• 통합자연과학논문집
• /
• 제4권2호
• /
• pp.113-120
• /
• 2011

Brassinosteroids (BRs) are plant steroid hormones that play essential roles in growth and development. Mutations in BR-signaling pathways cause defective in growth and development like dwarfism, male sterility, abnormal vascular development and photomorphogenesis. Transition from vegetative to reproductive growth is a critical phase change in the development of a flowering plant. In a screen of activation-tagged Arabidopsis, we identified a mutant named abz126 that displayed longer hypocotyls when grown in the dark on MS media containing brassinazole (Brz), an inhibitor of BRs biosynthesis. We have cloned the mutant locus using adapter ligation PCR walking and identified that a single T-DNA had been integrated into the ninth exon of the GIGANTEA (GI) gene, involved in controling flowering time. This insertion resulted in loss-of-function of the GI gene and caused the following phenotypes: long petioles, tall plant height, many rosette leaves and late flowering. RT-PCR assays on abz126 mutant showed that the T-DNA insertion in GIGANTEA led to the loss of mRNA expression of the GI gene. In the hormone dose response assay, abz126 mutant showed: 1) an insensitivity to paclobutrazole (PAC), 2) an altered response with 6-benzylaminopurine (BAP) and 3) insensitive to Brassinolide (BL). Based on these results, we propose that the late flowering and tall phenotypes displayed by the abz126 mutant are caused by a loss-of-function of the GI gene associated with brassinosteroid hormone signaling.

• Journal of Plant Biotechnology
• /
• 제30권4호
• /
• pp.411-419
• /
• 2003

GC-MS/SIM를 통하여 옥수수 유식물 신초로부터 해 castasterone (CS)과 6-deoxocastasterone (6-deoxoCS)을 동정하였다. 또한 BRs의 생합성 전구물질인 24$\alpha$-methylcholesterol과 24$\alpha$-methylcholestanol이 동정하였다. 이들 BRs와 생합성 전구물질은 BRs의 생합성과정 중 후기 C6산화과정에 속하는 화합물로서, 옥수수 유식물 줄기에서는 BRs의 생합성 과정으로 후기 C6산화과정이 주된 생합성 과정임을 알 수 있었다. 다음으로 옥수수 유식물 줄기내의 BRs가 발아와 더불어 종자에서부터 이동해 온 것인지, 아니면 줄기 생장 시 줄기 자체가 생합성된 것인지를 조사하기 위하여 몇몇 중요한 후기 C6산화과정의 반응을 촉매하는 효소의 활성이 유식물 줄기에 존재하는지를 조사하였다. 그 결과 후기 C6산화과정에 포함되는 24$\alpha$-methylcholestanol에 서 6-deoxocathasterone (6-deoxoCT), 6-deoxoteasterone (6-deoxoTE) 에서 6-deoxo-3-dehydroteasterone (6-deoxo-3-DHT)을 거쳐 6-deoxotyphasterol (6-deoxoTY),그리고 6-deoxoCS에서 CS로의 과정을 촉매하는 24$\alpha$-methylcholestanol 22(R)-hydroxlyase, 6-deoxoTE dehydrogenase/6-deoxo-3-DHT reductase, 6-deoxoCS oxidase의 활성이 옥수수 유식물 줄기에 존재하고 있음이 확인되었다. 이는 유식물 줄기 생장 시 필요한 BRs가 발아종자로부터 이동되는 것이 아니라, 유식물 줄기 자체에서 생합성 될 가능성이 높음을 나타내는 결과라 할 수 있다. 또한 옥수수 유식물 줄기에서 CS를 brassinolide(BL)로 전환하는 BL synthase의 활성이 검출되지 않아, 옥수수 유식물 줄기에서의 활성형 BR은 BL이 아닌 CS임을 밝혔다.

• 한국식물생명공학회:학술대회논문집
• /
• 한국식물생명공학회 2002년도 추계학술대회
• /
• pp.69-75
• /
• 2002

Sterols play two major roles in plants: a bulk component in biological membranes and precursors of plant steroid hormones. Physiological effects of plant steroids, brassinosteroids (BRs), include cell elongation, cell division, stress tolerance, and senescence acceleration. Arabidopsis mutants that carry genetic defects in BR biosynthesis or its signaling display characteristic phenotypes, such as short robust inflorescences, dark-green round leaves, and sterility. Currently there are more than 100 dwarf mutants representing 7 genetic loci in Arabidopsis. Mutants of 6 loci, dwf1/dim1/cbb1, cpd/dwf3, dwf4, dwf5, det2/dwf6, dwf7 are rescued by exogenous application of BRs, whereas bri1/dwf2 shares phenotypes with the above 6 loci but are resistant to BRs. These suggest that the 6 loci are defective in BR biosynthesis, and the one locus is in BR signaling. Biochemical analyses, such as intermediate feeding tests, examining the levels of endogenous BR, and molecular cloning of the genes revealed that dwf7, dwf5, and dwf1 are defective in the three consecutive steps of sterol biosynthesis, from episterol to campesterol via 5-dehydroepisterol. Similarly, det2/dwf6, dwf4, and cpd/dwf3 were shown to be blocked in $D^4$ reduction, 22a-hydroxylation, and 23 a-hydroxylation, respectively. A signaling mutant bri1/dwf2 carries mutations in a Leucine-rich repeat receptor kinase. Interestingly, the bri1 mutant was shown to accumulate significant amount of BRs, suggesting that signaling and biosynthesis are dynamically coupled in Arabidopsis. Thus It is likely that transgenic plants over-expressing the rate-limiting step enzyme DWF4 as well as blocking its use by BRI1 could dramatically increase the biosynthetic yield of BRs. When applied industrially, BRs will boost new sector of plant biotechnology because of its potential use as a precursor of human steroid hormones, a novel lead compound for cholesterol-lowering effects, and a various application in plant protection.

• 한국식물생명공학회:학술대회논문집
• /
• 한국식물생명공학회 2002년도 춘계학술대회
• /
• pp.69-75
• /
• 2002

Sterols play two major roles in plants: a bulk component in biological membranes and precursors of plant steroid hormones. Physiological effects of plant steroids, brassinosteroids (BRs), include cell elongation, cell division, stress tolerance, and senescence acceleration. Arabidopsis mutants that carry genetic defects in BR biosynthesis or its signaling display characteristic phenotypes, such as short robust inflorescences, dark-green round leaves, and sterility. Currently there are more than 100 dwarf mutants representing 7 genetic loci in Arabidopsis. Mutants of 6 loci, dwf1/dim1/cbb1, cpd/dwf3, dwf4, dwf5, det2/dwf6, dwf7 are rescued by exogenous application of BRs, whereas bri1/dwf2 shares phenotypes with the above 6 loci but are resistant to BRs. These suggest that the 6 loci are defective in BR biosynthesis, and the one locus is in BR signaling. Biochemical analyses, such as intermediate feeding tests, examining the levels of endogenous BR, and molecular cloning of the genes revealed that dwf7, dwf5, and dwf1 are defective in the three consecutive steps of sterol biosynthesis, from episterol to campesterol via 5-dehydroepisterol. Similarly, det2/dwf6, dwf4, and cpd/dwf3 were Shown to be blocked in $D^4$ reduction, 22a-hydroxylation, and 23 a-hydroxylation, respectively. A signaling mutant bri1/dwf2 carries mutations in a Leucine-rich repeat receptor kinase. Interestingly, the bri1 mutant was shown to accumulate significant amount of BRs, suggesting that signaling and biosynthesis are dynamically coupled in Arabidopsis. Thus it is likely that transgenic plants over-expressing the rate-limiting step enzyme DWF4 as well as blocking its use by BRI1 could dramatically increase the biosynthetic yield of BRs. When applied industrially, BRs will boost new sector of plant biotechnology because of its potential use as a precursor of human steroid hormones, a novel lead compound for cholesterol-lowering effects, and a various application in plant protection.

• Journal of Plant Biotechnology
• /
• 제29권2호
• /
• pp.139-144
• /
• 2002

Sterols play two major roles in plants: a bulk component in biological membranes and precursors of plant steroid hormones. Physiological effects of plant steroids, brassinosteroids (BRs), include cell elongation, cell division, stress tolerance, and senescence acceleration. Arabidopsis mutants that carry genetic defects in BR biosynthesis or its signaling display characteristic phenotypes, such as short robust inflorescences, dark-green round leaves, and sterility. Currently there are more than 100 dwarf mutants representing 7 genetic loci in Arabidopsis. Mutants of 6 loci, dwf1/dim1/cbb1, cpd/dwf3, dwf4, dwf5, det2/dwf6, dwf7 are rescued by exogenous application of BRs, whereas bri1/dwf2 shares phenotypes with the above 6 loci but are resistant to BRs. These suggest that the 6 loci are defective in BR biosynthesis, and the one locus is in BR signaling. Biochemical analyses, such as intermediate feeding tests, examining the levels of endogenous BR, and molecular cloning of the genes revealed that dwf7, dwf5, and dwf1 are defective in the three consecutive steps of sterol biosynthesis, from episterol to campesterol via 5-dehydroepisterol. Similarly, det2/dwf6, dwf4, and cpd /dwf3 were shown to be blocked in D$^4$reduction, 22a-hydroxylation, and 23 a-hydroxylation, respectively. A signaling mutant bril/dwf2 carries mutations in a Leucine-rich repeat receptor kinase. Interestingly, the bri1 mutant was shown to accumulate significant amount of BRs, suggesting that signaling and biosynthesis are dynamically coupled in Arabidopsis. Thus it is likely that transgenic plants over-expressing the rate-limiting step enzyme DWF4 as well as blocking its use by BRIl could dramatically increase the biosynthetic yield of BRs. When applied industrially, BRs will boost new sector of plant biotechnology because of its potential use as a precursor of human steroid hormones, a novel lead compound for cholesterol-lowering effects, and a various application in plant protection.

• Journal of Plant Biotechnology
• /
• 제34권3호
• /
• pp.243-251
• /
• 2007

BRs의 생합성 경로는 $C_{28}-BRs$ 생합성 경로 외에 $C_{27}-BRs$ 생합성 경로가 존재함이 확인되었고, 최근 $C_{29}-BRs$의 생합성 경로가 존재함이 보고되어 BRs의 생합성 과정이 복잡하게 연결되어 있을 가능성이 예상되었다. 이에 $C_{29}-BRs$인 28-homoTE와 28-homoTY 등이 동정된 벼의 유식물을 대상으로 하여 $C_{29}-BR$인 25-homoCS의 대사 과정을 조사하였다. 그 결과 in vitro 효소변환 연구를 통해 28-homoCS은 CS와 26-nor-28-homoCS으로 전환됨을 확인 할 수 있었으며, 그 역반응인 CS에서 28-homoCS로 또는 26-nor-28-homoCS에서 28-homoCS로의 전환은 일어나지 않음을 알 수 있었다. 이는 $C_{29}-BRs$인 28-homoCS은 C-28위치의 demethylation에 의해 보다 강한 활성의 $C_{28}-BRs$인 CS로 생합성 되는 과정과 C-26 위치의 demethylation에 의해 26-nor-28-homoCS으로 생분해 되는 과정이 존재함을 최초로 확인하였다. 한편, $C_{28}-BRs$인 CS에서 BL로의 전환과 동일한 반응이 $C_{29}-BRs$에서도 일어나는지 확인하고자 하였으나 벼 유식물에서는 28-homoCS에서 28-homoBL로 전환되지 않음을 확인 할 수 있었다. 이는 $C_{29}-BRs$와 $C_{28}-BRs$의 생합성과정의 연결이 28-homoCS에서 CS를 통하고 있음을 알 수 있었다. 따라서 28-homoCS에서 CS로 전환되는 과정을 통하여 $C_{29}-BRs$ 또한 $C_{28}-BRs$ 와 동일한 과정을 거쳐 활성형의 CS로 전환됨을 확인 할 수 있었으며, BRs의 생합성은 $C_{27}-BRs$과 $C_{28}-BRs$의 생합성과정이 연결된 것처럼 $C_{29}-BRs$ 또한 $C_{28}-BRs$ 생합성 과정과 연결되어 있음을 확인 할 수 있었다. 즉, 활성형 BR인 CS은 $C_{27}-BRs$, $C_{28}-BRs$의 생합성 과정뿐만 아니라 $C_{29}-BRs$의 생합성 과정을 통하여 생성되는 과정이 식물체내에 존재함을 확인 할 수 있었다.