• Proceedings of the Botanical Society of Korea Conference
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• 1987.07a
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• pp.133-148
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• 1987

Gibberellin(GA) 3-$\beta$ hydroxylation is very important for the shoot elogation in the higher plants, since only 3$\beta$-hydryoxylated GAs promote shoot elogation in several plants. Fluctuation of 3$\beta$-hydryoxylase activity was examined during seed maturation using two cultivars of , P. vulgaris, Kentucky Wonder (normal) and Masterpiece (dwarf). Very immature seeds of both cultivars contain high level of 3$\beta$-hydroxylase activity (per mg protein). Both cultivars showed maximum of enzyme activity (per seed) in the middle of their maturation process. Gibberellin 3$\beta$-hydroxylase catalyzing the hydroxylation of GA20 to GA1 was purified 313-fold from very early immature seeds of P. vulgaris. Crude soluble enzyme extracts were purified by 15% methanol precipitation, hydrophobic interaction chromatogrphy, DEAE ion exchange column chromatography and gel filtration HPLC. The 3$\beta$-hydroxylase activity was unstable and lost much of its activity duting the purification. The molecular weight of purified enzyme was extimated to be 42, 000 by gel filtration HPLC and SDS-PAGE. The enzyme exhibited maximum activity at pH 7.7. The Km values for [2.3-3H] GA20 and [2.3-3H]GA9 were 0.29 $\mu$M and 0.33 $\mu$M, respectively. The enzyme requires 2-oxoglutarate as a cosubstrate; the Km value for 2-oxoglutarate was 250 $\mu$M using 3H GA20 as a substrate. Fe2+ and ascorbate significantly activated the enzyme at all purification steps, while catalase and BSA activated the purified enzyme only. The enzyme was inhibited by divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Effects of several GAs and GA anaogues on the putrified 3$\beta$-hydroxylase were examined using [3H]GA9 and GA20 as a substrates. Among them, GA5, GA9, GA15, GA20 and GA44 inhibited the enzyme activity. [13C, 3H] GA20 was converted by the partially purified enzyme preparation to [13C, 3H]GA1, GA5 and GA6, which were identified by GC-MS, GA9 was converted only GA4, GA15 and GA44 were converted to GA37 and GA38, respectively. GA5 was epoxidized to GA6 by the preparation. This suggests that 3$\beta$-hydroxylation of GA20 and epoxidation of GA5 are catalyzed by the same enzyme in P, vulgaris.

• Journal of Plant Biology
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• v.35 no.3
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• pp.185-190
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• 1992

Changes in activity of gibberellin $3{\beta}-hydroxylase$ which converts $[^3H]GA_{20}\;to\;GA_1$ were studied during seed maturation using partially purified enzyme preparations of two cultivars, Kentucky Wonder (normal) and Masterpiece (dwarf) of Phaseolus vulgaris. The specific activity of $3{\beta}-hydroxylase$ per seed reached maximum at 21 days after flowering and subsequently decreased during seed maturation in both cultivars. The ratios of conversion of $[17-^{13}C,\;^3H_2]GA_{20}\;to\;GA_1.\;GA_5,\;and\;GA_6$ by the same amount of $3{\beta}-hydroxylase$ were almost identical. Epoxidation of $GA_5\;to\;GA_6$ is also catalyzed by the partially purified $3{\beta}-hydroxylase$ preparation(Kobayashi et aI., 1991) and the conversion was inhibited by the substrates of $3{\beta}-hydroxylase$. These results suggest that the same enzyme might catalyze $3{\beta}-hydroxylase{\;}of{\;}GA_{20}\;to\;GA_1$ and epoxidation of $GA_5\;to\;GA_6$..

• Journal of Life Science
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• v.14 no.4
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• pp.644-649
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• 2004

During plant development, active gibberellins (GAs) control many aspects of plant growth and development including seed germination, stem elongation, flower induction, anther development and seed growth. To understand the biosynthesis and functional role of active GAs in high plants, this study investigated GA 3$\beta$-hydroxylase gene en-coding $GA_1$ and$GA_4$ catalizing last step in GA biosynthetic pathway. The antisense GA 3$\beta$-hydroxylase gene was inserted into expression vector, pIG121-Hm. Calli derived from mature seeds of rice (Oryza satiiva L. cv. Donjinbyeo) were co-cultivated with Agrohacterium tumefaciens EHA101 earring a pIG121-Hm containing hygromycin resistance ($Hyg^r$) and antisense GA 3$\beta$-hydroxylase gene. Seventeen transgenic plants obtained inhibiting GA 3$\beta$-hydroxylase. Transgenic plants had shorter plant height more than that of the Dongjinbyeo. Stable integration of antisense GA 3$\beta$-hydroxylase gene was confirmed by polymerase chain reaction of genomic DNA isolated from the leaf organs of the $T_o$ generation.

• Journal of Microbiology and Biotechnology
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• v.22 no.10
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• pp.1375-1380
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• 2012

We previously isolated a rhizobacterium (Bacillus subtilis IJ-31) and demonstrated that its associated allelochemicals could indicate plant growth retardation. However, little is known about how the growth of plants is regulated by B. subtilis IJ-31 and its allelochemicals. In this study, we investigated whether plant growth retardation in this relationship occurred through the inhibition of gibberellin (GA) biosynthesis. GA $3{\beta}$-hydroxylase activity was found to be inhibited by B. subtilis IJ-31 and hydrocinnamic acid (HCA), which is one of the allelochemicals produced by B. subtilis IJ-31. Additionally, thin layer chromatography (TLC) demonstrated that B. subtilis IJ-31 culture broth and HCA both inhibit GA $3{\beta}$-hydroxylase (MBP-GA4) activity. The retardation of plants by HCA was then confirmed in vivo and in vitro using a Ryegrass and Arabidopsis growth retardation assay. Furthermore, treatment with either B. subtilis IJ-31 culture extract or its allelochemicals resulted in significant down-regulation of XTR9 gene expression in Arabidopsis. Overall, we identified the functional mechanism of plant growth retardation by B. subtilis IJ-31 and its allelochemicals.

• Journal of Plant Biology
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• v.35 no.3
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• pp.191-195
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• 1992

Changes in activity of gibberellin C20-hydroxylase which converts $[^{14}C]GA_{12}\;to\;GA_{l5}$ were studied during seed maturation using the partially purified enzyme preparations of two cultivars, Kentucky Wonder (normal) and Masterpiece (dwarf) of Phaseolus vulgaris. The preparations obtained by methanol precipitation and hydrophobic interaction chromatography efficiently converted; $GA_{l2}\;to\;GA_{4},\;via\;GA_{15},\;GA_{24}\;and\;GA_{9};\;GA_{20}\;to\;GA_{1},\;GA_{5}\;and\;GA_{6}$. The activities of C20-hydroxylase which converts $GA_{l2}\;to\;GA_{l5}$ were almost the same in both cultivars. The C20-hydroxylase activity per protein reached a maximum at the very early immature seed stage, followed by a subsequent rapid decrease during seed maturation, whereas the enzyme specific activity per seed reached a maximum at 21 days after flowering, and showed a similar fiuctuation to that of the 313-hydroxylase which converts $GA_{20}\;to\;GA_{l}$ during seed maturation.ration.