• Korean Journal of Plant Tissue Culture
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• v.28 no.5
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• pp.249-254
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• 2001

Effects of ultraviolet stress and elicitors, cellulase and jasmonic acid (JA), for the production of capsidiol, sesquiterpenoid phytoalexin, in seedlings and suspension cultures of pepper (Capsicum annum L. cv, Soobicho) were examined. Extracellular capsidiol in the medium of suspension cultures was absent from control cells, but accumulated in the elicitor treated cells with 0.05 $\mu\textrm{g}$/mL of cellulase or 0.1 $\mu\textrm{g}$/mL JA. Elicited cells gradually decreased their viability and eventually died within 48 hours of elicitor treatment by the toxicity of capsidiol accumulated in the culture medium. Capsidiol production in the leaves of pepper seedlings was markedly increased by the treatment of ultraviolet stress and reached maximum level at 48 hours of irradiation. Infiltration of elicitors, 0.05 $\mu\textrm{g}$/mL cellulase or 1.0 $\mu\textrm{g}$/mL JA, to the surface of leaf or fruit, stimulated the elicitation of the cells which resulted in the production of capsidiol and expansion of pathogene-like lesion around the elicitor treated region.

• Korean Journal of Plant Resources
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• v.21 no.2
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• pp.139-143
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• 2008

Enzyme activity and expression of cytochrome P450 gene involved in the pathway of capsidiol biosynthesis were compared in five different solanaceae plants such as red pepper, green pepper, tobacco, potato and egg plant. Base on genomic DNA and/or RT-PCR results, four solanaceae plants such as red pepper, green pepper, tobacco and egg plant possess P450 gene in the genome and specifically expressed by elicitor treatment. However, potato was appeared to have neither P450 nor cyclase gene in the genome. P450 genes did not show any expression in the plants under normal condition, but showed highly specific expression under elicitation condition in various organs and tissue such as leaf, root, stem and culture cells.

• Journal of Life Science
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• v.11 no.6
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• pp.603-611
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• 2001

Phytolalexins are produced in plants affected by various environmental factors such as fungal infection treatment with many chemicals and irradiation by ultraviolet light. When pepper and tobacco bel suspension cultures were grown on a basal MS medium supplemented with 2,4-D(1mg/$\ell$, benzyl adenine(0.001 mg/$\ell$) and 100$\mu$ M jasmonic acid, the production of capsidiol was observed. The total of compound found in pepper plant were around seventy and thirty of them were located intissue-specific manner. 1-propanethiol, $\alpha$-D-xylofuranoside, phenol, hexadecanonic acid ethyl tridecanoate, phytol, linoleic acid and capsidiol are those which have change the production level by treatments, such as the inoculation of Phytophthora capsici Leonian, the metalaxyl treatment and the UV-B irradiation, respectively. The content of capsidiol on inoculation of P. capsici with metalxyl suspension in soil were higher than those of P.capsici without metalaxyl. When the soil dernch of metalaxyl treatment (1$\mu\textrm{g}$/${mu}ell$)was delayed after inoculation, the content of capsidiol were higher than that of before. Irrradiated UB-B the production on capsidiol was identified only at leaf, and contents were the highest for 24 hrs incubation after 20 minutes irradiation.

• Journal of Life Science
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• v.9 no.4
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• pp.408-413
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• 1999

Extracellular capsidiol, sesquiterpenoid phytoalexin, in the medium of pepper (Capsicum annuum L.) suspension cells was not identified from control cells, but highly accumulated in the elicitor-induced cells within 6 hours after the addition of 0.05$\mu\textrm{g}$/$m\ell$ cellulase. Capsidiol production in elicitor-induced cells was markedly suppressed by cytochrome P450 inhibitors, such as ancymidol and ketoconazole demonstrating that biosynthesis of capsidiol is catalyzed by at least on hydroxylation enzyme in the biochemical pathway. Based on protein electrophoresis, two bands, 23.0kDa and 27.5kDa, were identified as newly synthesized polypeptides in the elicitor-induced suspension cells, suggesting that pepper cells which were subjected to elicitor treatment activate specific gene(s) for capsidiol biosynthesis in cultured cells.

• Korean Journal of Plant Tissue Culture
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• v.25 no.3
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• pp.141-146
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• 1998

Feeding experiment of [$^3$H] 5-epi-aristolochene (5-EAS) demonstrated in suspension cultures of tobacco (Nicotiana tabacum L.) that 5-EAS hydroxylase activity was absent from control cells, but induced to a maximum level within 18 h after the addition of cellulase, and was very similar to induction pattern of sesquiterpene cyclase. This result suggest that the conversion of 5-EAS to capsidiol is catalyzed by at least one elicitor-inducible hydroxylase. Cytochrome P450 inhibitors, ancymidol and ketoconazole, suppressed the elicitor-induced capsidiol accumulation by inhibiting hydroxylase activity, suggesting that the hydroxylase may be a Cyt P450 type enzyme.

• Journal of Plant Biotechnology
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• v.30 no.2
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• pp.201-206
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• 2003

Differential responses of red pepper plant and cultured cells to enhanced ${\gamma}$-ray($^{60}$ Co) and ultraviolet(UV) stress were investigated. In seed treatment, 1 Gy of ${\gamma}$-ray increased seedling dry weight up to 19.1%, but 50 Gy treatment markedly ingibited seed germination and subsequent growth of seedling. UV treatment to seed did not change the germination ability of seeds and the growth of seedlings regardless of duration of UV treatment until 24 hrs. In case of UV treatment to seedlings, plant injury was seriously progressed even after the seedlings were returned to no UV condition, and eventually all the leaves showed chlorosis by the stress. However, progress of plant injury by ${\gamma}$-ray stress slower than that caused by UV stress, and even at the high dose of ${\gamma}$-ray 50 Gy, did not caused the cholrosis of stressed plant leaf. Amount of electrolytes leakage from plant leaf by UV treatment for 24hrs was increased up to 28.8 folds in comparison with untreated control, whereas that of 50 Gy of ${\gamma}$-ray was increased only 1.2 folds. UV stress induced the production of capsidiol, antimicrobial phytoalexin, by activation of gene expression involved in capsidiol biosynthesis, such as sesquiterpene cyclase and cyclase and cytochrome P450 hydroxylase in the leaf and cultured cell, but ${\gamma}$-ray stress induced neither the production of capsidiol nor expression of the genes.

• Journal of Life Science
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• v.13 no.6
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• pp.879-888
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• 2003

In order to measure the enzyme activity of 5-epi-aristolochene hydroxylase, one of cytochrome P450 (P450) enzymes in eicitor-treated pepper cell, we used in vivo assay method and demonstrated a dramatic suppression of the activity by P450-inhibitors, ancymidol and ketocornazole. Using RT-PCR method with degenerate primer of the well conserved domains found within most P450-enzymes, and using cDNA library screening method, one distinct cDNA, being designated P450Hy01, was successfully isolated from elicitor-treated pepper cells. P450Hy01 mRNA was all induced in elicitor-treated cells whereas never induced in control cells. Moreover, levels of P450Hy01 expression were highly correlated with the levels of extracellular capsidiol production by different elicitors in cell cultures. P450Hy01 transcript was also induced by several other elicitors such as, cellulase, arachidonic acid, jasmonic acid, yeast extract as well as UV stress. P450Hy01 sequence contained high probability amino acid matches to known Plant P450 genes and ORF with a conserved FxxGxRxCxG heme-binding domain. P450Hy01 cDNA showed 98% of homology in sequence of nucleotide as well as amino acid to 5-epi-aristolochene-1, 3-hydroxylase (5EAl, 3H) which has been isolated in tobacco cells, suggesting that P450Hy01 is prominent candidate gene for P450-enzyme encoding 5EAl, 3H in pepper cell.

• Journal of Life Science
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• v.8 no.5
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• pp.604-613
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• 1998

The last enzyme of the sesquiterpen phytoalexin capsidiol synthesis in tobacco cell, 5-epi-aristolochene hydro-xylase which convert 5-epi-aristolochene (EAS) to capsidiol, was cloned by a reverse transcription polymerase chain reaction strategy and cDNA library screening. Cloned CYP-B3 contained high probability amino acid matches to known plant cytochrome P450 sequences and open reading frame with the conserved FxxGxRxCxG heme-binding region. Transcripts of CYP-B3 were not detected in control cells, but induced in elicitor-treated cells. Furthermore, CYP-B3 transcripts were induced by fungal extracts and cellulase but not by other stimuli(chilling, heat shock and 2,4-D). Induction of CYP-B3 transcripts by elicitor treatment was not affected by ancymidol and ketoconazole treat-ments suggesting that an inhibition of hydroxylase activity by Cyt P450 inhibitors resulting from post translational processing event.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2003.04a
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• pp.98-98
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• 2003

• Proceedings of the Korean Society of Life Science Conference
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• 2001.09a
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• pp.30-30
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• 2001