up and utilizes sodium (Na) as an alternative element to K. In this study, we cloned a gene responsible for cultivar differences in shoot Na accumulation using a map-based cloning method. The responsible gene OsHKT2;1 encodes an Na transporter associated with Na uptake in root tissues, and its expression level was positively correlated with Na uptake potential in 11 rice cultivars. We found that OsHKT2;1 overexpression promoted shoot Na accumulation under low K supply and proposed that OsHKT2;1 expression level is a key factor in the Na accumulation potential in rice cultivars. However, under sufficient K supply, OsHKT2;1-overexpressing rice plants accumulated Na in roots but not in shoots. This result suggests that Na transfer from root to shoot may be regulated by another Na transporter.

In this study sequential extraction was used to fractionate cadmium (Cd) and zinc (Zn) from soils into six operationally defined groups; water soluble, buffer-exchangeable, carbonate, FeMn oxide, organic, and residual. Soil samples from agricultural areas surrounding Pha Te village, Mae Sot District, Tak Province, Thailand, were classified into four categories; forest soil, upland soil, upper-paddy soil and lower-paddy soil. Total soil Cd and Zn concentrations ranged from 0.63 to 30.4 mg kg-1 and 14.4 to 594 mg kg-1, respectively. Cd and Zn concentrations were higher in the upper- and lower-paddy soil (5.93 to 30.4 mg kg-1 for Cd and 286 to 594 mg kg-1 for Zn). These soils are considered as polluted. Cd in the polluted soil was dominantly associated with the buffer-exchangeable and carbonate-bound (40 to 70 % of total Cd), while in non-polluted soils; the residual fraction was dominant (50 to 80 % of the total Cd). The major proportion of Zn (37 to 46 % of total Zn) in the non-polluted soil and the upper-paddy soil occurred in the residual fraction. On the other hand, the major proportion of total Zn in the lower-paddy soil was associated with FeMn oxides (36 % of total Zn). The results show that mobility and potential bioavailability of Cd and Zn (61 and 25 %) in polluted soil were higher than in non-polluted soils (15 and 19 % in Cd and Zn, respectively). Metal distribution in different chemical fractions in these soils depended on the respective total metal concentrations.

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.