Background
Bikini is a strong activator of Brassinosteroid (BR) signaling [1] and an inhibitor of glycogen synthase kinase 3 proteins (GSK3) [2].
BRs are a group of polyhydroxylated steroid hormones implicated in multiple developmental processes, including stem elongation, leaf expansion, vascular development, seed germination, and resistance to biotic and abiotic stresses. Genetic defects in biosynthesis or perception of BRs resulted in dwar?sm, dark-green and curled leaves, reduced seed germination and fertility, and de-etiolation in the dark. [1]
Bikinin is the ?rst nonsteroidal molecule to modulate the BR signaling cascade downstream of the BRI1 receptor. Bikinin directly targeted BIN2 and activated BR-dependent gene expression. Application of bikini partially restored the phenotype of the bri1 loss-of-function mutant and the bin2 gain-of-function mutant to wild-type. Bikinin clearly rescued the dwar?sm of the hypocotyls and the petioles of the bin2 gain-of-function mutant. Bikinin are fully rescuing the BR-related phenotypes of this mutation while having no effect on the auxin-related phenotypes. [1]
Bikinin treatment also induced tracheary element differentiation in Nicotiana benthamiana leaf disks, this leaf-disk culture system may be applicable to different plant species. [2]
参考文献:
[1]. De Rybel B, Audenaert D, Vert G et al. Chemical inhibition of a subset of Arabidopsis thaliana GSK3-like kinases activates brassinosteroid signaling. Chem Biol. 2009 Jun 26;16(6):594-604.
[2]. Kondo Y, Fujita T, Sugiyama M, Fukuda H. A Novel System for Xylem Cell Differentiation in Arabidopsis thaliana. Mol Plant. 2014 Dec 13. pii: S1674-2052(14)00026-4.
Protocol
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Kinase experiment:
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ASKs are expressed as GST-fusion proteins in Escherichia coli BL21. In vitro kinase assays are performed by incubating 50 ng GST-fusion protein, 10 μg myelin basic protein (MBP) as a substrate and 0.15 MBq γ-[32P]-ATP as co-substrate at 25°C for 30 min. The reaction buffer consists of 20 mM HEPES/KOH pH 7.4, 15 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol and 1 μM cold ATP. The reaction products are separated by SDS-PAGE and the amount of radioactivity incorporated into MBP quantified using an Amersham storage phosphor imager screen and a Biorad Molecular Imager FX[2].
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参考文献:
[1]. Deng XG, et al. Role of brassinosteroid signaling in modulating Tobacco mosaic virus resistance in Nicotiana benthamiana. Sci Rep. 2016 Feb 3;6:20579.
[2]. Rozhon W, et al. Bikinin-like inhibitors targeting GSK3/Shaggy-like kinases: characterisation of novel compounds and elucidation of their catabolism in planta. BMC Plant Biol. 2014 Jun 19;14:172.
[3]. De Rybel B, et al. Chemical inhibition of a subset of Arabidopsis thaliana GSK3-like kinases activates brassinosteroid signaling. Chem Biol. 2009 Jun 26;16(6):594-604.
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