<Emphasis Type="Italic">White clover mosaic virus</Emphasis>-induced gene silencing in pea

Double-stranded RNAs formed in secondary structures and replicative intermediates of viral genomes are thought to strongly elicit RNA silencing. This phenomenon is known as virus-induced gene silencing (VIGS). VIGS is a powerful tool for modifying gene expression in host plants. We constructed a virus vector based on White clover mosaic virus (WClMV) and demonstrated VIGS of phytoene desaturase (PDS) in pea. Photobleaching of tissues, caused by VIGS of PDS, was observed in restricted areas of upper leaves and stems. We confirmed that the PDS mRNA and subgenomic RNAs of WClMV were reduced in the photobleached tissues.     

Development of an on-site plum pox virus detection kit based on immunochromatography

Sharka disease, caused by plum pox virus (PPV), is the most serious viral disease of stone fruit trees. Among the eight known strains of the virus, PPV-D is the most important due to its recent global spread. Although enzyme-linked immunosorbent assay (ELISA) is the most common approach for diagnosing sharka, it involves time-consuming steps and requires expensive equipment and trained technicians. In this study, an on-site PPV detection kit based on immunochromatography was developed using polyclonal antibodies against the coat protein (CP) of a PPV-D isolate. The immunochromatographic (IC) assay kit was as sensitive as a commercial ELISA system for detecting Japanese PPV-D isolates. Moreover, it was easy to use (a one-step procedure), and results could be obtained on-site within 15 min without special laboratory equipment. The IC assay kit detected the virus from every aerial part of symptomatic Japanese apricot trees. In a detailed study of viral localization in leaves, the most suitable plant parts for use in the IC assay were symptomatic mesophyll tissues and the region from the petiole to the main vein. A positive reaction was also observed using the CP of other major (PPV-M and PPV-Rec) and minor (PPV-EA, PPV-W, and PPV-T) strains.     

Volatile chemicals identified in extracts from leaves of Japanese mugwort (Artemisia princeps pamp.)

Extracts from leaves of Japanese mugwort (Artemisia princeps Pamp.) were obtained using two methods: steam distillation under reduced pressure followed by dichloromethane extraction (DRP) and simultaneous purging and extraction (SPSE). A total of 192 volatile chemicals were identified in the extracts obtained by both methods using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). They included 47 monoterpenoids (oxygenated monoterpenes), 26 aromatic compounds, 19 aliphatic esters, 18 aliphatic alcohols, 17 monoterpenes (hydrocarbon monoterpenes), 17 sesquiterpenes (hydrocarbon sesquiterpenes), 13 sesquiterpenoids (oxygenated sesquiterpenes), 12 aliphatic aldehydes, 8 aliphatic hydrocarbons, 7 aliphatic ketones, and 9 miscellaneous compounds. The major volatile constituents of the extract by DRP were borneol (10.27 ppm), alpha-thujone (3.49 ppm), artemisia alcohol (2.17 ppm), verbenone (1.85 ppm), yomogi alcohol (1.50 ppm), and germacren-4-ol (1.43 ppm). The major volatile constituents of the extract by SPSE were 1,8-cineole (8.12 ppm), artemisia acetate (4.22 ppm), alpha-thujone (3.20 ppm), beta-caryophyllene (2.39 ppm), bornyl acetate (2.05 ppm), borneol (1.80 ppm), and trans-beta-farnesene (1. 78 ppm).     

Effect of nitrogen deposition on CH4 uptake in forest soils in Hokkaido,Japan

Abstract

It has been well documented by short-term artificial experiments that the CH⁴ uptake is inhibited by N input, especially NH^{4 p+}-N input. To investigate the effect of the natural N input by throughfall and other factors on the CH⁴ uptake in forest soils, we measured the CH⁴ uptake rates for 6 months during the snow-free period of the year and N input by throughfall throughout the year at 10 sites in Hokkaido, Japan, from 1997 to 2002. Water filled pore space (WFPS) and pH values in the soils varied widely among the sites (38-93% and 3.9-6.2, respectively). The rates of NH^{4 p+}-N and NH^{3 p-}-N inputs ranged from 1.3 to 6.9 kg N ha^p-1 year^p-1 and from 0.8 to 2.9 kg N ha^p-1 year^p-1, respectively. The NH^{4 p+}-N input was generally higher than the NH^{3 p-}-N input. Total N input by throughfall amounted to 2.3-9.4 kg N ha^p-1 year^p-1. The highest CH⁴ uptake rate occurred within the period from July to September (41-215 μg CH⁴ m^p-2 h^p-1) each year at most sites. CH⁴ uptake rate was relatively low (~50 μg CH⁴ M-2 h^p-1) at northern sites, while a high CH⁴ uptake rate was observed throughout the year 100 (? CH⁴ m^p-2 h^p-1) at southern sites. The mean CH⁴ uptake rates were significantly different among the sites. Cumulative CH⁴ uptake ranged from 1.4 to 6.6 kg CH⁴ ha^p-1 [184 d]^p-1 with a mean values of 3.22 ± 1.36 kg CH⁴ ha^p-1 [184 d]^p-1. Cumulative CH⁴ uptake increased with increasing temperature and decreased with an increase in precipitation (Rain), NH^{4 p+}-N input (TF^NH4) WFPS, soil total C (TC), and total N (TN). There was a quadratic relationship between the CH⁴ uptake and NH^{3 p-}-N input (TF^NO3), soil pH, and C / N ratio in soil. A regression equation was obtained as follows to predict the CH⁴ uptake in forest soils: Cumulative CH⁴ uptake = 0.47 / Rain + 0.38 / TF^NH4 + 0.34 / TC - 0.30 / TF^N03 (R ^p2 = 0.74, p = 0.0001). This equation indicates that atmospheric N input into forest soils is one of the main factors that control cumulative CH⁴ uptake with precipitation, total carbon content in soil in Hokkaido, Japan.     

Prenylated flavanones isolated from flowers of Azadirachta indica (the neem tree) as antimutagenic constituents against heterocyclic amines

Four prenylated flavanones were isolated from the methanol extract of the flowers of Azadirachta indica (the neem tree) as potent antimutagens against Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole) in the Salmonella typhimurium TA98 assay by activity-guided fractionation. Spectroscopic properties revealed that those compounds were 5,7,4'-trihydroxy-8-prenylflavanone (1), 5,4'-dihydroxy-7-methoxy-8-prenylflavanone (2), 5,7,4'-trihydroxy-3',8-diprenylflavanone (3), and 5,7,4'-trihydroxy-3',5'-diprenylflavanone (4). All isolated compounds were found for the first time in this plant. The antimutagenic IC(50) values of compounds 1-4 were 2.7 +/- 0.1, 3.7 +/- 0.1, 11.1 +/- 0.1, and 18.6 +/- 0.1 microM in the preincubation mixture, respectively. These compounds also similarly inhibited the mutagenicity of Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine). All of the compounds 1-4 strongly inhibited ethoxyresorufin O-dealkylation activity of cytochrome P450 1A isoforms, which catalyze N-hydroxylation of heterocyclic amines. However, compounds 1-4 did not show significant inhibition against the direct-acting mutagen NaN(3). Thus, the antimutagenic effect of compounds 1-4 would be mainly based on the inhibition of the enzymatic activation of heterocyclic amines.     

Tomatidine, a tomato sapogenol, ameliorates hyperlipidemia and atherosclerosis in apoE-deficient mice by inhibiting acyl-CoA:cholesterol acyl-transferase (ACAT)

It was previously revealed that esculeoside A, a new glycoalkaloid, and esculeogenin A, a new aglycon of esculeoside A, contained in ripe tomato ameliorate atherosclerosis in apoE-deficent mice. This study examined whether tomatidine, the aglycone of tomatine, which is a major tomato glycoalkaloid, also shows similar inhibitory effects on cholesterol ester (CE) accumulation in human monocyte-derived macrophages (HMDM) and atherogenesis in apoE-deficient mice. Tomatidine significantly inhibited the CE accumulation induced by acetylated LDL in HMDM in a dose-dependent manner. Tomatidine also inhibited CE formation in Chinese hamster ovary cells overexpressing acyl-CoA:cholesterol acyl-transferase (ACAT)-1 or ACAT-2, suggesting that tomatidine suppresses both ACAT-1 and ACAT-2 activities. Furthermore, the oral administration of tomatidine to apoE-deficient mice significantly reduced levels of serum cholesterol, LDL-cholesterol, and areas of atherosclerotic lesions. The study provides the first evidence that tomatidine significantly suppresses the activity of ACAT and leads to reduction of atherogenesis.     

Evaluation of the effects of spores and their heat-treated residues from different Bacillus strains on the initial growth of rice plants

This study evaluated the effects of the spores and their heat-treated residues from different strains of Bacillus species (B. pumilus, B. altitudinis, and B. megaterium) on the early growth of paddy rice cultivars, including Hitomebore (short-grain japonica rice), Takanari (high-yielding indica rice), and two new lines, TULK-143-6 and LTAT-29. The spores of seven Bacillus strains positively affected Hitomebore root growth, while, the root volume of TULK-143-6 with inoculation of B. pumilus TUAT1 and JM52, and root length and root surface area of LTAT-29 with inoculation of B. megaterium MAFF301694 were increased significantly. In contrast with Hitomebore, Takanari root growth was significantly inhibited by the spores of six Bacillus strains. Surprisingly, inoculations with the spore residues from all tested Bacillus strains increased the root dry weight of Hitomebore, with the effects of four bacterial strains being significant. Furthermore, there were more Bacillus spores than vegetative cells at different time points during the initial rice growth stage, and most plant samples mainly consisted of Bacillus spores. Thus, the spores of Bacillus species likely promote rice root development.     

Identification and some properties of anthocyanin isolated from Zuiki, stalk of Colocasia esculenta

Zuiki, a stalk of taro (Colocasia esculenta), is a traditional vegetable in Japan. Raw zuiki is often boiled and vinegared to eat. The surface color of zuiki is reddish. Here, we isolated a red pigment from zuiki and identified it as cyanidin 3-rutinoside using instrumental analyses. The color of zuiki disappeared by boiling, but the zuiki turned red again in an acetic acid solution. It seems that the cyanidin 3-rutinoside that exists on the surface of zuiki elutes in boiling water and then, the pigment that seeps out from the inside of the zuiki is exposed to an acid solution, and its surface turns red again. The radical scavenging activity of purified zuiki anthocyanin was 114 mg equivalent to BHT/g. About half of the anthocyanin in fresh zuiki was washed out by boiling, and the radical scavenging activity of zuiki was definitely reduced.