Brassinosteroids (BRs) are plant hormones of steroid nature, regulating various developmental and adaptive processes. The perception, transport, and signaling of BRs are actively studied nowadays via a wide range of biochemical and genetic tools. However, most of the knowledge about BRs intracellular localization and turnover relies on the visualization of the receptors or cellular compartments using dyes or fluorescent protein fusions. We have previously synthesized a conjugate of epibrassinolide with green fluorescent dye BODIPY (eBL-BODIPY). Here we present a detailed assessment of the compound bioactivity and its suitability as probe for in vivo visualization of BRs. We show that eBL-BODIPY rapidly penetrates epidermal cells of Arabidopsis thaliana roots and after long exposure causes physiological and transcriptomic responses similar to the natural hormone.

5 P Kukhar Institute of Bioorganic Chemistry and Petrochemistry National Academy of Sciences of Ukraine 02094 Kyiv Ukraine

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republic

Institute of Bioorganic Chemistry National Academy of Sciences of Belarus Kuprevich Str 5 2 220141 Minsk Belarus

Institute of Ecology and Environmental Sciences of Paris Paris Est University UPEC 94010 Créteil France

Institute of Experimental Botany of the Czech Academy of Sciences Rozvojová 263 165 02 Prague Czech Republic

UMR 7025 CNRS GEC Génie Enzymatique et Cellulaire Centre de Recherches Rue Personne de Roberval CS 60319 Alliance Sorbonne Universités Université de Technologie de Compiègne 60203 Compiègne CEDEX France

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Clouse S.D., Sasse J.M. Brassinosteroids: Essential regulators of plant growth and development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998;49:427–451. doi: 10.1146/annurev.arplant.49.1.427. PubMed DOI

Yang C.-J., Zhang C., Lu Y.-N., Jin J.-Q., Wang X.-L. The mechanisms of brassinosteroids’ action: From signal transduction to plant development. Mol. Plant. 2011;4:588–600. doi: 10.1093/mp/ssr020. PubMed DOI

Nam K.H., Li J. BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell. 2002;110:203–212. doi: 10.1016/S0092-8674(02)00814-0. PubMed DOI

Li J., Wen J., Lease K.A., Doke J.T., Tax F.E., Walker J.C. BAK1, an arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell. 2002;110:213–222. doi: 10.1016/S0092-8674(02)00812-7. PubMed DOI

The Molecular Circuitry of Brassinosteroid Signaling-Belkhadir-2015-New Phytologist-Wiley Online Library. [(accessed on 17 February 2021)]; Available online: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.13269. PubMed DOI

Russinova E., Borst J.-W., Kwaaitaal M., Caño-Delgado A., Yin Y., Chory J., de Vries S.C. Heterodimerization and endocytosis of arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1) Plant Cell. 2004;16:3216–3229. doi: 10.1105/tpc.104.025387. PubMed DOI PMC

Li J., Nam K.H. Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase. Science. 2002;295:1299–1301. doi: 10.1126/science.1065769. PubMed DOI

Ma X., Xu G., He P., Shan L. SERKing coreceptors for receptors. Trends Plant Sci. 2016;21:1017–1033. doi: 10.1016/j.tplants.2016.08.014. PubMed DOI

Wang X., Kota U., He K., Blackburn K., Li J., Goshe M.B., Huber S.C., Clouse S.D. Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling. Dev. Cell. 2008;15:220–235. doi: 10.1016/j.devcel.2008.06.011. PubMed DOI

Wang X., Chory J. Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science. 2006 doi: 10.1126/science.1127593. PubMed DOI

Wang X., Li X., Meisenhelder J., Hunter T., Yoshida S., Asami T., Chory J. Autoregulation and homodimerization are involved in the activation of the plant steroid receptor BRI1. Dev. Cell. 2005;8:855–865. doi: 10.1016/j.devcel.2005.05.001. PubMed DOI

Lin W., Lu D., Gao X., Jiang S., Ma X., Wang Z., Mengiste T., He P., Shan L. Inverse modulation of plant immune and brassinosteroid signaling pathways by the receptor-like cytoplasmic kinase BIK1. Proc. Nat. Aada. Sci. USA. 2013;110:12114–12119. doi: 10.1073/pnas.1302154110. PubMed DOI PMC

Kim T.-W., Guan S., Burlingame A.L., Wang Z.-Y. The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2. Mol. Cell. 2011;43:561–571. doi: 10.1016/j.molcel.2011.05.037. PubMed DOI PMC

Tang W., Kim T.-W., Oses-Prieto J.A., Sun Y., Deng Z., Zhu S., Wang R., Burlingame A.L., Wang Z.-Y. BSKs mediate signal transduction from the receptor kinase BRI1 in arabidopsis. Science. 2008;321:557–560. doi: 10.1126/science.1156973. PubMed DOI PMC

Kim T.-W., Guan S., Sun Y., Deng Z., Tang W., Shang J.-X., Sun Y., Burlingame A.L., Wang Z.-Y. Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors. Nat. Cell Biol. 2009;11:1254–1260. doi: 10.1038/ncb1970. PubMed DOI PMC

Yin Y., Wang Z.-Y., Mora-Garcia S., Li J., Yoshida S., Asami T., Chory J. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell. 2002;109:181–191. doi: 10.1016/S0092-8674(02)00721-3. PubMed DOI

Tang W., Yuan M., Wang R., Yang Y., Wang C., Oses-Prieto J.A., Kim T.-W., Zhou H.-W., Deng Z., Gampala S.S., et al. PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1. Nat. Cell Biol. 2011;13:124–131. doi: 10.1038/ncb2151. PubMed DOI PMC

He J.-X., Gendron J.M., Sun Y., Gampala S.S.L., Gendron N., Sun C.Q., Wang Z.-Y. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science. 2005;307:1634–1638. doi: 10.1126/science.1107580. PubMed DOI PMC

Yin Y., Vafeados D., Tao Y., Yoshida S., Asami T., Chory J. A new class of transcription factors mediates brassinosteroid-regulated gene expression in arabidopsis. Cell. 2005;120:249–259. doi: 10.1016/j.cell.2004.11.044. PubMed DOI

Aerts N., Mendes M.P., Wees S.C.M.V. Multiple levels of crosstalk in hormone networks regulating plant defense. Plant J. 2021;105:489–504. doi: 10.1111/tpj.15124. PubMed DOI PMC

Sokołowska K., Kizińska J., Szewczuk Z., Banasiak A. Auxin conjugated to fluorescent dyes—A tool for the analysis of auxin transport pathways. Plant Biol. 2014;16:866–877. doi: 10.1111/plb.12144. PubMed DOI

Auxin Transport Sites are Visualized in Planta Using Fluorescent Auxin Analogs|PNAS. [(accessed on 17 February 2021)]; Available online: https://www.pnas.org/content/111/31/11557.abstract. PubMed

Benson C.L., Kepka M., Wunschel C., Rajagopalan N., Nelson K.M., Christmann A., Abrams S.R., Grill E., Loewen M.C. Abscisic acid analogs as chemical probes for dissection of abscisic acid responses in arabidopsis thaliana. Phytochemistry. 2015;113:96–107. doi: 10.1016/j.phytochem.2014.03.017. PubMed DOI

Fluorescence-Labeled Abscisic Acid Possessing Abscisic Acid-Like Activity in Barley Aleurone Protoplasts: Bioscience, Biotechnology, and Biochemistry: Volume 61, No. 7. [(accessed on 17 February 2021)]; Available online: https://www.tandfonline.com/doi/abs/10.1271/bbb.61.1198. DOI

Lace B., Prandi C. Shaping small bioactive molecules to untangle their biological function: A focus on fluorescent plant hormones. Mol. Plant. 2016;9:1099–1118. doi: 10.1016/j.molp.2016.06.011. PubMed DOI

Synthesis and Cytokinin Activity of Fluorescent 7-Phenylethynylimidazo[4,5-b]Pyridine and Its Riboside. [(accessed on 17 February 2021)];J. Agric. Food Chem. Available online: https://pubs.acs.org/doi/abs/10.1021/jf0000225. PubMed DOI

Liu S., Wang W.-H., Dang Y.-L., Fu Y., Sang R. Rational design and efficient synthesis of a fluorescent-labeled jasmonate. Tetrahedron Lett. 2012;53:4235–4239. doi: 10.1016/j.tetlet.2012.06.006. DOI

Gamoh K., Takatsuto S. A boronic acid derivative as a highly sensitive fluorescence derivatization reagent for brassinosteroids in liquid chromatography. Anal. Chim. Acta. 1989;222:201–204. doi: 10.1016/S0003-2670(00)81893-0. DOI

Gamoh K., Omote K., Okamoto N., Takatsuto S. High-Performance liquid chromatography of brassinosteroids in plants with derivatization using 9-phenanthreneboronic acid. J. Chromatogr. A. 1989;469:424–428. doi: 10.1016/S0021-9673(01)96481-7. DOI

Winter J., Schneider B., Meyenburg S., Strack D., Adam G. Monitoring brassinosteroid biosynthetic enzymes by fluorescent tagging and HPLC analysis of their substrates and products. Phytochemistry. 1999;51:237–242. doi: 10.1016/S0031-9422(98)00760-2. DOI

Borisevich N.A., Raichenok T.F., Khripach V.A., Zhabinskii V.N., Ivanova G.V. Solution electronic spectra of brassinosteroid and a synthesized conjugate of a steroid and a fluorescent label. J. Appl. Spectrosc. 2008;75:75–79. doi: 10.1007/s10812-008-9009-6. DOI

Borisevich N.A., Bagnich S.A., Raichenok T.F., Knyukshto V.N., Baranovskii A.V., Zhabinskii V.N. Luminescence of biologically active 24-epicastasterone and a model compound. J. Appl. Spectrosc. 2008;75:187–191. doi: 10.1007/s10812-008-9033-6. DOI

Raichenok T.F., Litvinovskaya R.P., Zhabinskii V.N., Raiman M.E., Kurtikova A.L., Minin P.S. Synthesis and spectral and luminescence properties of new conjugates of brassinosteroids for immunofluorescence analysis. Chem. Nat. Compd. 2012;48:267–271. doi: 10.1007/s10600-012-0218-0. DOI

Lv T., Zhao X.-E., Zhu S., Ji Z., Chen G., Sun Z., Song C., You J., Suo Y. Development of an efficient HPLC fluorescence detection method for brassinolide by ultrasonic-assisted dispersive liquid–liquid microextraction coupled with derivatization. Chromatographia. 2014;77:1653–1660. doi: 10.1007/s10337-014-2767-9. DOI

Malachowska-Ugarte M., Sperduto C., Ermolovich Y.V., Sauchuk A.L., Jurášek M., Litvinovskaya R.P., Straltsova D., Smolich I., Zhabinskii V.N., Drašar P., et al. Brassinosteroid-BODIPY conjugates: Design, synthesis, and properties. Steroids. 2015;102:53–59. doi: 10.1016/j.steroids.2015.07.002. PubMed DOI

Synthetic Protocol for AFCS: A Biologically Active Fluorescent Castasterone Analog Conjugated to an Alexa Fluor 647 Dye.-Abstract-Europe PMC. [(accessed on 23 February 2021)]; Available online: https://europepmc.org/article/med/28124242. PubMed

Litvinovskaya R.P., Savchuk A.L., Kuprienko O.S., Sviridov O.V., Khripach V.A. Competitive lanthanide immunofluorescent assay of endogenous brassinosteroids in plants. Chem. Nat. Compd. 2018;54:1106–1113. doi: 10.1007/s10600-018-2566-x. DOI

Khripach V., Zhabinskii V., Antonchick A., Litvinovskaya R., Drach S., Sviridov O., Pryadko A., Novik T., Matveentsev V., Schneider B. A new type of modified brassinosteroids for enzyme-linked immunosorbent assay. Nat. Prod. Commun. 2008;3:735–748. doi: 10.1177/1934578X0800300513. DOI

Irani N., Rubbo S., Mylle E., Begin J., Schneider-Pizoń J., Hniliková J., Sisa M., Buyst D., Vilarrasa-Blasi J., Szatmári A.-M., et al. Fluorescent castasterone reveals bri1 signaling from the plasma membrane. Nat. Chem. Biol. 2012;8:583–589. doi: 10.1038/nchembio.958. PubMed DOI

Hurski A.L., Kukel A.G., Liubina A.I., Baradzenka A.G., Straltsova D., Demidchik V., Drašar P., Zhabinskii V.N., Khripach V.A. Regio- and stereoselective C–H functionalization of brassinosteroids. Steroids. 2019;146:92–98. doi: 10.1016/j.steroids.2019.03.010. PubMed DOI

Ulrich G., Ziessel R., Harriman A. The chemistry of fluorescent bodipy dyes: Versatility unsurpassed. Angew. Chem. Int. Ed. 2008;47:1184–1201. doi: 10.1002/anie.200702070. PubMed DOI

Paës G. Fluorescent probes for exploring plant cell wall deconstruction: A review. Molecules. 2014;19:9380–9402. doi: 10.3390/molecules19079380. PubMed DOI PMC

He K., Gou X., Yuan T., Lin H., Asami T., Yoshida S., Russell S.D., Li J. BAK1 and BKK1 regulate brassinosteroid-dependent growth and brassinosteroid-independent cell-death pathways. Curr. Biol. 2007;17:1109–1115. doi: 10.1016/j.cub.2007.05.036. PubMed DOI

Divi U.K., Rahman T., Krishna P. Gene expression and functional analyses in brassinosteroid-mediated stress tolerance. Plant Biotechnol. J. 2016;14:419–432. doi: 10.1111/pbi.12396. PubMed DOI PMC

Besseau S., Li J., Palva E.T. WRKY54 and WRKY70 co-operate as negative regulators of leaf senescence in arabidopsis thaliana. J. Exp. Bot. 2012;63:2667–2679. doi: 10.1093/jxb/err450. PubMed DOI PMC

Li J., Zhong R., Palva E.T. WRKY70 and its homolog WRKY54 negatively modulate the cell wall-associated defenses to necrotrophic pathogens in Arabidopsis. PLoS ONE. 2017;12:e0183731. PubMed PMC

Irani N.G., Di Rubbo S., Russinova E. In vivo imaging of brassinosteroid endocytosis in arabidopsis. In: Otegui M.S., editor. Plant Endosomes: Methods and Protocols. Springer; New York, NY, USA: 2014. pp. 107–117. Methods in Molecular Biology. PubMed

Hacham Y., Holland N., Butterfield C., Ubeda-Tomas S., Bennett M.J., Chory J., Savaldi-Goldstein S. Brassinosteroid perception in the epidermis controls root meristem size. Development. 2011;138:839–848. doi: 10.1242/dev.061804. PubMed DOI PMC

Fridman Y., Elkouby L., Holland N., Vragović K., Elbaum R., Savaldi-Goldstein S. Root growth is modulated by differential hormonal sensitivity in neighboring cells. Genes Dev. 2014;28:912–920. doi: 10.1101/gad.239335.114. PubMed DOI PMC

Cheng Y., Zhu W., Chen Y., Ito S., Asami T., Wang X. Brassinosteroids control root epidermal cell fate via direct regulation of a MYB-BHLH-WD40 complex by GSK3-like kinases. Elife. 2014;3:e02525. doi: 10.7554/eLife.02525. PubMed DOI PMC

Kuppusamy K.T., Chen A.Y., Nemhauser J.L. Steroids are required for epidermal cell fate establishment in arabidopsis roots. Proc. Nat. Aada. Sci. USA. 2009;106:8073–8076. doi: 10.1073/pnas.0811633106. PubMed DOI PMC

Lanza M., Garcia-Ponce B., Castrillo G., Catarecha P., Sauer M., Rodriguez-Serrano M., Páez-García A., Sánchez-Bermejo E., Tc M., Leo del Puerto Y., et al. Role of actin cytoskeleton in brassinosteroid signaling and in its integration with the auxin response in plants. Dev. Cell. 2012;22:1275–1285. doi: 10.1016/j.devcel.2012.04.008. PubMed DOI

Li L., Xu J., Xu Z.-H., Xue H.-W. Brassinosteroids stimulate plant tropisms through modulation of polar auxin transport in brassica and arabidopsis. Plant Cell. 2005;17:2738–2753. doi: 10.1105/tpc.105.034397. PubMed DOI PMC

Planas-Riverola A., Gupta A., Betegón-Putze I., Bosch N., Ibañes M., Caño-Delgado A.I. Brassinosteroid signaling in plant development and adaptation to stress. Development. 2019:146. doi: 10.1242/dev.151894. PubMed DOI PMC

Retzer K., Akhmanova M., Konstantinova N., Malínská K., Leitner J., Petrášek J., Luschnig C. Brassinosteroid signaling delimits root gravitropism via sorting of the arabidopsis PIN2 auxin transporter. Nat. Commun. 2019;10:5516. doi: 10.1038/s41467-019-13543-1. PubMed DOI PMC

Durbak A., Yao H., McSteen P. Hormone signaling in plant development. Curr. Opin. Plant Biol. 2012;15:92–96. doi: 10.1016/j.pbi.2011.12.004. PubMed DOI

Ibañes M., Fàbregas N., Chory J., Caño-Delgado A.I. Brassinosteroid signaling and auxin transport are required to establish the periodic pattern of arabidopsis shoot vascular bundles. Proc. Nat. Aada. Sci. USA. 2009 doi: 10.1073/pnas.0906416106. PubMed DOI PMC

Brassinosteroids Interact with Auxin to Promote Lateral Root Development in Arabidopsis. Plant Physiol. [(accessed on 17 February 2021)]; Available online: http://www.plantphysiol.org/content/134/4/1624. PubMed PMC

Goda H., Sawa S., Asami T., Fujioka S., Shimada Y., Yoshida S. Comprehensive comparison of auxin-regulated and brassinosteroid-regulated genes in arabidopsis. Plant Physiol. 2004;134:1555–1573. doi: 10.1104/pp.103.034736. PubMed DOI PMC

Nemhauser J.L., Mockler T.C., Chory J. Interdependency of brassinosteroid and auxin signaling in arabidopsis. PLoS Biol. 2004;2:e258. doi: 10.1371/journal.pbio.0020258. PubMed DOI PMC

Kretynin S.V., Kolesnikov Y.S., Derevyanchuk M.V., Kalachova T.A., Blume Y.B., Khripach V.A., Kravets V.S. Brassinosteroids application induces phosphatidic acid production and modify antioxidant enzymes activity in tobacco in calcium-dependent manner. Steroids. 2019:108444. doi: 10.1016/j.steroids.2019.108444. PubMed DOI

Schaller F., Biesgen C., Müssig C., Altmann T., Weiler E.W. 12-oxophytodienoate reductase 3 (opr3) is the isoenzyme involved in jasmonate biosynthesis. Planta. 2000;210:979–984. doi: 10.1007/s004250050706. PubMed DOI

Kitanaga Y., Jian C., Hasegawa M., Yazaki J., Kishimoto N., Kikuchi S., Nakamura H., Ichikawa H., Asami T., Yoshida S., et al. Sequential regulation of gibberellin, brassinosteroid, and jasmonic acid biosynthesis occurs in rice coleoptiles to control the transcript levels of anti-microbial thionin genes. Biosci. Biotechnol. Biochem. 2006;70:2410–2419. doi: 10.1271/bbb.60145. PubMed DOI

Gan L., Wu H., Wu D., Zhang Z., Guo Z., Yang N., Xia K., Zhou X., Oh K., Matsuoka M., et al. Methyl jasmonate inhibits lamina joint inclination by repressing brassinosteroid biosynthesis and signaling in rice. Plant Sci. 2015;241:238–245. doi: 10.1016/j.plantsci.2015.10.012. PubMed DOI

Peng Z., Han C., Yuan L., Zhang K., Huang H., Ren C. Brassinosteroid enhances jasmonate-induced anthocyanin accumulation in arabidopsis seedlings. J. Integr. Plant Biol. 2011;53:632–640. doi: 10.1111/j.1744-7909.2011.01042.x. PubMed DOI

Zhang S., Wei Y., Lu Y., Wang X. Mechanisms of brassinosteroids interacting with multiple hormones. Plant Signal Behav. 2009;4:1117–1120. doi: 10.4161/psb.4.12.9903. PubMed DOI PMC

The Antagonistic Regulation of Abscisic Acid-Inhibited Root Growth by Brassinosteroids is Partially Mediated via Direct Suppression of ABSCISIC ACID INSENSITIVE 5 Expression by BRASSINAZOLE RESISTANT 1-Yang-2016-Plant, Cell &Environment-Wiley Online Library. [(accessed on 17 February 2021)]; Available online: https://onlinelibrary.wiley.com/doi/full/10.1111/pce.12763. PubMed DOI

Divi U.K., Rahman T., Krishna P. Brassinosteroid-mediated stress tolerance in arabidopsis shows interactions with abscisic acid, ethylene and salicylic acid pathways. Bmc. Plant Biol. 2010;10:151. doi: 10.1186/1471-2229-10-151. PubMed DOI PMC

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Lin F., Krishnamoorthy P., Schubert V., Hause G., Heilmann M., Heilmann I. A dual role for cell plate-associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis. EMBO J. 2019;38:e100303. doi: 10.15252/embj.2018100303. PubMed DOI PMC

Leontovyčová H., Kalachova T., Trdá L., Pospíchalová R., Lamparová L., Dobrev P.I., Malínská K., Burketová L., Valentová O., Janda M. Actin depolymerization is able to increase plant resistance against pathogens via activation of salicylic acid signalling pathway. Sci. Rep. 2019;9:10397. doi: 10.1038/s41598-019-46465-5. PubMed DOI PMC