THOUSAND-GRAIN WEIGHT 6, which is an IAA-glucose hydrolase, preferentially recognizes the structure of the indole ring.

An indole-3-acetic acid (IAA)-glucose hydrolase, THOUSAND-GRAIN WEIGHT 6 (TGW6), negatively regulates the grain weight in rice. TGW6 has been used as a target for breeding increased rice yield. Moreover, the activity of TGW6 has been thought to involve auxin homeostasis, yet the details of this putative TGW6 activity remain unclear. Here, we show the three-dimensional structure and substrate preference of TGW6 using X-ray crystallography, thermal shift assays and fluorine nuclear magnetic resonance (19F NMR). The crystal structure of TGW6 was determined at 2.6 Å resolution and exhibited a six-bladed ß-propeller structure. Thermal shift assays revealed that TGW6 preferably interacted with indole compounds among the tested substrates, enzyme products and their analogs. Further analysis using 19F NMR with 1,134 fluorinated fragments emphasized the importance of indole fragments in recognition by TGW6. Finally, docking simulation analyses of the substrate and related fragments in the presence of TGW6 supported the interaction specificity for indole compounds. Herein, we describe the structure and substrate preference of TGW6 for interacting with indole fragments during substrate recognition. Uncovering the molecular details of TGW6 activity will stimulate the use of this enzyme for increasing crop yields and contributes to functional studies of IAA glycoconjugate hydrolases in auxin homeostasis.

Generation of major glutelin-deficient (GluA, GluB, and GluC) semi-dwarf Koshihikari rice line.

KEY MESSAGE: We generated a new Koshihikari rice line with a drastically reduced content of glutelin proteins and higher lodging resistance by using new and conventional plant breeding techniques. Using CRISPR/Cas9-mediated genome editing, we generated mutant rice with drastically decreased contents of major glutelins. A Koshihikari rice mutant line, a123, lacking four glutelins (GluA1, GluA2, GluB4, and GluB5) was used as a host, and another five major glutelin genes (GluA3, GluB1a, GluB1b, GluB2, and GluC) were knocked out through two iterations of Agrobacterium-mediated transformation. Mutant seeds were deficient in the GluA family, GluB family, and GluC, and the line obtained was named GluABC KO. Glutelin content was much lower in GluABC KO than in the existing low-glutelin rice mutant LGC-1. A null segregant of GluABC KO was selected using new-generation sequencing and backcrossing, and the sd-1 allele for the semi-dwarf trait was introduced to increase lodging resistance.

A Rice Ancestral Genetic Resource Conferring Ideal Plant Shapes for Vegetative Growth and Weed Suppression.

To maximize crop growth, crops need to capture sunlight efficiently. This property is primarily influenced by the shape of the crops such as the angle, area, and arrangement of leaves. We constructed a rice (Oryza sativa L.) inbred line that displayed an ideal transition of plant shapes in terms of sunlight receiving efficiency. During vegetative growth, this line exhibited tiller spreading with increased tiller number, which formed a parabolic antenna-like structure. The architecture probably improved light reception efficiency of individuals compared with the recurrent parent. The line achieved not only acceleration of the vegetative growth, but also significant suppression of weed growth under the canopy. The increased light reception efficiency of the line has consequently reduced the amount of incident light to the ground and supplied significant competitiveness against weeds. The spread tillers became erect from the entry of the reproductive growth phase, adaptively sustaining light reception efficiency in thicker stands. The line carries a small chromosomal segment from Oryza rufipogon Griff., a putative progenitor of Asian cultivated rice. The introduced chromosome segment had little effect on grain yield and quality. Our results shed light on potentials hidden in the wild rice chromosome segment to achieve the valuable traits.

Nucleotide Sequence of HIV-1-Positive Specimen Reference Panel for Evaluation of HIV In Vitro Diagnostics in Japan.

HIV-1 subtype/circulating recombinant form (CRF) distribution of HIV-1-positive specimens for evaluating HIV in vitro diagnostics (IVDs) was examined and compared with the HIV-1 epidemic in Japan. The nucleotide sequences of the gag-pol region of 173 plasma specimens (84, provided in 2007, and 89 in 2013-2015) were determined. HIV-1 subtype/CRF classification was performed based on the phylogenetic analyses of the sequences. The subtype/CRF distribution resulting in this study was similar to that of a previous epidemiological report. Three CRF02_AG and one unique recombinant form, including subtype G and A regions, were observed in the 2013 and 2014 specimens, except in the 2007 specimens. The reference panel consisting of these specimens was practical for the evaluation of HIV IVDs in Japan.

Evaluation of Geenius HIV-1/2 Confirmatory Assay for the confirmatory and differential diagnosis of HIV-1/HIV-2 in Japan and reliability of the Geenius Reader in the diagnosis of HIV-2.

BACKGROUND: NEW LAV BLOT I and II (LAV I and LAV II), they were only option for human immunodeficiency virus (HIV) confirmatory test, following HIV screening test using HIV Ag/Ab combination test in Japan. We evaluated the performance of Geenius HIV-1/2 Confirmatory Assay (Geenius), both as a confirmatory test and for differentiation between HIV-1 and HIV-2, in comparison with LAV I and LAV II. METHODS: Eighty-nine HIV-1-positive plasma specimens, one anti-HIV-1 low-titer performance panel, 10 seroconversion panels, and two anti-HIV-1/2 combo performance panels were tested. The results were read with the Geenius Reader and by visual reading. RESULTS: All 89 HIV-1-positive plasma specimens were identified as HIV-1-positive using Geenius; this 100% success rate was superior to that with LAV I (95.5% using WHO criteria, 98.9% using CDC criteria). The HIV-1-positive specimens showed low cross-reactivity with HIV-2 lines in Geenius. The sensitivity of Geenius for HIV-1 detection was the same as or greater than that of LAV I, but less than that of Genscreen HIV Ag-Ab ULT, in our analysis of the commercial performance and seroconversion panels. In contrast, five of the 13 HIV-2-positive specimens that had been identified as HIV-positive untypable by visual reading because of their cross-reactivity to HIV-1 lines were successfully identified by the Geenius Reader as HIV-2-positive with cross-reactivity. CONCLUSIONS: Geenius provides strong performance for HIV confirmatory tests and HIV-1 differentiation tests. However, when visual reading is used, its performance in HIV-2 differentiation is less reliable. Because HIV-2 infection has been sporadically reported in Japan, the use of the Geenius Reader is preferable to ensure more reliable HIV-1/HIV-2 differentiation.

Erythrocyte Alloimmunity and Genetic Variance: Results from the Collaborative Study of Alloimmunity to Antigen Diversity in Asian Populations (All ADP).

As an East-Asian international study, we evaluated erythrocyte alloimmunity by gender and history of transfusion or pregnancy. In total, data from more than 1,826,000 patients were analyzed, from whom 26,170 irregular erythrocyte antibodies were detected in 22,653 cases. Antibody frequencies in these cases were as follows: anti-E, 26.8%; anti-Lea, 20.0%; anti-P1, 7.1%; anti-M, 6.4%; anti-Mia, 5.6%; anti-c + E, 5.6%; anti-Leb, 4.6%; anti-D, 2.8%; anti-Fyb, 2.6%; anti-Lea+Leb, 2.5%; anti-Dia, 2.0%; and others. For pregnant patients, anti-D (12.7%) was statistically more frequent. For transfused patients, anti-E (37.3%), anti-c + E (9.5%), anti-C + e (3.3%) and anti-Jka (3.1%) were significantly more frequent.

A Japanese multi-institutional collaborative study of antigen-positive red blood cell (RBC) transfusions in patients with corresponding RBC antibodies.

BACKGROUND AND OBJECTIVES: It is sometimes difficult to obtain antigen-negative red blood cells (RBCs) for patients with antibodies against RBCs. However, the frequency and severity of the adverse reactions have not been well elucidated. Here, we conducted a multi-institutional collaborative study to clarify the background, frequency and clinical significance of antigen-positive RBC transfusions to patients with the respective antibodies. MATERIALS AND METHODS: The survey included the background of patients, antigens on RBCs transfused, total amount of antigen-positive RBCs transfused, results from antibody screen and direct antiglobulin tests, specificity of antibodies, adverse reactions and efficacies. All antibodies were surveyed regardless of their clinical significance. RESULTS: In all, 826 cases containing 878 antibodies were registered from 45 institutions. The main reasons for antigen-positive RBC transfusions included 'negative by indirect antiglobulin test' (39%) and 'detection of warm autoantibodies' (25%). In 23 cases (3% of total), some adverse reactions were observed after antigen-positive RBC transfusion, and 25 antibodies (9 of 119 clinically significant and 16 of 646 insignificant antibodies) were detected. Non-specific warm autoantibodies were detected in 9 cases, anti-E in 5 cases, 2 cases each of anti-Lea , anti-Jra or cold alloantibodies, and 1 case each of anti-Dib , anti-Leb or anti-P1. Other antibodies were detected in 2 further cases. Five (22%) of these 23 cases, who had anti-E (3 cases) or anti-Jra (2 cases), experienced clinically apparent haemolysis. CONCLUSIONS: Adverse reactions, especially haemolysis, were more frequently observed in cases with clinically significant antibodies than those with clinically insignificant antibodies (P < 0·001).

Evaluation of in vitro screening and diagnostic kits for hepatitis B virus infection.

BACKGROUND: For the diagnosis of hepatitis B virus (HBV) infection, the detection and quantification of hepatitis B surface antigen (HBsAg) and HBV DNA are used. Several kits are available for this purpose, and there is a growing need for the evaluation of these kits because their performance may be affected by HBV genotype- or strain-specific polymorphisms. OBJECTIVES AND STUDY DESIGN: In this study, we used International Standards and the established regional reference panel to evaluate the performance of two HBV DNA quantitative kits, five HBsAg qualitative kits, seven HBsAg quantitative kits and three rapid immune-chromatographic tests for HBsAg. RESULTS: The quantification values of two HBV DNA quantitative kits exhibited excellent correlation. In the evaluation of HBsAg qualitative and quantitative kits, the titers of several specimens in the HBV-positive panel were below the detection limits of a few kits, and the specimens were determined as HBV-negative. Notably, the quantitative kit results exhibited low correlation values. However, when these data were analyzed for each genotype, the correlations improved. These results suggest that the HBsAg quantification data are influenced by HBV genotypes. The novel rapid immune-chromatographic test exhibited the comparable level of sensitivity to the HBsAg quantitative kits. CONCLUSIONS: We evaluated the performance of kits for the detection of HBV infection. The HBV DNA quantification data correlated with an excellent agreement, whereas the HBsAg quantification data were affected by HBV genotype. Such evaluations will be useful for estimating the quality of currently available and new HBV assay kits, and for the quality control of these kits.

How elevated CO2 affects our nutrition in rice, and how we can deal with it.

Increased concentrations of atmospheric CO2 are predicted to reduce the content of essential elements such as protein, zinc, and iron in C3 grains and legumes, threatening the nutrition of billions of people in the next 50 years. However, this prediction has mostly been limited to grain crops, and moreover, we have little information about either the underlying mechanism or an effective intervention to mitigate these reductions. Here, we present a broader picture of the reductions in elemental content among crops grown under elevated CO2 concentration. By using a new approach, flow analysis of elements, we show that lower absorption and/or translocation to grains is a key factor underlying such elemental changes. On the basis of these findings, we propose two effective interventions-namely, growing C4 instead of C3 crops, and genetic improvements-to minimize the elemental changes in crops, and thereby avoid an impairment of human nutrition under conditions of elevated CO2.

Partial loss-of-function of NAL1 alters canopy photosynthesis by changing the contribution of upper and lower canopy leaves in rice.

Little is known about the genetic basis of leaf and canopy photosynthesis. Here we aimed to detect novel quantitative trait loci (QTL) controlling photosynthesis by increasing leaf nitrogen content (LNC) per leaf area and analysed its effect on leaf and canopy photosynthesis. To identify QTL that increase photosynthetic rate in leaves, we screened chromosome segment substitution lines (CSSLs) of Oryza sativa ssp. japonica cultivar Koshihikari and O. sativa ssp. indica cultivar Nona Bokra using LNC per leaf area as the phenotype indicator. Locus leaf nitrogen content on chromosome four (qLNC4) is associated with increased LNC and photosynthetic rate per leaf area. Moreover, a non-synonymous amino acid substitution was identified in the NARROW LEAF 1 (NAL1) gene located in the qLNC4 region. This NAL1 allele increases LNC and photosynthetic rate per leaf area in flag leaves but does not increase whole-leaf photosynthesis. This NAL1 allele also increases light capture and whole-leaf nitrogen content of the lower leaves and is associated with slower senescence in flag leaves. These results suggest that this NAL1 allele does not increase whole-leaf photosynthesis but plays a role in regulating spatial and temporal trade-offs among traits at the whole-plant level.

Elevated atmospheric CO2 levels affect community structure of rice root-associated bacteria.

A number of studies have shown that elevated atmospheric CO2 ([CO2]) affects rice yields and grain quality. However, the responses of root-associated bacteria to [CO2] elevation have not been characterized in a large-scale field study. We conducted a free-air CO2 enrichment (FACE) experiment (ambient + 200 µmol.mol(-1)) using three rice cultivars (Akita 63, Takanari, and Koshihikari) and two experimental lines of Koshihikari [chromosome segment substitution and near-isogenic lines (NILs)] to determine the effects of [CO2] elevation on the community structure of rice root-associated bacteria. Microbial DNA was extracted from rice roots at the panicle formation stage and analyzed by pyrosequencing the bacterial 16S rRNA gene to characterize the members of the bacterial community. Principal coordinate analysis of a weighted UniFrac distance matrix revealed that the community structure was clearly affected by elevated [CO2]. The predominant community members at class level were Alpha-, Beta-, and Gamma-proteobacteria in the control (ambient) and FACE plots. The relative abundance of Methylocystaceae, the major methane-oxidizing bacteria in rice roots, tended to decrease with increasing [CO2] levels. Quantitative PCR revealed a decreased copy number of the methane monooxygenase (pmoA) gene and increased methyl coenzyme M reductase (mcrA) in elevated [CO2]. These results suggest elevated [CO2] suppresses methane oxidation and promotes methanogenesis in rice roots; this process affects the carbon cycle in rice paddy fields.

Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield.

Increases in the yield of rice, a staple crop for more than half of the global population, are imperative to support rapid population growth. Grain weight is a major determining factor of yield. Here, we report the cloning and functional analysis of THOUSAND-GRAIN WEIGHT 6 (TGW6), a gene from the Indian landrace rice Kasalath. TGW6 encodes a novel protein with indole-3-acetic acid (IAA)-glucose hydrolase activity. In sink organs, the Nipponbare tgw6 allele affects the timing of the transition from the syncytial to the cellular phase by controlling IAA supply and limiting cell number and grain length. Most notably, loss of function of the Kasalath allele enhances grain weight through pleiotropic effects on source organs and leads to significant yield increases. Our findings suggest that TGW6 may be useful for further improvements in yield characteristics in most cultivars.

Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species.

BACKGROUND: Genotype analysis using multiple single nucleotide polymorphisms (SNPs) is a useful but labor-intensive or high-cost procedure in plant research. Here we describe an alternative genotyping method that is suited to multi-sample or multi-locus SNP genotyping and does not require electrophoresis or specialized equipment. RESULTS: We have developed a simple method for multi-sample or multi-locus SNP genotyping using allele-specific primers (ASP). More specifically, we (1) improved the design of allele-specific primers, (2) established a method to detect PCR products optically without electrophoresis, and (3) standardized PCR conditions for parallel genomic assay using various allele-specific primers. As an illustration of multi-sample SNP genotyping using ASP, we mapped the locus for lodging resistance in a typhoon (lrt5). Additionally, we successfully tested multi-locus ASP-PCR analysis using 96 SNPs located throughout the genomes of rice (Oryza sativa) cultivars 'Koshihikari' and 'Kasalath', and demonstrated its applicability to other diverse cultivars/subspecies, including wild rice (O. rufipogon). CONCLUSION: Our ASP methodology allows characterization of SNPs genotypes without electrophoresis, expensive probes or specialized equipment, and is highly versatile due to the flexibility in the design of primers. The method could be established easily in any molecular biology laboratory, and is applicable to diverse organisms.

Evidence for separate translocation pathways in determining cadmium accumulation in grain and aerial plant parts in rice.

BACKGROUND: Cadmium (Cd) translocation and accumulation in the grain and aerial plant parts of rice (Oryza sativa L.) is an important aspect of food safety and phytoextraction in areas with contaminated soil. Because control of Cd translocation and accumulation is likely to be determined by the plants genetics, the Cd contents of grain and the aerial parts of rice may be manipulated to improve food safety and for phytoextraction ability. This study studied Cd translocation and accumulation and their genetic control in aerial parts of rice to provide a starting point for improving food safety and phytoextraction in Cd-contaminated soils. RESULTS: In the japonica rice cultivar "Nipponbare", Cd accumulated in leaves and culms until heading, and in culms and ears after heading. Two quantitative trait loci (QTLs) from indica cv. "Kasalath", qcd4-1 and qcd4-2, affect Cd concentrations in upper plant parts just before heading. Three near-isogenic lines (NILs) with qcd4-1 and qcd4-2 were selected from the "Nipponbare" background, and were analyzed for the effects of each QTL, and for interactions between the two QTLs. From the results compared between "Nipponbare" and each NIL, neither QTL influenced total Cd accumulation in aerial parts at 5 days after heading, but the interaction between two QTLs increased Cd accumulation. At 35 days after heading, qcd4-2 had increased Cd accumulation in the aerial plant parts and decreased translocation from leaves other than flag leaf, but interaction between the two QTLs increased translocation from leaves. NILqcd4-1,2 accumulated higher concentrations of Cd in brown rice than "Nipponbare". CONCLUSION: Three types of Cd translocation and accumulation patterns demonstrated by NILs suggested that the accumulation of Cd in leaves and culms before heading, and translocation from them after heading are responsible for Cd accumulation in grain. Cd translocation from roots to culms and ears after heading may direct Cd to the aerial organs without influencing brown rice accumulation.

Improvement of lodging resistance with QTLs for stem diameter in rice (Oryza sativa L.).

Varietal differences among ten rice cultivars showed that stem diameter is a key factor in lodging resistance (measured in terms of pushing resistance). Two near-isogenic lines (NILs) were selected from a series of chromosome segment substitution lines developed between cultivars Nipponbar and Kasalath, one containing a single stem diameter QTL (sdm8; NIL114), and another with four stem diameter QTLs (sdm1, sdm7, sdm8, sdm12; NIL28). Compared with the Nipponbare control, stem diameters were larger in NIL114 and NIL28 by about 7 and 39%, respectively. Pushing resistance in NIL28 was significantly greater than in Nipponbare, but NIL114 was similar to Nipponbare. The two NILs had greater weight of lower stem and culm wall thickness than Nipponbare. NIL28 had higher plant height, which is a negative effect on lodging resistance, than Nipponbare. The non-structural carbohydrate contents of NIL stems were higher than that of Nipponbare, whereas the silicon contents were lower in the NILs, and cellulose contents were lower only in NIL28. The basal internodes of the two NILs were significantly stiffer than those of Nipponbare. These results suggest that increasing stem diameter in rice breeding programs would improve lodging resistance, although the combination of multiple QTLs would be necessary to produce thicker stems with higher pushing resistance, whereas the higher plant height could also result from the combination of multiple QTLs.

Time-related identification of chromosome regions affecting plant elongation in rice (Oryza sativa L.).

We investigated the time-related changes of Chromosome Regions that Affect Traits (CRATs) for elongation rate in rice (Oryza sativa L.) using chromosome segment substitution lines (CSSLs) carrying a single chromosome segment of the cultivar Kasalath (indica) in a Koshihikari (japonica) genetic background. The growth period of rice was partitioned into eight stages (each lasting 5-7days) from 18days after transplanting, and the elongation rate was determined as the increase of total plant height per time at each growth stage. CRATs for plant elongation rate were determined based on graphical genotype data of CSSLs that showed a significantly higher or lower elongation rate than Koshihikari. In total, 23 CRATs for plant elongation rate were detected, and different CRATs acted at different growth stages. Fifteen CRATs increased the elongation rate through Kasalath alleles, and eight increased it through Koshihikari alleles. These results suggest that plant height in rice is regulated in a stage-specific manner by a variety of genetic mechanisms that control plant elongation rate. Kasalath alleles of PE1-9 increased the elongation rate at an early growth stage (18-25days after transplanting), while Koshihikari alleles of PE8-3 decreased the elongation rate at a late growth stage (68-74days after transplanting). In a line that contained both of these CRATs, the elongation rate at the early growth stage was increased without affecting plant height at harvesting. We conclude that stage-specific optimization of plant height in rice may be achieved by combining CRATs that control plant elongation at specific stages.

New target for rice lodging resistance and its effect in a typhoon.

We demonstrated the new target for lodging resistance in rice (Oryza sativa L.) by the analysis of physiological function of a locus for lodging resistance in a typhoon (lrt5) with the near isogenic line under rice "Koshihikari" genetic background (tentatively named S1). The higher lodging resistance of S1 was observed during a typhoon in September 2004 (28 days after heading), when most other plants in "Koshihikari" became lodged. Visual observations showed that bending of the upper stems triggered lodging during the typhoon; the upper stem of "Koshihikari" buckled completely, whereas that of S1 remained straight. In addition to the strong rain and winds during the typhoon, the weight of the buckled upper plant parts increased the pressure on adjacent plants and caused a domino effect in "Koshihikari". Young's modulus, an indicator of the rigidity of the culm, was significantly higher in S1 than in "Koshihikari". In the upper culm, the starch content in S1 was 4.8 times the value in "Koshihikari", and senescence was delayed in the upper leaves of S1. These results suggest that the rigidity of the upper culm by the higher starch content (as a result of delayed senescence in the upper leaves) may be responsible for the higher lodging resistance during a typhoon in rice.

Quantitative trait loci for sucrose, starch, and hexose accumulation before heading in rice.

We studied the storage of sucrose, starch, and hexose before heading in rice (Oryza sativa L.) plants by quantitative trait locus (QTL) analysis with a population of backcross inbred lines (BILs) of japonica cv. Nipponbare x indica cv. Kasalath. Carbohydrates are accumulated in the rice plant before heading and are translated to the panicle after heading. A higher capacity for accumulation is thus a main target for improvement in yield. The form of carbohydrate (sucrose, starch, or hexose) differs depending on the organ in which it is stored. There was no correlation between starch and sucrose or hexose contents in BILs, and the positions of QTLs controlling starch differed from those for sucrose and hexose accumulation. These results suggest that the genetic control of accumulation differs between starch and sugars. QTLs that control the ratio of sucrose to starch content were detected, suggesting the existence of a mechanism(s) that determines this ratio. On chromosome 1, sucrose-phosphate synthase 1, the key enzyme in sucrose synthesis was close to the peaks of the likelihood odds ratios in QTLs for sucrose or hexose content. These results suggest that SPS1 is related to conversion of carbohydrate to sucrose as accumulated form in a plant before heading.