Chang-Sheng Kuoh

Alkaloidal and other constituents from the root bark of Clausena excavata

Ten new carbazole alkaloids, clausine-M, -N, -O, -P, -Q, -R, -S, -U, -V and clausenatine-A, together with 39 known compounds were isolated and identified from the acetone extract of the root bark of Clausena excavata. Their structures have been elucidated by spectroscopic analyses. This is the first report of a decarboxylated carbazole alkaloid from a natural source. The presence of a C23-skeleton carbazole alkaloid in a plant of Clausena genus is also reported for the first time.

Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway

BackgroundFloral scent is one of the important strategies for ensuring fertilization and for determining seed or fruit set. Research on plant scents has hampered mainly by the invisibility of this character, its dynamic nature, and complex mixtures of components that are present in very small quantities. Most progress in scent research, as in other areas of plant biology, has come from the use of molecular and biochemical techniques. Although volatile components have been identified in several orchid species, the biosynthetic pathways of orchid flower fragrance are far from understood. We investigated how flower fragrance was generated in certain Phalaenopsis orchids by determining the chemical components of the floral scent, identifying floral expressed-sequence-tags (ESTs), and deducing the pathways of floral scent biosynthesis in Phalaneopsis bellina by bioinformatics analysis.ResultsThe main chemical components in the P. bellina flower were shown by gas chromatography-mass spectrometry to be monoterpenoids, benzenoids and phenylpropanoids. The set of floral scent producing enzymes in the biosynthetic pathway from glyceraldehyde-3-phosphate (G3P) to geraniol and linalool were recognized through data mining of the P. bellina floral EST database (dbEST). Transcripts preferentially expressed in P. bellina were distinguished by comparing the scent floral dbEST to that of a scentless species, P. equestris, and included those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase. In addition, EST filtering results showed that transcripts encoding signal transduction and Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, were detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpointed 66% of the biosynthetic steps from G3P to geraniol, linalool and their derivatives.ConclusionThis systems biology program combined chemical analysis, genomics and bioinformatics to elucidate the scent biosynthesis pathway and identify the relevant genes. It integrates the forward and reverse genetic approaches to knowledge discovery by which researchers can study non-model plants.

A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2–4D motif

SUMMARY Geranyl diphosphate (GDP) is the precursor of monoterpenes, which are the major floral scent compounds in Phalaenopsis bellina. The cDNA of P. bellina GDP synthase (PbGDPS) was cloned, and its sequence corresponds to the second Asp-rich motif (SARM), but not to any aspartate-rich (Asp-rich) motif. The recombinant PbGDPS enzyme exhibits dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and a yeast two-hybrid assay and gel filtration revealed that PbGDPS was able to form a homodimer. Spatial and temporal expression analyses showed that the expression of PbGDPS was flower specific, and that maximal PbGDPS expression was concomitant with maximal emission of monoterpenes on day 5 post-anthesis. Homology modelling of PbGDPS indicated that the Glu-rich motif might provide a binding site for Mg(2+) and catalyze the formation of prenyl products in a similar way to SARM. Replacement of the key Glu residues with alanine totally abolished enzyme activity, whereas their mutation to Asp resulted in a mutant with two-thirds of the activity of the wild-type protein. Phylogenetic analysis indicated that plant GDPS proteins formed four clades: members of both GDPS-a and GDPS-b clades contain Asp-rich motifs, and function as homodimers. In contrast, proteins in the GDPS-c and GDPS-d clades do not contain Asp-rich motifs, but although members of the GDPS-c clade function as heterodimers, PbGDPS, which is more closely related to the GDPS-c clade proteins than to GDPS-a and GDPS-b proteins, and is currently the sole member of the GDPS-d clade, functions as a homodimer.

Cytotoxic and antiplatelet aggregation principles from Aglaia elliptifolia

Two related 1H-2,3,3a,8b-tetrahydrocyclopenta[b]benzofurans, aglafolin (1a) and rocaglamide (2), isolated from the stems of Aglaia elliptifolia, showed significant cytotoxicity in six cancer cell lines. Aglafolin (1a) was also found to completely block platelet aggregation caused by arachidonic acid and platelet-activating factor at 100 microM and 2 ng/mL, respectively.

Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in Oncidium

AbstractTraditional breeding processes to genetically modify the long reproductive cycle and slow seed maturation of orchids have limits. We developed a more efficient protocol using particle bombardment to produce transgenic plants of Oncidium Sharry Baby ‘OM8’ (Orchidaceae). Pretreating protocorm-like bodies (PLBs) with 0.5 M sucrose for 2 h increased single-cell embryogenesis 3- to 4-fold; however, shoot formation was suppressed. In addition, new PLBs were regenerated from the entire sucrose-pretreated PLBs, whereas in untreated PLBs, this occurred only from the bases. Pretreated PLBs were bombarded with pSPFLP containing genes encoding a sweet pepper ferredoxin-like protein (pflp), hygromycin phosphotransferase (hpt) and β-glucuronidase (GUS) driven by the cauliflower mosaic virus 35S promoter. Pretreated PLBs showed a 14.8-fold increase in GUS expression over the untreated PLBs 40 days after bombardment. The presence of pflp and hpt transgenes in the 40 putatively stably transformed lines that produced 113 clones was confirmed by PCR analysis. Six lines (eight clones) were positive for both pflp and hpt transgenes. In addition, clones derived from these lines were either all positive or all negative for the two transgenes, which suggests homogeneity in pretreated PLBs with more single-cell embryogenesis. Thus, sucrose pretreatment enhanced the regeneration of PLBs, single-cell embryogenesis and efficiency of transformation.

Coumarins, acridone alkaloids and a flavone from Citrus grandis

Abstract An acetone extract of root bark of Citrus grandis Osbeck gave a new flavone, honyucitrin, and a new coumarin, honyudisin, together with nine known coumarins and 11 acridone alkaloids. Their structures were determined by spectral methods and some chemical transformations. The antimicrobial activity of the compounds was also examined.

Annoquinone-A, an antimicrobial and cytotoxic principle from Annona montana

Abstract A new naturally occurring phenanthrene-1,4-quinone, annoquinone-A, along with parietin (physcion) and β-sitostenone were isolated from the stem bark of Annona montana . The structure of annoquinone-A was elucidated by spectral methods and synthesis. Annoquinone-A demonstrated potent antimicrobial activity against Bacillus subtilis and Micrococcus luteus as well as cytotoxicity in the KB ( ED 50 = 0.16 μg/ml) tissue culture assay. β-Sitostenone also showed significant cytotoxicity.

Development of simple sequence repeats (SSR) markers in Setaria Italica (poaceae) and cross-amplification in related species

Foxtail millet is one of the world’s oldest cultivated crops. It has been adopted as a model organism for providing a deeper understanding of plant biology. In this study, 45 simple sequence repeats (SSR) markers of Setaria italica were developed. These markers showing polymorphism were screened in 223 samples from 12 foxtail millet populations around Taiwan. The most common dinucleotide and trinucleotide repeat motifs are AC/TG (84.21%) and CAT (46.15%). The average number of alleles (Na), the average heterozygosities observed (Ho) and expected (He) are 3.73, 0.714, 0.587, respectively. In addition, 24 SSR markers had shown transferability to six related Poaceae species. These new markers provide tools for examining genetic relatedness among foxtail millet populations and other related species. It is suitable for germplasm management and protection in Poaceae.

Coumarins of the flowers of Murraya paniculata

Abstract Four new coumarins, omphalocarpin, (−)-murracarpin, murrayacarpin-A and -B together with known coumarins, scopolin, scopoletin, 5,7-dimethoxy-8-(3′-methyl-2′-oxobutyl)coumarin, (±)-murracarpin and mupanidin have been isolated from the flowers of Murraya paniculata var. omphalocarpa . Their structures have been characterized from spectral analysis, chemical transformation and/or synthesis.

Tetranortriterpenoid insect antifeedants from Severinia buxifolia

Abstract Two new tetranortriterpenoids, severinolide and cycloseverinolide, together with four known compounds, were isolated and characterized from the root bark of Severinia buxifblia. Severinolide, atalantin and cycloepiatalantin showed significant antifeeding effects against Plutella xylostalla.