• 생명과학회지
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• 제21권7호
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• pp.969-975
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• 2011

본 연구에서는 식물뿌리의 굴중성 반응에 있어서 식물호르몬과 AtEXPA3 유전자와의 관계를 밝히고자 하였다. AtEXPA3 유전자의 RT-PCR을 통한 발현분석 결과 잎, 근출엽, 뿌리 꽃 등 생장이 활발한 조직에서 발현률이 높게 나타났으며, 굴중성 자극과 BRs, IAA에 의해서도 발현이 증가되었다. 또한 ethylene 생합성 저해제인 AVG를 처리하면 굴중성 반응이 현저히 억제되었는데 이는 ethylene 그 자체도 BR과 IAA처럼 굴중성을 촉진시키는 활성을 갖고 있음을 의미한다. 한편 호르몬을 처리하지 않은 AtEXPA3 RNAi mutant에서 굴중성 반응이 억제되는 현상은 애기장대 뿌리의 생장에 관여하는 AtEXPA3의 조절 인자로 BRs, auxin, ethylene 등의 식물호르몬이 관여하고 있음을 나타낸다. 아울러 BRs signaling mutant에서 변화된 굴중성(bri1-301, bak1에서 감소, BRI-GFP에 서 증가) 반응의 감소와 증가는 굴중성 반응이 BRs의 신호전달 과정을 통하여 일어남을 의미한다. 결론적으로 애기장대 뿌리의 굴중성 반응은 식물호르몬에 의한 AtEXPA3 유전자의 발현 증가의 결과로 인해 애기장대 뿌리의 생장이 촉진되어 나타나는 결과라 하겠다.

• Molecules and Cells
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• 제37권11호
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• pp.795-803
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• 2014

To withstand ever-changing environmental stresses, plants are equipped with phytohormone-mediated stress resistance mechanisms. Salt stress triggers abscisic acid (ABA) signaling, which enhances stress tolerance at the expense of growth. ABA is thought to inhibit the action of growth-promoting hormones, including brassinosteroids (BRs). However, the regulatory mechanisms that coordinate ABA and BR activity remain to be discovered. We noticed that ABA-treated seedlings exhibited small, round leaves and short roots, a phenotype that is characteristic of the BR signaling mutant, brassinosteroid insensitive1-9 (bri1-9). To identify genes that are antagonistically regulated by ABA and BRs, we examined published Arabidopsis microarray data sets. Of the list of genes identified, those upregulated by ABA but downregulated by BRs were enriched with a BRRE motif in their promoter sequences. After validating the microarray data using quantitative RT-PCR, we focused on RD26, which is induced by salt stress. Histochemical analysis of transgenic Arabidopsis plants expressing RD26pro:GUS revealed that the induction of GUS expression after NaCl treatment was suppressed by co-treatment with BRs, but enhanced by co-treatment with propiconazole, a BR biosynthetic inhibitor. Similarly, treatment with bikinin, an inhibitor of BIN2 kinase, not only inhibited RD26 expression, but also reduced the survival rate of the plant following exposure to salt stress. Our results suggest that ABA and BRs act antagonistically on their target genes at or after the BIN2 step in BR signaling pathways, and suggest a mechanism by which plants fine-tune their growth, particularly when stress responses and growth compete for resources.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2002년도 추계학술대회
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• pp.69-75
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• 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
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• 한국식물생명공학회 2002년도 춘계학술대회
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• pp.69-75
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• 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
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• 제29권2호
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• pp.139-144
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• 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.

• 통합자연과학논문집
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• 제1권1호
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• pp.24-31
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• 2008

Brassinosteroids (BRs) are a special class of plant steroid hormones that are essential for normal growth and development. Part of confusion is whether BRs are unique to plants, because they have overlapping physiological roles with other better-studied hormones and with physiological responses caused by light. In systems designed to assay for cytokinins, the effects of BRs vary. We measured hypocotyl length for testing the ability of brassinolide (BL) to rescue double mutant between det2 and the photoreceptor null mutant phytochrome B (phyB). PHYB involved in controlling hypocotyl elongation in increased concentration of BL whereas phyBdet2 double mutant just partially rescue to phyB in white and red light indicated the involvement of BRs in PHYB regulated cell elongation. BRs regulated hypocotyl growth was delayed by BAP, a cytokinin treatment but inhibitory effects of BAPs on hypocotyl growth was slightly recovered by BL. The result indicated that the mode of action of BR and cytokinin is independent or sequential in the downstream light-regulated response control on hypocotyl elongation and also light modulated the action of BR and cytokinin in some extent.

• 통합자연과학논문집
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• 제4권2호
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• pp.113-120
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• 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.

• Molecules and Cells
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• 제27권2호
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• pp.183-190
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• 2009

The plasma membrane-localized BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1) are a well-known receptor pair involved in brassinosteroids (BR) signaling in Arabidposis. The formation of a receptor complex in response to BRs and the subsequent activation of cytoplasmic domain kinase activity share mechanistic characteristics with animal receptor kinases. Here, we demonstrate that BRI1 and BAK1 are BR-dependently phosphorylated, and that phosphorylated forms of the two proteins persist for different lengths of time. Mutations of either protein abolished phosphorylation of the counterpart protein, implying transphosphorylation of the receptor kinases. To investigate the specific amino acids critical for formation of the receptor complex and activation of BAK1 kinase activity, we expressed several versions of BAK1 in yeast and plants. L32E and L46E substitutions resulted in a loss of binding of BAK1 to BRI1, and threonine T455 was essential for the kinase activity of BAK1 in yeast. Transgenic bri1 mutant plants overexpressing BAK1(L46E) displayed reduced apical dominance and seed development. In addition, transgenic wild type plants overexpressing BAK1(T455A) lost the phosphorylation activity normally exhibited in response to BL, leading to semi-dwarfism. These results suggest that BAK1 is a critical component regulating the duration of BR efficacy, even though it cannot directly bind BRs in plants.

• Journal of Plant Biotechnology
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• 제42권4호
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• pp.277-283
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• 2015

Plant genome analyses, including Arabidopsis thaliana showed a large gene family of plant receptor kinases with various extracellular ligand-binding domain. Now intensively studies to understand physiological and cellular functions for higher plant receptor kinases in diverse and complex biological processes including plant growth, development, ligands perception including steroid hormone and plant-microbe interactions. Brassinosteroids (BRs) as a one of well know steroid hormone are plant growth hormones that control biomass accumulation and also tolerance to many biotic and abiotic stress conditions and hence are of relevance to agriculture. BRI1 receptor kinase, which is localized in plasma membrane in the cell sense BRs and it bind to a receptor protein known as BRASSINOSTEROID INSENSITIVE 1 (BRI1). Recently, we reported that BRI1 and its co-receptor, BRI1-ASSOCIATED KINASE (BAK1) autophosphorylated on tyrosine residue (s) in vitro and in vivo and thus are dual-specificity kinases. Other plant receptor kinases are also phosphorylated on tyrosine residue (s). Post-translational modifications (PTMs) can be studied by altering the residue modified by directed mutagenesis to mimic the modified state or to prevent the modification. These approaches are useful to not only characterize the regulatory role of a given modification, but may also provide opportunities for plant improvement.

• Journal of Plant Biotechnology
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• 제43권1호
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• pp.30-36
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• 2016

Brassinosteroids (BRs) are essential plant steroid hormones required for cell elongation, plant growth, development and abiotic and biotic stress tolerance. BRs are recognized by BRI1 receptor kinase that is localized in the plasma membrane, and the BRI1 protein will eventually autophosphorylate in the intracellular domain and transphosphorylate BAK1, which is a co-receptor in Arabidopsis thaliana. However, little is known of the role OsBRI1 receptor kinase plays in Oryza sativa, monocotyledonous plants, compared to that in Arabidopsis thaliana, dicotyledonous plants. As such, we have studied OsBRI1 receptor kinase in vitro and in vivo with recombinant protein and transgenic plants, whose phenotypes were also investigated. A OsBRI1 cytoplasmic domain (CD) recombinant protein was induced in BL21 (DE3) E.coli cells with IPTG, and purified to obtain OsBRI1 recombinant protein. Based on Western blot analysis with phospho-specific pTyr and pThr antibodies, OsBRI1 recombinant protein and OsBRI1-Flag protein were phosphorylated on Threonine residue(s), however, not on Tyrosine residue(s), both in vitro and in vivo. This is particularly intriguing as AtBRI1 protein was phosphorylated on both Ser/Thr and Tyr residues. Also, the OsBRI1 full-length gene was expressed in, and rescued, bri1-5 mutants, such as is seen in normal wild-type plants where AtBRI1-Flag rescues bri1-5 mutant plants. Root growth in seedlings decreased in Ws2, AtBRI1, and 3 independent OsBRI1 transgenic seedlings and had an almost complete lack of response to brassinolide in the bri1-5 mutant. In conclusion, OsBRI1, an orthologous gene of AtBRI1, can mediate normal BR signaling for plant growth and development in Arabidopsis thaliana.