Construction of TUATinsecta database that integrated plant and insect database for screening phytophagous insect metabolic products with medicinal potential.

Phytophagous insect larvae feed on plants containing secondary metabolic products with biological activity against other predatory organisms. Phytophagous insects can use their specialised metabolic systems to covert these secondary metabolic products into compounds with therapeutic properties useful to mankind. Some Asians drink tea decoctions made from phytophagous insect frass which is believed to be effective against inflammatory diseases. However, insects that can convert plant-derived secondary metabolic products into useful human therapeutic agents remain poorly studied. Here, we constructed the TUATinsecta database by integrating publicly plant/insect datasets for the purpose of selecting insect species. Using TUAT-insecta we selected the Asian swallowtail butterfly, Papilio xuthus larvae fed on several species of Rutaceous plants and examined whether the plant-derived secondary metabolites, especially those present in frass, were chemically altered or not. We extracted metabolic products from frass using three organic solvents with different polarities, and evaluated solvent fractions for their cytotoxic effects against several human cell lines. We found that chloroform frass extracts from P. xuthus larvae fed on Poncirus trifoliata leaves contained significant cytotoxic activity. Our findings demonstrate that screening of insect species using the 'TUATinsecta' database provides an important pipeline for discovering novel therapeutic agents that might be useful for mankind.

Construction of Models for Nondestructive Prediction of Ingredient Contents in Blueberries by Near-infrared Spectroscopy Based on HPLC Measurements.

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared spectroscopy is used to predict nondestructively total sugar, total organic acid, and total anthocyanin content in each blueberry. The technique is expected to enable the selection of only delicious blueberries from all harvested ones. The near-infrared absorption spectra of blueberries are measured with the diffuse reflectance mode at the positions not on the calyx. The ingredient contents of a blueberry determined by high-performance liquid chromatography are used to construct models to predict the ingredient contents from observed spectra. Partial least squares regression is used for the construction of the models. It is necessary to properly select the pretreatments for the observed spectra and the wavelength regions of the spectra used for analyses. Validations are necessary for the constructed models to confirm that the ingredient contents are predicted with practical accuracies. Here we present a protocol to construct and validate the models for nondestructive prediction of ingredient contents in blueberries by near-infrared spectroscopy.

Removal of redundant contigs from de novo RNA-Seq assemblies via homology search improves accurate detection of differentially expressed genes.

BACKGROUND: For plant species with unsequenced genomes, cDNA contigs created by de novo assembly of RNA-Seq reads are used as reference sequences for comparative analysis of RNA-Seq datasets and the detection of differentially expressed genes (DEGs). Redundancies in such contigs are evident in previous RNA-Seq studies, and such redundancies can lead to difficulties in subsequent analysis. Nevertheless, the effects of removing redundancy from contig assemblies on comparative RNA-Seq analysis have not been evaluated. RESULTS: Here we describe a method for removing redundancy from raw contigs that were primarily created by de novo assembly of Arabidopsis thaliana RNA-Seq reads. Specifically, the contigs with the highest bit scores were selected from raw contigs by a homology search against the gene dataset in the TAIR10 database. The two existing methods for removal of redundancy based on contig length or clustering analysis used to eliminate redundancies from raw contigs. Contig number was reduced most effectively with the method based on homology search. In a comparative analysis of RNA-Seq datasets, DEGs detected in contigs that underwent redundancy removal via the homology search method showed the highest identity to the DEGs detected when the TAIR10 gene dataset was used as an exact reference. Redundancy in raw contigs could also be removed by a homology search against integrated protein datasets from several plant species other than A. thaliana. DEGs detected using contigs that underwent such redundancy-removed also showed high homology to DEGs detected using the TAIR10 gene dataset. CONCLUSION: Here we describe a method for removing redundant contigs within raw contigs; this method involves a homology search against a gene or protein database. In principal, this method can be used with unsequenced plant genomes that lack a well-developed gene database. Redundant contigs were not removed adequately via either of two existing methods, but our method allowed for removal of all redundant contigs. To our knowledge, this is the first reported improvement in accurate detection of DEGs via comparative RNA-Seq analysis that involved preparation of a non-redundant reference sequence. This method could be used to rapidly and cost-effectively detect useful genes in unsequenced plants.

Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model.

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-ß/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of Ki-67(+) proliferating cells within GST-P(+) foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P(+) foci (area) to 48.9-49.4% and Ki-67(+) cells to 55.5-61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN(-) foci, phospho-Akt substrate(+) foci, phospho-Smad3(-) foci and Smad4(-) foci in GST-P(+) foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN(-) foci in GST-P(+) foci to 59.8-72.2% and Smad4(-) foci to 62.4-71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate(+) foci in GST-P(+) foci to 75.2-75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-ß/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells.

Length of the dark period affects flower opening and the expression of circadian-clock associated genes as well as xyloglucan endotransglucosylase/hydrolase genes in petals of morning glory (Ipomoea nil).

KEY MESSAGE: We isolated differentially expressed and dark-responsive genes during flower development and opening in petals of morning glory. Flower opening usually depends on petal expansion and is regulated by both genetic and environmental factors. Flower opening in morning glory (Ipomoea nil) is controlled by the dark/light regime just prior to opening. Opening was normal after 8- or 12-h dark periods but progressed very slowly after a 4-h dark period or in continuous light. Four genes (InXTH1-InXTH4) encoding xyloglucan endotransglucosylase/hydrolases (XTHs) and three genes (InEXPA1-InEXPA3) encoding alpha-expansins (EXPAs) were isolated. The expression patterns of InXTH2, InXTH3, and InXTH4 in petals were closely correlated with the rate of flower opening controlled by the length of the dark period prior to opening, but those of the EXPA genes were not. The expression pattern of InXTH1 gene was closely correlated with petal elongation. Suppression subtractive hybridization was used to isolate dark-responsive genes accompanying flower opening. The expressions of ten isolated genes were associated with the length of the dark period prior to flower opening. One gene was highly homologous to Arabidopsis pseudo-response regulator7, which is associated with the circadian clock and phytochrome signaling; another to Arabidopsis REVEILLE1, which affects the output of the circadian clock. Other genes were related to light responses, plant hormone effects and signal transduction. The possible roles of these genes in regulation of flower opening are discussed.

Expression of an AtNAP gene homolog in senescing morning glory (Ipomoea nil) petals of two cultivars with a different flower life span.

AtNAP, a NAC family transcription factor, has been shown to promote leaf senescence in Arabidopsis. We isolated an AtNAP homolog in morning glory (Ipomoea nil), designated InNAP, and investigated its expression during petal senescence. We used two cultivars, one showing a normal short flower life span (cv. Peking Tendan) and another a longer life span (cv. Violet). InNAP was highly expressed in both cultivars. Expression was high before that of the senescence marker gene InSAG12. InNAP and InSAG12 expression was high in cv. Peking Tendan before cv. Violet. The expression of both genes was therefore temporally related to the onset of the visible senescence symptoms. An inhibitor of ethylene action (silver thiosulphate, STS) delayed petal senescence in cv. Peking Tendan but had no effect in cv. Violet. STS treatment had no clear effect on the InNAP expression in petals of both cultivars, suggesting that endogenous ethylene may not be necessary for its induction. These data suggest the hypothesis that InNAP plays a role in petal senescence, independent of the role of endogenous ethylene.

Flower color alteration in Lotus japonicus by modification of the carotenoid biosynthetic pathway.

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding beta-carotene ketolase (4,4'-beta-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.

Physiological changes in gentian axillary buds during two-step preculturing with sucrose that conferred high levels of tolerance to desiccation and cryopreservation.

BACKGROUND AND AIMS: Induction of dehydration tolerance is a key to achieving high survival rates in cryopreservation of plant specimens. It has been reported previously that two-step preculturing with sucrose effectively increased desiccation tolerance in axillary buds of gentian (Gentiana scabra), which allow the buds to survive cryopreservation. This study is aimed at characterizing each step of this preculturing and to elucidate physiological changes induced during this preculturing. METHODS: In standard two-step preculture, excised gentian axillary buds were incubated for 11 d on MS medium with 0.1 m sucrose at 25 degrees C (first step: mild osmotic stress was given) and the subsequent incubation on MS medium with 0.4 m and 0.7 m sucrose for 1 d each (second step). The levels of abscisic acid (ABA), proline and soluble sugars in gentian buds during the preculture were determined. Effects of various combinations of two-step preculturing and of exogenous ABA and proline were studied. KEY RESULTS: During the first preculture step, there was a transient increase in ABA content peaking on day 4, which declined to a background level at the end of the first and second step preculturing. Proline level increased steadily during the first preculture step and increased further in the second preculture step. Incubating buds with medium containing proline, instead of the two-step preculturing, did not allow them to survive desiccation. Incubating buds with ABA instead of 0.1 m sucrose-preculturing effectively increased desiccation tolerance only when it was followed by the second preculture step. Fluridone, an ABA synthesis inhibitor included in the two-step preculture medium, reduced desiccation tolerance of the buds. The normal first-step preculture increased the levels of soluble sugars 2.4-fold, especially sucrose and raffinose. Buds treated with the second preculture step had greatly increased sucrose levels. CONCLUSIONS: These observations lead to the hypothesis that the first preculture step involves ABA-mediated cellular changes and the second step induces loading of sucrose in the gentian buds.