Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (Mr) of 102,069 Da, similar in size to its Mr of about 100,000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3'-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.

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Summary Sesame (Sesamum indicum) seeds contain a large number of lignans, phenylpropanoid‐related plant specialized metabolites. (+)‐Sesamin and (+)‐sesamolin are major hydrophobic lignans, whereas (+)‐sesaminol primarily accumulates as a water‐soluble sesaminol triglucoside (STG) with a sugar chain branched via β1→2 and β1→6‐O‐glucosidic linkages [i.e. (+)‐sesaminol 2‐O‐β‐d‐glucosyl‐(1→2)‐O‐β‐d‐glucoside‐(1→6)‐O‐β‐d‐glucoside]. We previously reported that the 2‐O‐glucosylation of (+)‐sesaminol aglycon and β1→6‐O‐glucosylation of (+)‐sesaminol 2‐O‐β‐d‐glucoside (SMG) are mediated by UDP‐sugar‐dependent glucosyltransferases (UGT), UGT71A9 and UGT94D1, respectively. Here we identified a distinct UGT, UGT94AG1, that specifically catalyzes the β1→2‐O‐glucosylation of SMG and (+)‐sesaminol 2‐O‐β‐d‐glucosyl‐(1→6)‐O‐β‐d‐glucoside [termed SDG(β1→6)]. UGT94AG1 was phylogenetically related to glycoside‐specific glycosyltransferases (GGTs) and co‐ordinately expressed with UGT71A9 and UGT94D1 in the seeds. The role of UGT94AG1 in STG biosynthesis was further confirmed by identification of a STG‐deficient sesame mutant that predominantly accumulates SDG(β1→6) due to a destructive insertion in the coding sequence of UGT94AG1. We also identified UGT94AA2 as an alternative UGT potentially involved in sugar–sugar β1→6‐O‐glucosylation, in addition to UGT94D1, during STG biosynthesis. Yeast two‐hybrid assays showed that UGT71A9, UGT94AG1, and UGT94AA2 were found to interact with a membrane‐associated P450 enzyme, CYP81Q1 (piperitol/sesamin synthase), suggesting that these UGTs are components of a membrane‐bound metabolon for STG biosynthesis. A comparison of kinetic parameters of these UGTs further suggested that the main β‐O‐glucosylation sequence of STG biosynthesis is β1→2‐O‐glucosylation of SMG by UGT94AG1 followed by UGT94AA2‐mediated β1→6‐O‐glucosylation. These findings together establish the complete biosynthetic pathway of STG and shed light on the evolvability of regio‐selectivity of sequential glucosylations catalyzed by GGTs.

Aphanomyces root rot, caused by Aphanomyces cochlioides Drechs., is one of the most serious diseases of sugar beet (Beta vulgaris L.). Identification and characterization of resistance genes is a major task in sugar beet breeding. To ensure the effectiveness of marker-assisted screening for Aphanomyces root rot resistance, genetic analysis of mature plants' phenotypic and molecular markers' segregation was carried out. At a highly infested field site, some 187 F(2) and 66 F(3) individuals, derived from a cross between lines 'NK-310mm-O' (highly resistant) and 'NK-184mm-O' (susceptible), were tested, over two seasons, for their level of resistance to Aphanomyces root rot. This resistance was classified into six categories according to the extent and intensity of whole plant symptoms. Simultaneously, two selected RAPD and 159 'NK-310mm-O'-coupled AFLP were used in the construction of a linkage map of 695.7 cM. Each of nine resultant linkage groups was successfully anchored to one of nine sugar beet chromosomes by incorporating 16 STS markers. Combining data for phenotype and molecular marker segregation, a single QTL was identified on chromosome III. This QTL explained 20% of the variance in F(2) population (in the year 2002) and 65% in F(3) lines (2003), indicating that this QTL plays a major role in the Aphanomyces root rot resistance. This is the first report of the genetic mapping of resistance to Aphanomyces-caused diseases in sugar beet.

Sesame (Sesamum indicum L.) is an important oilseed crop, but is negatively affected by continuous cropping. There is still a lack of information on the effect of continuous cropping on soil chemical properties and mineral nutrition related to sesame growth and yield decline. Therefore, we investigated sesame growth and yield, nutrient concentration and soil chemical properties on five fields with continuous cropping history: non-continuous cropping (Year 0) and durations of two, four, five and six years on an upland field converted paddy in Tottori, Japan. Results show that plant height significantly decreased by 18.76%, 15.22%, and 13.64% in the Year 4, Year 5 and Year 6 fields, respectively, compared to Year 0. The effect of continuous cropping was more pronounced on the 1000-seed weight decline than seed yield. Compared to Year 0, seed yield decreased by 52.86% in Year 2 with no significant differences among the Year 2, Year 4, Year 5 and Year 6 fields, whereas the 1000-seed weight decreased by 6.68% and 12.20% in the Year 2 and Year 5 fields, respectively, compared to Year 0. Plant leaf tissue N concentration significantly decreased in the Year 2, Year 4 and Year 6 fields compared to Year 0, whereas leaf tissue K concentration decreased in the Year 6 field. The increase in duration of continuous cropping years gradually altered soil chemical properties. Soil pH, exchangeable Ca and Mg and cation exchange capacity (CEC) gradually increased in the long duration of continuous cropping, whereas total N and C, exchangeable NH4+-N, urease, dehydrogenase and catalase activities decreased. Our study suggested that the decrease in soil available N and enzyme activities, and decrease in K nutrition due to competitive ion effect as a result of increase in soil Ca and Mg could possibly contribute to the growth and yield decline of continuous sesame on upland field converted paddy.

Lamivudine is effective in suppressing replication of hepatitis B virus (HBV). However, the emergence of HBV variants resistant to lamivudine is a concern. Lamivudine resistance has been attributed mainly to a substitution of isoleucine or valine for methionine at residue 550 (M550I or M550V) in the catalytic site of the virus polymerase. A substitution of methionine for leucine at residue 526 (L526M) has also been identified. To examine such virus genotypic mutations in Japanese patients, we studied five patients with chronic hepatitis B, who showed HBV breakthrough while on a 1-year lamivudine treatment. The entire nucleotide and deduced amino acid sequences of the proposed reverse transcriptase domain of the polymerase gene were determined on HBV DNA amplified by polymerase chain reaction from patient sera collected at the start and at the end of therapy. The HBV sequences from all five patients were of genotype C. In four patients, a substitution of valine or isoleucine for leucine at residue 426, which has not been reported previously, emerged in combination with M550I. One also harbored L526M. In the remaining patient, an alteration of leucine to methionine at residue 428 co-occurred with M550V. Longitudinal study of the mutations showed that the two or three mutations in each patient emerged almost simultaneously 4 weeks before or at the time of breakthrough and were replaced by wild-type virus after completing the therapy. Our results indicate that occurrence of HBV polymerase mutations at residue 426 in combination with M550I is frequent in Japanese or genotype C virus-in- fected patients who develop resistance to lamivudine.

Sesame is an important oilseed crop cultivated worldwide. However, research has focused on biochar effects on grain crops and vegetable and there is still a scarcity of information of biochar addition on sesame. This study was to assess the effect of biochar addition on sesame performance, with a specific emphasis on growth, yield, leaf nutrient concentration, seed mineral nutrients, and soil physicochemical properties. A field experiment was conducted on an upland field converted from paddy at Tottori Prefecture, Japan. Rice husk biochar was added to sesame cropping at rates of 0 (F), 20 (F+20B), 50 (F+50B) and 100 (F+100B) t ha −1 and combined with NPK fertilization in a first cropping and a second cropping field in 2017. Biochar addition increased plant height, yield and the total number of seeds per plant more in the first cropping than in the second cropping. The F+50B significantly increased seed yield by 35.0% in the first cropping whereas the F+20B non-significantly increased seed yield by 25.1% in the second cropping. At increasing biochar rates, plant K significantly increased while decreasing Mg whereas N and crude protein, P and Ca were non-significantly higher compared to the control. Soil porosity and bulk density improved with biochar addition while pH, exchangeable K, total N, C/N ratio and CEC significantly increased with biochar, but the effect faded in the second cropping. Conversely exchangeable Mg and its plant tissue concentration decreased due to competitive ion effect of high K from the biochar. Biochar addition is effective for increasing nutrient availability especially K for sesame while improving soil physicochemical properties to increase seed yield, growth and seed mineral quality.Agronomy 2019, 9, 55 2 of 20 (13.5%), ash (5%) [2], and mineral components, such as K (815 mg/100 g), P (647 mg/100 g), Mg (579 mg/100 g) and Ca (415 mg/100 g) [3]. This contributes to its health and nutritional benefits. Therefore, demand for sesame seeds is increasing due to the increasing knowledge on their dietary and health benefits, but there has been limited research on sesame evidenced by low yield in most growing areas hence hampering its adoption and expansion in the world [4]. Although sesame has been reconsidered a local specialty crop in Japan [5], the production of sesame is still low. For instance, the Food and Agriculture Organization (FAO) in 2016 estimated that 11 tons of sesame seeds were produced from an area of 21 hectares [6]. With the increase in abandoned paddy fields estimated at 360,000 ha by the year 2010, farmers were encouraged to convert such fields into cultivation of upland crops, such as wheat and soybeans [7,8], including sesame. However, crop yield on upland fields converted from paddy may decrease due to declining soil fertility status of the paddy soils that could require soil amendment with organic materials [9].Biochar is a soil amendment produced from thermal decomposition of organic materials through pyrolysis and it has the potential to increase crop yields [10,11]....

We evaluated the fatty acid compositions in relation to yield and soil nutrients from four fields A, B, C, and D with continuous monocropping histories of 0–3 years, respectively, in Japan from 2015 to 2016. Results showed that, in both evaluation years, seed yield did not significantly differ among the fields although field A produced the highest mean seed yield and 1000-seed weight. Between fields A and C, 1000-seed weight showed significant differences. The contents of seed-saturated fatty acids lauric and myristic decreased in only fields C and D whereas oleic, linoleic, and linolenic acids increased in field D. Only field A produced the highest contents of lauric and myristic acids whereas field D produced the highest contents of linoleic and linolenic acids. The soil total N and exchangeable K contents tended to decrease as exchangeable Mg content significantly increased on the fields with long duration of cropping, fields C and D. Principal component analysis revealed significant positive correlations between soil exchangeable K, and total N contents with 1000-seed weight and lauric acid, as exchangeable Mg content was related with oleic, linoleic, and linolenic acids. Therefore, the high oleic, linoleic, and linolenic acids from field D were mainly attributed to high soil exchangeable Mg content, whereas the high 1000-seed weight, lauric acid and myristic acid were due to the high soil exchangeable K content in field A. Overall, the fatty acid composition quality on the long-duration continuously monocropped fields could show high economic value at the expense of yield under this management practice in continuous monocropping.