Baik Hwang

Cloning of a cDNA probably encoding oxidosqualene cyclase associated with asiaticoside biosynthesis from Centella asiatica (L.) Urban

A homology-based PCR method was used to clone a cDNA encoding oxidosqualene cyclase from Centella asiatica, which produces a large quantity of triterpene saponins such as asiaticoside and madecassoside. Sequence analysis of one clone found sequences related to β-amyrin synthase. An open reading frame in the full-length clone was named CabAS (Centella asiatica putative β-amyrin synthase). On the basis of amino acid sequence, CabAS appears to be an enzyme (β-amyrin synthase) that synthesizes β-amyrin. Southern analysis showed that the C. asiatica genome contains one copy of the CabAS gene. Northern blot analysis demonstrated that the CabAS gene is expressed in leaves with no detectable transcript in other plant tissues, consistent with the organ-specific accumulation of the asiaticoside. Up-regulation of expression of CabAS by methyl jasmonate in leaves was also demonstrated.

Isolation of cDNAs differentially expressed in response to drought stress and characterization of the Ca-LEAL1 gene encoding a new family of atypical LEA-like protein homologue in hot pepper (Capsicum annuum L. cv. Pukang)

Abstract Among various abiotic stresses, water deficit is one of the most severe environmental factors responsible for the reduction of crop yield in many parts of the world. By means of the mRNA differential display technique, seven cDNAs (pCa-DIs for Capsicum annuum drought induced) have been isolated that are rapidly induced when hot pepper plants are subjected to water stress (5–20% loss of fresh weight). For all of the isolated Ca-DIs , database search provided significant sequence similarity to previously described genes from different plant species. The predicted proteins encoded by the Ca-DI genes are putatively involved in processes as diverse as primary metabolism, protein degradation, cell wall modification and stress response, suggesting the complexity of cellular responses to drought stress in hot pepper plants. Particularly, we analyzed the detailed structural property and expression pattern of the Ca-DI4 ( Ca-LEAL1 ) gene. Sequence homology studies indicate that Ca-LEAL1 ( M r =19.3 kDa) belongs to a new family of atypical hydrophobic late embryogenesis-abundant-like (LEA-like) proteins. Expression analysis showed that Ca-LEAL1 was strongly activated by drought and salt stresses, and also in response to mechanical wounding in both local and systemic leaves. Moreover, the level of Ca-LEAL1 transcript was rapidly enhanced by exogenous application of ABA and ethylene. These results are consistent with the notion that an atypical hydrophobic Ca-LEAL1 protein is subject to control by diverse environmental factors and that ethylene, in conjunction with ABA, plays an important role in the regulation of the stress gene in hot pepper plants. The possible physiological functions of Ca-LEAL1 as well as other Ca-DI proteins in the adaptive process against drought stress are discussed

Polyacetylenes in hairy roots of Platycodon grandiflorum

Abstract Hairy roots of Korean balloon flower (Platycodon grandiflorum) were induced from root tissues infected with Agrobacterium rhizogenes ATCC 15834. Growth and polyacetylene (lobetyol, lobetyolin and lobetyolinin) production of 10 hairy roots clones cultured in 1 4 Gamborg B5 (B5) liquid medium were determined. One selected hairy root clone (D6) grew well in hormone-free B5 liquid medium and showed maximum content of polyacetylenes at week 6 for lobetyol (0.375% dry wt) and at week 7 for lobetyolin and lobetyolinin (3.030 and 0.206% dry wt, respectively) whose levels were much higher than those of the intact plant root (lobetyol: 0.019%; lobetyolin: 0.077% dry wt; lobetyolinin was not detected).

Production of antioxidant compounds by culture of Panax ginseng C.A. Meyer hairy roots: I. Enhanced production of secondary metabolite in hairy root cultures by elicitation.

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, established by infecting ginseng root discs with Agrobacterium rhizogenes, were used for secondary metabolite production. In this study, several elicitors [salicylic acid (SA), acetylsalicylic acid (ASA), yeast elicitor, and bacterial elicitor] were used to improve the productivity of useful metabolite in P. ginseng hairy root cultures. In SA elicitation, total ginseng saponin content increased slightly at lower elicitor dosages (0.1 to 0.5 mM). Also, the use of ASA as an elicitor resulted in the inhibition of biomass growth and an increase in total ginseng saponin content at every elicitor dosage (0.1 to 1.0 mM) by about 1.1 times. With yeast elicitor addition, hairy root growth was inhibited about 0.8-fold on a dry weight basis compared to the control, but total ginseng saponin content increased by about 1.17 times when compared to the control. The bacterial elicitor showed a slight inhibition of biomass growth, but total ginseng saponin content increased by about 1.23 times upon the addition of 1 mL.

Isolation and characterization of squalene synthase cDNA fromcentella asiatica (L) urban

We have cloned and characterized a gene for squalene synthase (SQS) fromCentella asiatica (L) Urban, a species that produces a large quantity of triterpene saponins such as asiaticoside and madecassoside. Its full-length cDNA clone was isolated by RACE PCR. The sequence ofpSQS contains an open reading frame of 1248 nucleotides, which code for 416 amino acids with a molecular mass of 47.3 kDa. Southern analysis revealed that one copy might exist in the C.asiatica genome. We also determined that 0.1 mM methyl jasmonate was sufficient to up-regulate those levels ofCaSQS mRNA.

Structure and expression of two cDNAs encoding S-adenosyl-l-methionine synthetase of rice (Oryza sativa L.)

Two cDNAs encoding rice (Oryza sativa L.) S-adenosyl-L-methionine synthetase (SAMS) have been cloned, sequenced and identified. The deduced protein sequences share a high homology (90-94%) with those of other plant SAMS and are 60-62% identical to yeast, rat and human SAMS. The rice SAMS genes are differentially regulated in a tissue-specific manner and by a salt stress, while they are coordinately expressed during growth of the rice cell culture.

Studies on mass production of transformed Panax ginseng hairy roots in bioreactor

The growth properties of Panax ginseng hairy roots transformed by Agrobacterium rhizogenes were compared between flask and aerated column or stirred bioreactor. In flask cultures, sucrose, initially 30 g/L, was nearly exhausted after 45 d of culture. The pH of the medium dropped from 5.5 to 4.96 after 10 d, but afterward it gradually increased to 6.4. After 45 d, hairy roots grew about 16-folds. The growth rate of hairy roots in air-bubble column or stirred bioreactor cultures was 1.13 (1.11) to 1.23 (1.20) g fresh wt (dry wt)/(g of cells x d), respectively. For both bioreactors, growth was about three times as high as in the flask cultivation.

Production of antioxidant compounds by culture of Panax ginseng C.A. Meyer hairy roots

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, established by infecting ginseng root discs with Agrobacterium rhizogenes, were used for secondary metabolite production. In this study, several elicitors [salicylic acid (SA), acetylsalicylic acid (ASA), yeast elicitor, and bacterial elicitor] were used to improve the productivity of useful metabolite in P. ginseng hairy root cultures. In SA elicitation, total ginseng saponin content increased slightly at lower elicitor dosages (0.1 to 0.5 mM). Also, the use of ASA as an elicitor resulted in the inhibition of biomass growth and an increase in total ginseng saponin content at every elicitor dosage (0.1 to 1.0 mM) by about 1.1 times. With yeast elicitor addition, hairy root growth was inhibited about 0.8-fold on a dry weight basis compared to the control, but total ginseng saponin content increased by about 1.17 times when compared to the control. The bacterial elicitor showed a slight inhibition of biomass growth, but total ginseng saponin content increased by about 1.23 times upon the addition of 1 mL.

Effect of growth regulators on asiaticoside production in whole plant cultures ofCentella asiatica (L) urban

We investigated the effects of growth regulators on whole-plant cultures derived from nodes ofCentella asiatica. A B5 liquid medium including 0.01 mg L-1 2,4-D resulted in decreased growth and asiaticoside production. Among the cytokinins tested (TDZ, BA, zeatin, and kinetin), TDZ was the best supplement for the promotion of asiaticoside biosynthesis. To directly estimate this effect, we measured asiaticoside content in the leaf, the main organ for synthesis. The addition of TDZ did not affect asiaticoside accumulation. Nevertheless, our results suggest that treatment with exogenous TDZ may enhance the production of asiaticoside in cultures simply through an increase in biomass.

Characterization of a dammarenediol synthase in Centella asiatica (L.) Urban.

To elucidate the exact function of CabAS in Centella asiatica, which was previously reported as a putative beta-amyrin synthase [Plant Cell Rep, 24:304-311, 2005], this gene was functionally expressed in the lanosterol synthase-deficient yeast mutant (erg7). After inducing the CabAS gene with galactose, a peak consistent with the dammarenediol standard was detected in LC/APCIMS analyses and the accumulated product was confirmed as dammarenediol. CabAS should therefore be renamed to C. asiatica dammarenediol synthase (CaDDS). The confirmation of this gene function may allow us to better understand the generation of numerous triterpene carbon skeletons.