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Kim, Gyung-Tae,Fujioka, Shozo,Kozuka, Toshiaki,Tax, Frans E.,Takatsuto, Suguru,Yoshida, Shigeo,Tsukaya, Hirokazu Blackwell Science Ltd 2005 The Plant journal Vol.41 No.5
<P>Summary</P><P>Brassinosteroids (BRs) are plant hormones that are essential for a wide range of developmental processes in plants. Many of the genes responsible for the early reactions in the biosynthesis of BRs have recently been identified. However, several genes for enzymes that catalyze late steps in the biosynthesis pathways of BRs remain to be identified, and only a few genes responsible for the reactions that produce bioactive BRs have been identified. We found that the <I>ROTUNDIFOLIA3</I> (<I>ROT3</I>) gene, encoding the enzyme CYP90C1, which was specifically involved in the regulation of leaf length in <I>Arabidopsis thaliana</I>, was required for the late steps in the BR biosynthesis pathway. ROT3 appears to be required for the conversion of typhasterol to castasterone, an activation step in the BR pathway. We also analyzed the gene most closely related to <I>ROT3</I>, <I>CYP90D1</I>, and found that double mutants for <I>ROT3</I> and <I>CYP90D1</I> had a severe dwarf phenotype, whereas <I>cyp90d1</I> single knockout mutants did not. BR profiling in these mutants revealed that CYP90D1 was also involved in BR biosynthesis pathways. <I>ROT3</I> and <I>CYP90D1</I> were expressed differentially in leaves of <I>A. thaliana</I>, and the mutants for these two genes differed in their defects in elongation of hypocotyls under light conditions. The expression of <I>CYP90D1</I> was strongly induced in leaf petioles in the dark. The results of the present study provide evidence that the two cytochrome P450s, CYP90C1 and CYP90D1, play distinct roles in organ-specific environmental regulation of the biosynthesis of BRs.</P>
Auxin stimulates <i>DWARF4</i> expression and brassinosteroid biosynthesis in Arabidopsis
Chung, Yuhee,Maharjan, Puna M.,Lee, Oksun,Fujioka, Shozo,Jang, Suyoun,Kim, Bokyung,Takatsuto, Suguru,Tsujimoto, Masafumi,Kim, Hobang,Cho, Seoae,Park, Taesung,Cho, Hyunwoo,Hwang, Ildoo,Choe, Sunghwa Blackwell Publishing Ltd 2011 The Plant journal Vol.66 No.4
<P><B>Summary</B></P><P>Brassinosteroids (BRs) are growth‐promoting steroidal hormones. Despite the importance of BRs in plant biology, the signal that initiates BR biosynthesis remains unknown. Among the enzymes involved in BR biosynthesis in Arabidopsis (<I>Arabidopsis thaliana</I>), DWARF4 catalyzes the rate‐determining step. Through both the histochemical analysis of <I>DWF4pro:GUS</I> plants and the direct measurement of endogenous BR content, we discovered that BR biosynthesis is stimulated by auxin. When <I>DWF4pro:GUS</I> was subjected to auxin dose–response tests and a time‐course analysis, GUS activity started to increase at an auxin concentration of 10 n<SMALL>m</SMALL>, rising noticeably after 1 h of auxin treatment. In addition, the analysis of the <I>DWF4pro:GUS</I> line in BR‐ and auxin‐mutant backgrounds revealed that the induction by auxin requires auxin‐signaling pathways but not BRs, which implies that auxin signaling directly controls BR biosynthesis. Furthermore, chromatin immunoprecipitation assays confirmed that auxin inhibits the binding of the transcriptional repressor, BZR1, to the <I>DWF4</I> promoter. A microarray analysis that was designed to examine the transcriptomes after treatment with auxin alone or auxin plus brassinazole (a BR biosynthetic inhibitor) revealed that genes previously characterized as being auxin responsive are not properly regulated when BR biosynthesis is disrupted by brassinazole. Therefore, our results support the idea that auxin regulates BR biosynthesis, and that auxin thus relies on synthesized BRs for some of its growth‐promoting effects in Arabidopsis.</P>
Bokyung Kim,최성화,Gyusik Kim,Shozo Fujioka,Suguru Takatsuto 한국분자세포생물학회 2012 Molecules and cells Vol.34 No.1
Sterols play crucial roles as membrane components and precursors of steroid hormones (e.g., brassinosteroids, BR). Within membranes, sterols regulate membrane per-meability and fluidity by interacting with other lipids and proteins. Sterols are frequently enriched in detergent-insoluble membranes (DIMs), which organize molecules involved in specialized signaling processes, including auxin transporters. To be fully functional, the two methyl groups at the C-4 position of cycloartenol, a precursor of plant sterols, must be removed by bifunctional 3-hy-droxysteroid dehydrogenases/C-4 decarboxylases (3HSD/D). To understand the role of 3HSD/D in Arabidopsis devel-opment, we analyzed the phenotypes of knock-out mu-tants and overexpression lines of two 3HSD/D genes (At1g47290 and At2g26260). Neither single nor double knock-out mutants displayed a noticeable phenotype; however, overexpression consistently resulted in plants with wrinkled leaves and short inflorescence internodes. Interestingly, the internode growth defects were opportunistic; even within a plant, some stems were more severely affected than others. Endogenous levels of BRs were not altered in the overexpression lines, suggesting that the growth defect is not primarily due to a flaw in BR biosynthesis. To determine if overexpression of the sterol biosynthetic genes affects the functions of membrane-localized auxin transporters, we subjected plants to the auxin efflux carrier inhibitor, 1-N-naphthylphthalamic acid (NPA). Where- as the gravity vectors of wild-type roots became randomly scattered in response to NPA treatment, those of the overexpression lines continued to grow in the direction of gravity. Overexpression of the two Arabidopsis 3HSD/D genes thus appears to affect auxin transporter activity, possibly by altering sterol composition in the membranes.
Kim, Ho Bang,Kwon, Mi,Ryu, Hojin,Fujioka, Shozo,Takatsuto, Suguru,Yoshida, Shigeo,An, Chung Sun,Lee, Ilha,Hwang, Ildoo,Choe, Sunghwa American Society of Plant Physiologists 2006 Plant Physiology Vol.140 No.2
<P>Mutants that are defective in brassinosteroid (BR) biosynthesis or signaling display severely retarded growth patterns due to absence of growth-promoting effects by BRs. Arabidopsis (Arabidopsis thaliana) DWARF4 (DWF4) catalyzes a flux-determining step in the BR biosynthetic pathways. Thus, it is hypothesized that the tissues of DWF4 expression may represent the sites of BR biosynthesis in Arabidopsis. Here we show that DWF4 transcripts accumulate in the actively growing tissues, such as root, shoot apices with floral clusters, joint tissues of root and shoot, and dark-grown seedlings. Conforming to the RNA gel-blot analysis, DWF4:beta-glucuronidase (GUS) histochemical analyses more precisely define the tissues that express the DWF4 gene. Examination of the endogenous levels of BRs in six and seven different tissues of wild type and brassinosteroid insensitive1-5 mutant, respectively, revealed that BRs are significantly enriched in roots, shoot tips, and joint tissues of roots and shoots. In addition, DWF4:GUS expression was negatively regulated by BRs. DWF4:GUS activity was increased by treatment with brassinazole, a BR biosynthetic inhibitor, and decreased by exogenous application of bioactive BRs. When DWF4:GUS was expressed in a different genetic background, its level was down-regulated in brassinazole resistant1-D, confirming that BRASSINAZOLE RESISTANT1 acts as a negative regulator of DWF4. Interestingly, in the brassinosteroid insensitive2/dwf12-1D background, DWF4:GUS expression was intensified and delocalized to elongating zones of root, suggesting that BRASSINOSTEROID INSENSITIVE2 is an important factor that limits DWF4 expression. Thus, it is likely that the DWF4 promoter serves as a focal point in maintaining homeostasis of endogenous bioactive BR pools in specific tissues of Arabidopsis.</P>