Putative genes involved in saikosaponin biosynthesis in Bupleurum species.

Alternative medicinal agents, such as the herb Bupleurum, are increasingly used in modern medicine to supplement synthetic drugs. First, we present a review of the currently known effects of triterpene saponins-saikosaponins of Bupleurum species. The putative biosynthetic pathway of saikosaponins in Bupleurum species is summarized, followed by discussions on identification and characterization of genes involved in the biosynthesis of saikosaponins. The purpose is to provide a brief review of gene extraction, functional characterization of isolated genes and assessment of expression patterns of genes encoding enzymes in the process of saikosaponin production in Bupleurum species, mainly B. kaoi. We focus on the effects of MeJA on saikosaponin production, transcription patterns of genes involved in biosynthesis and on functional depiction.

Methylation effect on chalcone synthase gene expression determines anthocyanin pigmentation in floral tissues of two Oncidium orchid cultivars.

The anthocyanin-biosynthetic pathway was studied in flowers of Oncidium Gower Ramsey with yellow floral color and mosaic red anthocyanin in lip crests, sepals and petals, and compared with the anthocyanin biosynthesis in flowers of Oncidium Honey Dollp, a natural somatoclone derived from tissue culture of Gower Ramsey, with a yellow perianth without red anthocyanins in floral tissues. HPLC analysis revealed that the red anthocyanin in lip crests of the Gower Ramsey cultivar comprised peonidin-3-O-glucoside, delphinidin-3-O-glucoside and cyanidin-3-O-glucoside, whereas Honey Dollp was devoid of anthocyanin compounds. Among the five anthocyanin-biosynthetic genes, OgCHS was actively expressed in lip crests of Gower Ramsey flowers, but no transcripts of OgCHS were detected in Honey Dollp floral tissues. Transient expression of OgCHS by bombardment confirmed that recovery of the OgCHS gene expression completed the anthocyanin pathway and produced anthocyanin compounds in lip crests of Honey Dollp flowers. Transcription factor genes regulating anthocyanin biosynthesis showed no distinctive differences in the expression level of OgMYB1, OgbHLH and OgWD40 between the two cultivars. A methylation assay revealed that the promoter of OgCHS was not methylated in Gower Ramsey, while a positive methylation effect was present in the upstream promoter region of OgCHS in Honey Dollp. Overall, our results suggest that the failure of anthocyanin accumulation in Honey Dollp floral tissues may be attributed to inactivation of the OgCHS gene resulting from the epigenetic methylation of 5'-upstream promoter region.

Differential expression of carotenoid-related genes determines diversified carotenoid coloration in floral tissues of Oncidium cultivars.

Three cultivars of Oncidium orchid with varied coloration, such as Oncidium Gower Ramsey (yellow), Sunkist (orange), and White Jade (white), were analyzed for carotenoid metabolites and gene expression of carotenoid-biosynthetic genes. The HPLC analysis revealed that yellow Gower Ramsey accumulates violaxanthin, 9-cis-violaxanthin and neoxanthin, orange Sunkist accumulates an additional beta-carotene, and White Jade is devoid of carotenoid compounds. Molecular characterization indicated that the three Oncidium cultivars exhibited varied expression pattern and level in carotenoid-biosynthetic pathway. Among them, high expression level of beta-hydroxylase (OgHYB) and zeaxanthin epoxidase (OgZEP) was displayed in yellow Gower Ramsey, relative to the down-regulation of OgHYB and OgZEP exhibited in orange Sunkist, which results in the accumulation of beta-carotene and orange coloration in floral tissues. However, White Jade is caused by the up-regulation of OgCCD1 (Carotenoid Cleavage Dioxygenase 1), which catabolizes carotenoid metabolites. Methylation assay of OgCCD1 promoter in White Jade and Gower Ramsey revealed that a high level of DNA methylation was present in OgCCD1 promoter region of Gower Ramsey. Transient expression of OgCCD1 in yellow lip tissues of Gower Ramsey by bombardment confirmed its function of disintegrating carotenoid compounds. Our results suggest an evolutionary significance that genetic variation of carotenoid-related genes in Oncidium generates the complexity of floral pigmentation and consequently provides the profound varieties in Oncidium population.

Development of an AVF Stenosis Assessment Tool for Hemodialysis Patients Using Robotic Ultrasound System.

With the aging population and lifestyle changes, the number of hemodialysis (HD) patients increases year by year. The arteriovenous fistula (AVF) is the gold standard vascular access used to access the blood for HD treatment. Since the status of stenosis affects HD efficiency, current clinical practices usually use a Doppler ultrasound imaging system to assess the parameters of the stenosis, such as the degree of stenosis (DOS). Unfortunately, this is a very time-consuming task. Furthermore, it is difficult to stably maintain the ultrasound probe for a prolonged period to give doctors clearer or reproducible images. In this study, a robotic ultrasound system (RUS) with ultrasound sequential imaging analysis was designed to evaluate the DOS of the AVF. The sequential imaging analysis was capable of image smoothing and vessel boundary detection. It enabled clinicians to mark the thickness of the plaque for further processing. Finally, the system was used to reconstruct 3D models of fistulas and calculated the DOS for clinical assessment. We also designed a pressure sensing module attached to the ultrasound probe to prevent the probe from coming loose, vibrating, and exerting abnormal pressure on the skin. In the phantom test, the results showed that the error of the DOS that was calculated by RUS was less than 3%. The results of clinical trials obtained from ten patients show that the error between the RUS and clinicians' measurement was about 10% and had a highly linear correlation (R Square > 0.95). In addition, the reproducibility error was about 3% and could effectively save about 46% of the time during clinical examinations.

RNA interference-based gene silencing of phytoene synthase impairs growth, carotenoids, and plastid phenotype in Oncidium hybrid orchid.

Phytoene synthase (PSY) is the first rate-limiting regulatory enzyme in the carotenoid biosynthesis pathway. In order to modify the floral color pattern by reducing carotenoid contents, a phytoene synthase-RNAi construct was delivered into protocorm-like body (PLB) of Oncidium hybrid orchid. The transgenic orchids show down-regulated level of PSY and geranyl synthase gene. They displayed semi-dwarf phenotype and brilliant green leaves. The microscopic anatomy revealed development-arrested plastids with rare grana. The total carotenoid content was decreased and the efficiency of the photosynthetic electron transport was declined. The chlorophyll level and the expression of chlorophyll biosynthetic genes, such as OgGLUTR and OgCS were dramatically reduced. HPLC analysis showed that the endogenous level of gibberellic acid and abscisic acid in the dwarf transformants are 4-fold lower than in wild type plants. In addition, chilling tolerance of the transgenic Oncidium plants was reduced. The data showed that down-regulation of PSY resulted in alterations of gene expression in enzymes involved in many metabolic pathways, such as carotenoid, gibberellic acid, abscisic acid and chlorophyll biosynthetic pathway as well as causes predominant defects in plant growth and development.

Differential expression of MYB gene (OgMYB1) determines color patterning in floral tissue of Oncidium Gower Ramsey.

The yellow coloration pattern in Oncidium floral lip associated with red sepal and petal tissues is an ideal model to study coordinate regulation of anthocyanin synthesis. In this study, chromatography analysis revealed that the red coloration in floral tissues was composed of malvidin-3-O-galactoside, peonidin-3-O-glucoside, delphinidin-3-O-glucoside and cyanidin-3-O-glucoside compounds. By contrary, these pigments were not detected in yellow lip tissue. Four key genes involved in anthocyanin biosynthetic pathway, i.e. chalcone synthase (OgCHS), chalcone isomerase (OgCHI), dihydroflavonol 4-reductase (OgDFR) and anthocyanidin synthase (OgANS) were isolated and their expression patterns were characterized. Northern blot analysis confirmed that although they are active during floral development, OgCHI and OgDFR genes are specifically down-regulated in yellow lip tissue. Bombardment with OgCHI and OgDFR genes into lip tissue driven by a flower-specific promoter, Pchrc (chromoplast-specific carotenoid-associated gene), demonstrated that transient expression of these two genes resulted in anthocyanin production in yellow lip. Further analysis of a R2R3 MYB transcription factor, OgMYB1, revealed that although it is actively expressed during floral development, it is not expressed in yellow lip tissue. Transient expression of OgMYB1 in lip tissues by bombardment can also induce formation of red pigments through the activation of OgCHI and OgDFR transcription. These results demonstrate that differential expression of OgMYB1 is critical to determine the color pattern of floral organ in Oncidium Gower Ramsey.

Characterization and promoter activity of chromoplast specific carotenoid associated gene (CHRC) from Oncidium Gower Ramsey.

Tissue-specific promoters are required for plant molecular breeding to drive a target gene in the appropriate location in plants. A chromoplast-specific, carotenoid-associated gene (OgCHRC) and its promoter (Pchrc) were isolated from Oncidium orchid and characterized. Northern blot analysis revealed that OgCHRC is specifically expressed in flowers, not in roots and leaves. Transient expression assay of Pchrc by bombardment transformation confirmed its differential expression pattern in floral tissues of different horticulture plants and cell-type location in conical papillate cells of adaxial epidermis of flower. These results suggest that Pchrc could serve as a useful tool in ornamental plant biotechnology to modify flower color.

Carbohydrate mobilization and gene regulatory profile in the pseudobulb of Oncidium orchid during the flowering process.

The pseudobulb of Oncidium orchid is a storage organ for supplying water, minerals and carbohydrates to the developing inflorescence. Different patterns of mannan, starch and pectin metabolism were observed in the pseudobulb of three developmental stages by histochemical staining and high performance anion exchange chromatographic (HPAEC) analysis. Copious pectin was strongly stained by ruthenium red in young pseudobulbs demonstrating that mannan and pectin were preferentially accumulated in the young pseudobulb sink at inflorescence pre-initiation stage. Concomitant with the emergence of the inflorescence, mannan and pectin decreased gradually and converted to starch. The starch, synthesized at the inflorescence developing stage, was eventually degraded at the floral development stage. A systematic survey on the subtractive EST (expression sequence tag) library of pseudobulb in the inflorescence pre-initiation stage revealed the presence of five groups of gene homologues related to sucrose, mannan, starch, pectin and other carbohydrate metabolism. The transcriptional level of 13 relevant genes related to carbohydrate metabolism was characterized from pseudobulbs of three different developmental stages. The specific activities of the enzymes encoded by these genes were also assayed. The expression profiles of these genes show that the transcriptional levels largely correlated with the enzyme activities, which were associated with the respective carbohydrate pools. These results demonstrated a novel functional profile of polysaccharide mobilization pathway as well as their relevant gene expression in the pseudobulb of Oncidium orchid during the flowering process.