Kyushu Okinawa Agricultural Research Center

Different colors, such as purple, brown, red and white, occur in the pericarp of rice. Here, two genes affecting proanthocyanidin synthesis in red- and brown-colored rice were elucidated. Genetic segregation analysis suggested that the Rd and A loci are identical, and both encode dihydroflavonol-4-reductase (DFR). The introduction of the DFR gene into an Rcrd mutant resulted in red-colored rice, which was brown in the original mutant, demonstrating that the Rd locus encodes the DFR protein. Accumulation of proanthocyanidins was observed in the transformants by the introduction of the Rd gene into the rice Rcrd line. Protein blot analysis showed that the DFR gene was translated in seeds with alternative translation initiation. A search for the Rc gene, which encodes a transacting regulatory factor, was conducted using available DNA markers and the Rice Genome Automated Annotation System program. Three candidate genes were identified and cloned from a rice RcRd line and subsequently introduced into a rice rcrd line. Brown-colored seeds were obtained from transgenic plants by the introduction of a gene containing the basic helix-loop-helix (bHLH) motif, demonstrating that the Rc gene encodes a bHLH protein. Comparison of the Rc locus among rice accessions showed that a 14-bp deletion occurred only in the rc locus.

ABSTRACT: The 80% ethanol extracts of 5 purple‐fleshed sweet potato cultivars were separated into 2 fractions, anthocyanins‐ and phenolic compounds‐rich fractions, to clarify the contribution of these constituents to the radical‐scavenging activity. The separation was accomplished with an ethyl acetate liquid/liquid extraction. 1,1‐Diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging activity in each fraction and the contributors varied according to the cultivars. The dominant DPPH radical‐scavengers in “Ayamurasaki” and “Kyushu‐132” were anthocyanins rather than phenolic compounds, while those in “Miyanou‐36” and “Bise” were phenolic compounds, such as chlorogenic acid. Furthermore, the high‐performance liquid chromatography analysis of anthocyanins showed that “Ayamurasaki” and “Kyushu‐132” were rich in anthocyanins with peonidin aglycon, whereas “Miyanou‐36,”“Bise,” and “Tanegashimamurasaki” contained cyanidin aglycon.

The extracts from white-, black-, and red-hulled rice were prepared by sequential extraction with six different polar solvents, and their radical-scavenging activities were measured by methods using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH*) and tert-butyl hydroperoxyl radical (t-BuOO*). The extracts prepared with highly polar solvents, methanol and deionized water, exhibited higher DPPH* and t-BuOO* scavenging activities in all three cultivars. In addition, the acetone extract from red-hulled rice exhibited a high DPPH* and t-BuOO* scavenging activity, while no such activity was detected for the acetone extracts from white- and black-hulled rice. The major components responsible for the radical scavenging in the acetone extract from red-hulled rice were identified as procyanidins by acidic hydrolysis, vanillin assay, and Sephadex LH-20 chromatography. GPC analysis of the acetylated procyanidins revealed that the average molecular weight is about 5000, in a range of about 500-18,000.

Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for pathogenicity of Fusarium oxysporum f. sp. melonis. Of the 1,129 REMI transformants tested, 13 showed reduced pathogenicity on susceptible melon cultivars. One of the mutants, FMMP95-1, was an arginine auxotroph. Structural analysis of the tagged site in FMMP95-1 identified a gene, designated ARG1, which possibly encodes argininosuccinate lyase, catalyzing the last step for arginine biosynthesis. Complementation of FMMP95-1 with the ARG1 gene caused a recovery in pathogenicity, indicating that arginine auxotrophic mutation causes reduced pathogenicity in this pathogen.

BACKGROUND AND AIMS: High night temperatures are more harmful to grain weight in rice than high day temperatures. Grain growth rate and growth duration were investigated to determine which was the cause of the decrease in final grain weight under high night temperatures. Endosperm cell number and cell sizes were also examined to determine which might cause the decrease in final grain weight. METHODS: Rice plants were grown outdoors in plastic pots and moved at heading time to three temperature-controlled glasshouses under high night temperature (HNT; 22/34 degrees C), high day temperature (HDT; 34/22 degrees C) and control conditions (CONT; 22/22 degrees C). Grains were sampled periodically, and the time-course of grain growth was divided into rate and duration by logistic regression analysis. Endosperm cell numbers and cell sizes were analysed by digitalized hand-tracing images of endosperm cross-sections. KEY RESULTS: The duration of grain growth was reduced by high temperature both day and night. However, the rate of grain growth was lower in HNT than in HDT. The number of cells in endosperm cross-sections in HNT was similar to that in HDT, and higher than that in CONT. The average cell area was smaller in HNT than in either CONT or HDT. The differences in average cell areas between HNT and HDT were greater at distances 60-80 % from the central point of endosperm towards the endosperm surface. CONCLUSIONS: The results show that HNT compared with HDT reduced the final grain weight by a reduction in grain growth rate in the early or middle stages of grain filling, and also reduced cell size midway between the central point and the surface of endosperm.

Many factors affect development of mammalian preimplantation embryos in vitro. It is well known that in vitro development of bovine embryos is highly affected by culture condition including energy source, growth factors, pH or gas environment. Many efforts have been made towards the suitable environments which can successfully support embryo development in vitro. For a rapid growth and differentiation, embryo requires energy by utilizing ATP, NADPH with oxygen molecules. These energy substrates are produced from the electron transport chain in the mitochondria. In addition to energy production, reactive oxygen species (ROS) are also generated as by-product of such energy production system. ROS production is sensitively controlled by the balance of oxidizing and reducing status and affected by several antioxidant enzymes such as superoxide dismutase (SOD), Catalase, glutathione peroxidase (GPx) or low molecular weight thiols such as glutathione (GSH). Imbalance of oxidation and reduction causes production of excess ROS, which causes the developmental arrest, physical DNA damage, apoptosis induction or lipid peroxidation. Environmental oxygen condition during embryo culture also highly affects embryo development as well as intracellular redox balance. Several studies have revealed that regulation of intra- and extra- cellular reducing environment by reducing excess ROS by using antioxidants, reducing oxygen concentration are effective for improving embryo development. Also, recent studies have demonstrated the difference in gene expression affected by oxidative stress. This review briefly summarizes the effects of ROS and the role of redox balance on preimplantation embryos for improving the efficiency of in vitro production of mammalian embryos.

Borate ester cross-linking of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II) is required for the growth and development of angiosperms and gymnosperms. Here, we report that the amounts of borate cross-linked RG-II present in the sporophyte primary walls of members of the most primitive extant vascular plant groups (Lycopsida, Filicopsida, Equisetopsida, and Psilopsida) are comparable with the amounts of RG-II in the primary walls of angiosperms. By contrast, the gametophyte generation of members of the avascular bryophytes (Bryopsida, Hepaticopsida, and Anthocerotopsida) have primary walls that contain small amounts (approximately 1% of the amounts of RG-II present in angiosperm walls) of an RG-II-like polysaccharide. The glycosyl sequence of RG-II is conserved in vascular plants, but these RG-IIs are not identical because the non-reducing L-rhamnosyl residue present on the aceric acid-containing side chain of RG-II of all previously studied plants is replaced by a 3-O-methyl rhamnosyl residue in the RG-IIs isolated from Lycopodium tristachyum, Ceratopteris thalictroides, Platycerium bifurcatum, and Psilotum nudum. Our data indicate that the amount of RG-II incorporated into the walls of plants increased during the evolution of vascular plants from their bryophyte-like ancestors. Thus, the acquisition of a boron-dependent growth habit may be correlated with the ability of vascular plants to maintain upright growth and to form lignified secondary walls. The conserved structures of pteridophyte, lycophyte, and angiosperm RG-IIs suggests that the genes and proteins responsible for the biosynthesis of this polysaccharide appeared early in land plant evolution and that RG-II has a fundamental role in wall structure.

Trials over two years were conducted using 1389 sweetpotato (Ipomoea batatas L.) genotypes collected from all over the world to characterize the polyphenolic composition in sweetpotato leaves. Wide variation was observed in relation to their total and individual leaf polyphenolic constituents. In all genotypes studied, the total polyphenol contents of sweetpotato leaf ranged from 1.42 to 17.1 g/100 g dry weight. The six different polyphenolic compounds were identified and quantified by NMR, FABMS, and RPHPLC analysis procedures. This is the first report of polyphenolic compositions in sweetpotato leaves. The relative levels of polyphenolic acids in sweetpotato leaves were as follows: 3,5-di-O-caffeoylquinic acid > 4,5-di-O-caffeoylquinic acid > chlorogenic acid (3-O-caffeoylquinic acid) > 3,4-di-O-caffeoylquinic acid > 3,4,5-tri-O-caffeoylquinic acid > caffeic acid. The highest 3,4,5-tri-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid occurred at 221 and 1183.30 mg/100 g dry weight, respectively.

A denaturing gradient gel electrophoresis (DGGE) method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil is presented. Five specific primers for 16S rDNA of methanogenic archaea, which were modified from the primers for archaea, were first evaluated by polymerase chain reaction and DGGE using genomic DNAs of 13 pure culture strains of methanogenic archaea. The DGGE analysis was possible with two primer pairs (0348aF-GC and 0691R; 0357F-GC and 0691R) of the five pairs tested although 16S rDNA of some non-methanogenic archaea was amplified with 0348aF-GC and 0691R. These two primer pairs were further evaluated for use in analysis of methanogenic archaeal community in Japanese paddy field soil. Good separation and quality of patterns were obtained in DGGE analysis with both primer pairs. A total of 41 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea. These results indicate that the procedure of DGGE analysis with the primer pair 0357F-GC and 0691R is suitable for investigating methanogenic archaeal community in paddy field soil.

The location of the 1:2 borate-diol ester cross-link in the dimer of the plant cell wall polysaccharide rhamnogalacturonan II (RG-II) has been determined. The ester cross-links the apiofuranosyl residue of the 2-O-methyl-d-xylose-containing side chains in each of the subunits of the dimer. The apiofuranosyl residue in each of the two aceric acid-containing side chains is not esterified. The site of borate esterification is identical in naturally occurring and inin vitro synthesized dimer. Pb²⁺, La³⁺, and Ca²⁺ increase dimer formationin vitro in a concentration- and pH-dependent manner. Pb²⁺ is the most effective cation. The dimer accounts for 55% of the RG-II when the monomer (0.5 mm) is treated for 5 min at pH 3.5 with boric acid (1 mm) and Pb²⁺ (0.5 mm); at pH 5 the rate of conversion is somewhat slower. Hg²⁺ does not increase the rate of dimer formation. A cation's charge density and its ability to form a coordination complex with RG-II, in addition to steric factors, may regulate the rate and stability of dimer formation in vitro. Our data provide evidence that the structure of RG-II itself determines which apiofuranosyl residues are esterified with borate and that in the presence of boric acid and certain cations, two RG-II monomers self-assemble to form a dimer.

The caffeoylquinic acid derivatives, 3-mono-O-caffeoylquinic acid (chlorogenic acid, ChA), 3,4-di-O-caffeoylquinic acid (3,4-diCQA), 3,5-di-O-caffeoylquinic acid (3,5-diCQA), 4,5-di-O-caffeoylquinic acid (4,5-diCQA) and 3,4,5-tri-O-caffeoylquinic acid (3,4,5-triCQA), and caffeic acid (CA) were isolated from the sweetpotato (Ipomoea batatas L.) leaf. We examined the antimutagenicity of these caffeoylquinic acid compounds to promote new uses of the sweetpotato leaf. These caffeoylquinic acid derivatives effectively inhibited the reverse mutation induced by Trp-P-1 on Salmonella typhimurium TA 98. The antimutagenicity of these derivatives was 3,4,5-triCQA > 3,4-diCQA = 3,5-diCQA = 4,5-diCQA > ChA in this order. There was no difference in the antimutagenicity of all dicaffeoylquinic acid derivatives. A comparison of the activities and structures of these compounds suggested that the number of caffeoyl groups bound to quinic acid played a role in the antimutagenicity of the caffeoylquinic acid derivatives. The sweetpotato leaves contained distinctive polyphenolic components with a high content of mono-, di-, and tricaffeoylquinic acid derivatives and could be a source of physiological functions.

Anthocyanins are polyphenolic ring-based flavonoids, and are widespread in fruits and vegetables of red-blue color. Epidemiological investigations and animal experiments have indicated that anthocyanins may contribute to cancer chemoprevention. The studies on the mechanism have been done recently at molecular level. This review summarizes current molecular bases for anthocyanidins on several key steps involved in cancer chemoprevention: (i) inhibition of anthocyanidins in cell transformation through targeting mitogen-activated protein kinase (MAPK) pathway and activator protein 1 (AP-1) factor; (ii) suppression of anthocyanidins in inflammation and carcinogenesis through targeting nuclear factor kappa B (NF- $\kappa$ B) pathway and cyclooxygenase 2 (COX-2) gene; (iii) apoptotic induction of cancer cells by anthocyanidins through reactive oxygen species (ROS) / c-Jun NH(2)-terminal kinase (JNK)-mediated caspase activation. These data provide a first molecular view of anthocyanidins contributing to cancer chemoprevention.

Studies on the physiological functionality of purple-fleshed sweet potatoes and their dominant anthocyanin pigments are described. The purple-fleshed sweet potato cultivar ‘Ayamurasaki’ contained anthocyanins, which consisted of mono- or di-acylated forms of cyanidin (YGM-1a, -1b, -2 and -3) and peonidin (YGM-4b, -5a, -5b and -6). It was also rich in anthocyanins with peonidin aglycon. The ‘Ayamurasaki’ extract and the purified YGM exhibited multiple physiological functions such as radical-scavenging, antimutagenic, angiotensin I-converting enzyme-inhibitory, and α-glucosidase-inhibitory activities in vitro. Moreover, they also showed an ameliorative effect on carbon tetrachloride-induced liver injury and decreased postprandial blood glucose levels in rats. In addition, their role in restoring the liver function and blood pressure levels to normal in volunteers with impaired hepatic function and/or hypertension was also confirmed. The acylated anthocyanins, which were the major radical scavengers in ‘Ayamurasaki’, were directly absorbed into the blood stream of rats and were present as intact acylated forms in the plasma, and could also enhance the plasma antioxidative capacity. Based on these evidences, the purple-fleshed sweet potato can be recommended as a superior source for the production of foods with health benefits. Some foods and beverages in Japan that utilize these characteristics of anthocyanin pigments are also introduced in this paper.

The toxicity of nine insecticides to predators of rice planthoppers was examined with first instars of four spider species, i.e. Pardosa pseudoannulata, Tetragnatha maxillosa, Ummeliata insecticeps and Gnathonarium exsiccatum, and females of the mirid bug Cyrtorhinus lividipennis and the dryinid wasp Haplogonatopus apicalis, by dipping the test arthropods into insecticide solution. Deltamethrin was the most toxic to the spiders, with LC50 at 0.033 to 1.1 ppm, followed by ethofenprox. The results indicate that the spiders are susceptible to synthetic pyrethroids. Insecticide susceptibilities of the spiders varied among species. T. maxillosa was more susceptible to seven insecticides, particularly to diazinon, than the other spiders, but P. pseudoannulata was more susceptible to phenthoate and carbaryl than T. maxillosa. Many insecticides, particularly phenthoate, imidacloprid and deltamethrin, were toxic to C. lividipennis. All insecticides tested were toxic to H. apicalis. We also evaluated the effect of insecticides on the spiders and C. lividipennis in paddy fields. Deltamethrin had a destructive effect on the spider populations and may have induced a resurgence of the Nilaparvata lugens population. Phenthoate reduced the abundance of lycosid spiders, and ethofenprox reduced the abundance of Tetragnatha. The C. lividipennis abundance decreased to a low level in all insecticide-treated plots except those treated by buprofezin.

Boron (B) deficiency results in inhibition of pumpkin (Cucurbia moschata Duchesne) growth that is accompanied by swelling of the cell walls. Monomeric rhamnogalacturonan II (mRG-II) accounted for 80% to 90% of the total RG-II in B-deficient walls, whereas the borate ester cross-linked RG-II dimer (dRG-II-B) accounted for more than 80% of the RG-II in control plants. The results of glycosyl residue and glycosyl linkage composition analyses of the RG-II from control and B-deficient plants were similar. Thus, B deficiency does not alter the primary structure of RG-II. The addition of (10)B-enriched boric acid to B-deficient plants resulted within 5 h in the conversion of mRG-II to dRG-II-(10)B. The wall thickness of the (10)B-treated plants and control plants was similar. The formation and possible functions of a borate ester cross-linked RG-II in the cell walls are discussed.

Pediococcus species isolated from forage crops were characterized, and their application to silage preparation was studied. Most isolates were distributed on forage crops at low frequency. These isolates could be divided into three (A, B, and C) groups by their sugar fermentation patterns. Strains LA 3, LA 35, and LS 5 are representative isolates from groups A, B, and C, respectively. Strains LA 3 and LA 35 had intragroup DNA homology values above 93.6%, showing that they belong to the species Pediococcus acidilactici. Strain LS 5 belonged to Pediococcus pentosaceus on the basis of DNA-DNA relatedness. All three of these strains and strain SL 1 (Lactobacillus casei, isolated from a commercial inoculant) were used as additives to alfalfa and Italian ryegrass silage preparation at two temperatures (25 and 48 degrees C). When stored at 25 degrees C, all of the inoculated silages were well preserved and exhibited significantly (P < 0.05) reduced fermentation losses compared to that of their control in alfalfa and Italian ryegrass silages. When stored at 48 degrees C, silages inoculated with strains LA 3 and LA 35 were also well preserved, with a significantly (P < 0.05) lower pH, butyric acid and ammonia-nitrogen content, gas production, and dry matter loss and significantly (P < 0.05) higher lactate content than the control, but silages inoculated with LS 5 and SL 1 were of poor quality. P. acidilactici LA 3 and LA 35 are considered suitable as potential silage inoculants.

The aim of this study was to use small unmanned aerial vehicles (UAVs) for determining high-resolution normalized difference vegetation index (NDVI) values. Subsequently, these results were used to assess their correlations with fertilizer application levels and the yields of rice and wheat crops. For multispectral sensing, we flew two types of small UAVs (DJI Phantom 4 and DJI Phantom 4 Pro)—each equipped with a compact multispectral sensor (Parrot Sequoia). The information collected was composed of numerous RGB orthomosaic images as well as reflectance maps with spatial resolution greater than a ground sampling distance of 10.5 cm. From 223 UAV flight campaigns over 120 fields with a total area coverage of 77.48 ha, we determined that the highest efficiency for the UAV-based remote sensing measurement was approximately 19.8 ha per 10 min while flying 100 m above ground level. During image processing, we developed and used a batch image alignment algorithm—a program written in Python language–to calculate the NDVI values in experimental plots or fields in a batch of NDVI index maps. The color NDVI distribution maps of wide rice fields identified differences in stages of ripening and lodging-injury areas, which accorded with practical crop growth status from aboveground observation. For direct-seeded rice, variation in the grain yield was most closely related to that in the NDVI at the early reproductive and late ripening stages. For wheat, the NDVI values were highly correlated with the yield ( R 2 = 0.601–0.809) from the middle reproductive to the early ripening stages. Furthermore, using the NDVI values, it was possible to differentiate the levels of fertilizer application for both rice and wheat. These results indicate that the small UAV-derived NDVI values are effective for predicting yield and detecting fertilizer application levels during rice and wheat production.

Climate change has been an increasingly significant factor behind fluctuations in the yield and quality of rice (Oryza sativa L.), particularly regarding chalky (white-back, basal-white, and milky-white) grain, immature thin grain, and cracked grain. The development and use of heat-tolerant varieties is an effective way to reduce each type of grain damage based on the existence of each varietal difference. Cultivation methods that increase the available assimilate supply per grain, such as deep-flood irrigation, are effective for diminishing the occurrence of milky-white grains under high temperature and low solar radiation conditions. The application of sufficient nitrogen during the reproductive stage is important to reduce the occurrence of most heat damage with the exception of milky-white grain. In regard to developing measures for heat-induced poor palatability of cooked rice, a sensory parameter, the hardness/adhesion ratio may be useful as an indicator of palatability within a relatively wide air–temperature range during ripening. Methods for heat damage to rice can be classified as either avoidance or tolerance measures. The timing of the measures is further divided into preventive and prompt types. The use of heat-tolerant varieties and late transplanting are preventive measures, whereas the application of sufficient nitrogen as a top dressing and irrigation techniques during the reproductive stage are prompt types which may function to lower the canopy temperature by enhancing evapotranspiration. Trials combining the different types of techniques will contribute towards obtaining more efficient and steady countermeasures against heat damage under conditions of climate change.

1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity of the 70% aqueous acetone extract from the seed coat of the brown soybean variety, Akita-Zairai, was investigated. The activity of the seed coat of Akita-Zairai was much higher than that of three other reddish-brown varieties, but lower than that of two black varieties, and was closely dependent on the content of phenolic compounds. In the LH20 column chromatography of Akita-Zairai, high DPPH radical-scavenging activities were detected in the fractions eluted with MeOH and 70% aqueous acetone. Proanthocyanidins were also detected in fractions showing high radical-scavenging activities. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis showed that the degree of polymerization (DP) of the procyanidins contained in the brown or black soybean seed coat was as high as DP30.

Abstract High temperature‐induced grain sterility in rice is becoming a serious problem in tropical rice‐growing ecosystems. We studied the mechanism of high temperature‐induced grain sterility of different rice ( Oryza sativa L) cultivars at two relative humidity (RH) levels. Four varieties of Indica and Japonica rice were exposed to over 85 % RH and 60 % RH at 36/30 °C, 34/30 °C, 32/24 °C and 30/24 °C day/night air temperatures from late booting to maturity inside sunlit phytotrons. Increasing both air temperature and RH significantly increased spikelet sterility while high temperature‐induced sterility decreased significantly with decreasing RH. Neither Indica nor Japonica rice types were superior to the other in the response of their spikelets to increased air temperature and RH. Increased spikelet sterility was due to increased pollen grain sterility which reduced deposition of viable pollen grains on stigma. Reduction in sterility with decreased RH was more due to decreased spikelet temperature than to air temperature. Thus the impact of RH should be considered when interpreting the effect of high temperature on grain sterility. Spikelet fertility was curvilinearly related to spikelet temperature. Grain sterility increased when spikelet temperature increased over 30 °C while it became completely sterile at 36 °C. The ability of a variety to decrease its spikelet temperature with decreasing RH could be considered as avoidance while the variability in spikelet sterility among varieties at a given spikelet temperature could be considered as true tolerance.