© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev151894. doi:10.1242/dev.151894 REVIEW Brassinosteroid signaling in plant development and adaptation to stress Ainoa Planas-Riverola1,*, Aditi Gupta1,*, Isabel Betegó n-Putze1, Nadja Bosch1, Marta Ibañes2,3 and Ana I. Caño-Delgado1,‡ ABSTRACT pathways under normal and environmentally challenging Brassinosteroids (BRs) are steroid hormones that are essential for scenarios, and in which tissues BR synthesis occurs (Caño- plant growth and development. These hormones control the division, Delgado and Blázquez, 2013; Vukasinovic and Russinova, 2018). elongation and differentiation of various cell types throughout the Over the past few decades, BR hormones have been shown to be entire plant life cycle. Our current understanding of the BR signaling essential for cell elongation and, as such, initial studies on hypocotyl pathway has mostly been obtained from studies using Arabidopsis elongation have been very rewarding in terms of understanding the thaliana as a model. In this context, the membrane steroid receptor transcriptional responses that trigger elongation (Clouse and Sasse, BRI1 (BRASSINOSTEROID INSENSITIVE 1) binds directly to the BR 1998). However, since the discovery that BRs also play a role in cell ligand, triggering a signal cascade in the cytoplasm that leads to the division (González-García et al., 2011; Hacham et al., 2011), studies transcription of BR-responsive genes that drive cellular growth. have switched focus in an attempt to understand how BRs modulate However, recent studies of the primary root have revealed distinct growth and development in plants, using the primary root of BR signaling pathways in different cell types and have highlighted cell- Arabidopsis thaliana as a model. In this context, techniques such as specific roles for BR signaling in controlling adaptation to stress. In this fluorescence-activated cell sorting (Brady et al., 2007), and tools that Review, we summarize our current knowledge of the spatiotemporal allow the local expression of signaling components (Marquès-Bueno control of BR action in plant growth and development, focusing on BR et al., 2016) and the visualization of cell-specific protein-protein functions in primary root development and growth, in stem cell self- interactions (Long et al., 2017), have been instrumental in elucidating renewal and death, and in plant adaption to environmental stress. novel BR signaling components and cell-specific signals (Fàbregas et al., 2013; Vilarrasa-Blasi et al., 2014; Vragović et al., 2015). More KEY WORDS: Brassinosteroid, Growth, Root, Stem cell, Stress recent work on BRs has also begun to decode the mechanisms by which BR-mediated signaling regulates adaptation to biotic (De Introduction Bruyne et al., 2014) and abiotic (Lozano-Durán and Zipfel, 2015; Brassinosteroids (BRs) are phytohormones that were originally Nolan et al., 2017a) stresses. Here, we review these recent advances discovered in Brassica napus pollen based on their ability to promote that aim to decipher the spatiotemporal control of BR action. First, we growth (Mitchell et al., 1970). Since their discovery, the main provide an overview of the BR signal transduction pathway and then components of the canonical BR signaling pathway have been discuss how BRs regulate root growth and development in a cell- identified through multiple genetic and biochemical screens (Vert specific fashion. We also highlight how BRs function within some of et al., 2005; Zhu et al., 2013). BR perception occurs at membrane- the most special cells of the plant, the root stem cells. Finally, we localized receptors and downstream cytosolic regulators transduce review our current understanding of the roles of BRs and their BR-mediated signals to the nucleus where they activate the crosstalk with other hormones in mediating adaptation to abiotic transcription of BR-responsive genes that drive cellular growth stresses, such as drought, temperature changes and salinity. (Belkhadir and Jaillais, 2015; Zhao and Li, 2012). Accordingly, mutations in genes encoding the main components of the BR Brassinosteroid ligand perception and signal transduction synthesis and signaling pathways result in severe dwarfism, impaired BR hormones are perceived extracellularly by members of the BRI1 organ growth and development, and limited plant fertility and yield (BRASSINOSTEROID INSENSITIVE 1) leucine-rich repeat (Li and Chory, 1997; Singh and Savaldi-Goldstein, 2015). Despite receptor-like kinase (LRR-RLK) family (Li and Chory, 1997; such knowledge of BR pathway components, many questions Wang et al., 2001). The BR hormone binds directly to a 93-amino- remain unclear, including how BRs function in a cell-specific acid region located within the extracellular domain of membrane- manner, how the BR pathway interacts with other hormonal bound BRI1 (Hothorn et al., 2011; Kinoshita et al., 2005; Sun et al., 2013). Direct binding triggers the formation of a BRI1- BAK1 [BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED 1 Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Barcelona E-08193, Spain. 2Departament de RECEPTOR KINASE 1, also known as SERK3 (SOMATIC DEVELOPMENT Fı́sica de la Matèria Condensada, Universitat de Barcelona, Barcelona 08028, EMBRYOGENESIS RECEPTOR KINASE 3)] heterodimer, which Spain. 3Universitat de Barcelona Institute of Complex Systems (UBICS), Universitat in turn initiates an intracellular phosphorylation relay cascade (Li de Barcelona, Barcelona 08028, Spain. *These authors contributed equally to this work and Nam, 2002; Russinova et al., 2004). The cascade (Fig. 1A) culminates in promotion of the activity and stability of the ‡ Author for correspondence (ana.cano@cragenomica.es) plant-specific transcription factors BZR1 (BRASSINAZOLE A.I.C., 0000-0002-8071-6724 RESISTANT 1) (Wang et al., 2002) and BES1 (BRI1-EMS- SUPPRESSOR 1) (Yin et al., 2002), which directly control the This is an Open Access article distributed under the terms of the Creative Commons Attribution transcription of thousands of BR-responsive genes and hence regulate a License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. plethora of developmental events in the plant (He et al., 2002; Sun et al., 1 REVIEW Development (2019) 146, dev151894. doi:10.1242/dev.151894 A BR signaling pathway whereas BRI1 is expressed nearly ubiquitously in the root (Friedrichsen and Chory, 2001) (Fig. 1B), the BRLs are found BRI1 only in some specific tissues (Fig. 1C,D). For example, BRL1 and BRL3 are localized in vascular stem cells, where they govern cell- +BR specific BR-response pathways (Caño-Delgado et al., 2004; Fàbregas et al., 2013; Salazar-Henao et al., 2016). Under native conditions, both BRL1 and BRL3 can heterodimerize with the BAK1 co- receptor, but not with BRI1, and form a complex (Fàbregas et al., BAK1 1 2013). These studies suggest that BRI1 and the BRLs are able to form BKI1 different receptor complexes in different cell types, thereby BSU1 performing different signaling roles, but the specific downstream components of the BRL1 and BRL3 pathways remain unknown. BIN2 The primary root as a model for deciphering cell-specific brassinosteroid signaling Owing to its simple and radial organization of cell types, the BZR1/BES1 BZR1/BES1 primary root of Arabidopsis provides an excellent model for PP2A 14-3-3 dissecting signaling mechanisms with cell-specific resolution Cytoplasmic (Dolan et al., 1993; Scheres et al., 1994). Indeed, a number of retention studies of the primary root have shown that BRs control specific cellular processes in distinct root cell types (Fig. 2). BRs play an important role in overall root development; both an excess and a lack of BRs are detrimental to primary root growth and development. On the one hand, mutants lacking BR compounds or BR receptors exhibit short roots, indicating that BRI1 signaling is BR target genes Degradation required for root growth (Chaiwanon and Wang, 2015; González- García et al., 2011; Hacham et al., 2011; Mussig et al., 2003). On the Growth and development other hand, short roots are also observed in bes1-D (gain-of-function) mutants, or in plants treated with high concentrations of BRs (González-García et al., 2011; Mussig et al., 2002). The short roots of B BRI1 C BRL1 D BRL3 mutants with impaired BR biosynthesis can be rescued by treatment with low concentrations of BR (Chaiwanon and Wang, 2015). Moreover, supporting the notion that BRs can promote root growth, it has been shown that wild-type roots treated with low concentrations of BRs increase their length (González-García et al., 2011; Mussig et al., 2003), although this enlargement is small and not always detectable (Chaiwanon and Wang, 2015). Altogether, these results suggest that, rather than controlling root growth in a linear fashion, Fig. 1. An overview of the BR signaling pathway. (A) Schematic of the BRI1 the correct balance of BR levels appears to be crucial for normal root signaling pathway. In the absence of BR, BIN2 phosphorylates BZR1 and growth and development (González-García et al., 2011). BES1 proteins, inactivating them by promoting their binding to 14-3-3 proteins, leading to their cytoplasmic retention and degradation. When BRI1 perceives Root growth also depends on cell proliferation at the meristem BR molecules, it heterodimerizes with BAK1, initiating an intracellular and on cell elongation prior to differentiation. BRs impinge on both phosphorylation relay cascade that ends with the dephosphorylation and of these processes. BRs modulate meristematic proliferation consequent activation of BZR1 and BES1. (B-D) Schematics of the root tissue- (González-García et al., 2011; Hacham et al., 2011) and have specific expression of BR receptors. BRI1 is expressed throughout the root (B), been proposed as key regulators in the optimal control of cell cycle whereas BRL1 (C) and BRL3 (D) exhibit a more discrete expression progression (González-García et al., 2011). BRs have been also pattern, being active mainly in the root stem cell niche area. Dark green represents high expression of the protein, whereas light green represents lower proposed to be crucial for optimal cell expansion (Chaiwanon and expression. Wang, 2015; Clouse and Sasse, 1998). Recent mathematical and computational modeling has further demonstrated that root growth features depend on the mechanism by which cell elongation 2010). When BRs are absent, the GSK3-like kinase BIN2 terminates, e.g. whether cells stop elongating according to their (BRASSINOSTEROID-INSENSITIVE 2) phosphorylates BZR1/ spatial position along the root, according to a time interval, and/or BES1 proteins and inactivates them, promoting their binding to 14-3-3 according to their cell size (Pavelescu et al., 2018). Quantification of DEVELOPMENT proteins and leading to their cytoplasmic retention and degradation cell length in single roots, together with mathematical and (Gampala et al., 2007; Li and Nam, 2002; Peng et al., 2008). This thereby computational modeling, suggests that the dominant mechanism inhibits their ability to bind DNA and causes pathway inactivation. for cell elongation termination is a size-based mechanism whereby Based on the presence of the extracellular BR-binding domain, root cells stop expanding when they reach a determined length, and there are three membrane-localized BRI1-like homologs named that BRI1 facilitates this mechanism (Pavelescu et al., 2018). In BRL1, BRL2 and BRL3 (BRI1-LIKE 1, 2 and 3). Whereas BRL1 addition, this suggests that BR signaling at least partially controls and BRL3 are functional BR receptors that, like BRI1, can bind to these three separate functions: cell division, cell elongation rate and steroid molecules with high affinity, BRL2 appears not to be a termination of cell elongation (Pavelescu et al., 2018). Indeed, functional BR receptor (Caño-Delgado et al., 2004). Furthermore, plants treated with high concentrations of BR increase expansion at 2 REVIEW Development (2019) 146, dev151894. doi:10.1242/dev.151894 depends on the cell-specific expression and site of action of BR receptors, which could promote specific signals and thus Differentiation contribute differentially to overall root development. Given that zone BRL receptors function in the phloem (Caño-Delgado et al., Stopping cell elongation by sizer mechanism 2004) and the recent proposed role for BRL3 in root mobilization of osmoprotectant metabolites to confer drought resistance (Fàbregas et al., 2018), we propose that that BR receptors expressed in the inner layers of the root may selectively promote Elongation Cell elongation growth under stress. zone BR signaling is also involved in the development of vascular tissues within the plant. Early studies in Zinnia elegans cells indicate that BR synthesis increases prior to, and is necessary for, Vascular differentiation tracheary element differentiation (Yamamoto et al., 2001), and in Arabidopsis suspension cultures BRs induce VND7-mediated xylem cell wall differentiation (Yamaguchi et al., 2010). In Meristem size Arabidopsis, BR-deficient plants harboring mutations in genes Cell cycle progression such as CPD (CONSTITUTIVE PHOTOMORPHOGENIC Meristematic DWARF) and DWF7 (DWARF 7) have abnormal xylem zone development (Choe et al., 1999; Szekeres et al., 1996). BR Stem cell niche renewal receptor mutants also exhibit abnormal vascular differentiation, a and maintenance process in which BRI1 and the BRLs have redundant functions (Caño-Delgado et al., 2004). In the primary root, BR suppresses radial vascular cell divisions (Fàbregas et al., 2013; Kang et al., 2017). In line with this, the brl1 brl3 bak1-3 triple mutant is hypersensitive to BR in the stele, showing greater stele narrowing Key Epidermis Endodermis QC cells than that of wild-type, bak1 or brl1 brl3 mutant plants upon BR Cortex Vasculature Stem cells treatment (Fàbregas et al., 2013). In addition, the wider stele of the bri1 brl1 brl3 triple mutant increases when BRI1 is expressed in the Fig. 2. BR functions in the primary root. BRs are involved in a variety of stele and decreases when BRI1 is expressed in the epidermis (Kang cell-specific processes that occur within the different zones of the root. These et al., 2017). Thus, the control of formative asymmetric divisions in include processes such as cell cycle division, cell elongation and cell the stele can be controlled both cell-autonomously and non-cell- differentiation. autonomously in an opposite manner, implying that the nature of the stele divisions might depend on the localization of the instructing the meristem and reduce the number of meristematic cells, but do signal. Conversely, the control of formative asymmetric cell not exhibit an increase in meristem cell length (Chaiwanon and divisions in the epidermis appears to be cell-autonomous, as Wang, 2015). expression of BRI1 in the epidermis restores the wider phenotype of The control of root growth by BR signaling is also spatially the bri1 brl1 brl3 triple receptor mutant (Kang et al., 2017). Of note, segregated throughout the root. BR signaling is not found BRs together with auxins are also involved in establishing the homogeneously throughout the root, with BZR1 being more periodic pattern of vascular bundles in the Arabidopsis shoot strongly activated at the transition from the meristem to the (Ibanes et al., 2009); the quantification of this pattern, together with elongation zones and in the elongation zone itself (Chaiwanon mathematical modeling, supports the notion that cell numbers, and Wang, 2015). Moreover, BR signaling induces target genes in which are controlled by BRs, are relevant for vascular patterning. the epidermis (the outer layer of the root) but mostly represses However, despite these various lines of evidence linking BRs genes in the stele (the inner layer) (Vragović et al., 2015), and vascular development, little is known about the contribution highlighting that BR signaling can elicit tissue-specific responses. of different BR receptors and downstream transcriptional players Based on these results, it has been proposed that BR signaling can in the formation of functional vascular tissues and overall function in a non-cell-autonomous manner, signaling from the organ growth. epidermis to inner cells (Hacham et al., 2011; Vragović et al., 2015). Interestingly, the differential expression of BRI1 between The role of brassinosteroid signaling in stem cell hair and non-hair epidermal cells controls the length of mature self-renewal and differentiation cells as well as their sensitivity to BR hormonal treatment The root stem cell niche comprises a small group of stem cells (Fridman et al., 2014). Furthermore, it was recently shown that located at the base of the meristem in the root apex. These cells are expressing BRI1 under the control of cell-specific promoters of essential for sustaining root growth, as they continuously provide DEVELOPMENT the protophloem (a component of the stele) such as pMAKR5 the precursors of more-specialized cells, and to replace tissues that (MEMBRANE-ASSOCIATED KINASE REGULATOR 5) and have been damaged (Dolan et al., 1993; Sabatini et al., 2003; van pCVP2 (COTYLEDON VASCULAR PATTERN 2) rescues the den Berg et al., 1997). The core of the niche contains a group of cells phenotypic defects of bri1 brl1 brl3 triple receptor mutants, with very low mitotic activity that are collectively known as the suggesting that a phloem-derived signal can non-autonomously quiescent center (QC). The QC maintains the undifferentiated state drive root growth (Kang et al., 2017). These results point to the of the surrounding stem cells (Sarkar et al., 2007; van den Berg complexity of BR signaling and highlight some level of et al., 1997) whilst maintaining its own stemness, but it can also act directionality – from inner to outside cell layers and vice versa as a reservoir of cells that can replenish damaged ones (Heyman – of BR signaling in the root. This signaling directionality likely et al., 2013; Vilarrasa-Blasi et al., 2014). As we discuss below, 3 REVIEW Development (2019) 146, dev151894. doi:10.1242/dev.151894 BRs play a key role in maintaining the identity and quiescence of factor that acts as a co-repressor of BRAVO is TPL (TOPLESS), QC cells (González-García et al., 2011), and thereby affect the which can bind to the BRAVO promoter as well as interact with maintenance of the root stem cell niche. BES1 via its ERF-associated amphiphilic repression (EAR) motif BR signaling acts within the root stem cell niche by (Espinosa-Ruiz et al., 2017). Future studies aiming to dissect the modulating BRAVO (BRASSINOSTEROIDS AT VASCULAR cell-specific gene regulatory networks controlled by BRAVO in AND ORGANIZING CENTER) (Vilarrasa-Blasi et al., 2014). the stem cell niche will be instrumental for uncovering how and This transcription factor, also named MYB56, belongs to the when QC cells divide. R2R3-MYB family and is expressed specifically in vascular BRs can also induce the expression of ERF115 (ETHYLENE initials and QC cells (Vilarrasa-Blasi et al., 2014). Phenotypic RESPONSE FACTOR 115), a transcription factor that belongs to the analyses have shown that BRAVO represses QC cell divisions ethylene response factor family and plays a key role in root growth (Fig. 3A), as bravo mutants show a significant increase in QC and development. Specifically, ERF115 acts as a limiting factor division frequency. However, when BR signaling is activated, for for QC divisions as it regulates the expression of PSK5 example following DNA damage (Fig. 3B), the BR downstream (PHYTOSULFOKINES 5), a peptide hormone that enhances the effector BES1 becomes activated and downregulates the levels of frequency of QC divisions (Heyman et al., 2016, 2013). Collectively, BRAVO transcript. It also heterodimerizes with BRAVO protein BR signaling represses BRAVO activity and, at the same time, itself, strongly inhibiting its action and promoting the division activates ERF115 to promote QC divisions when needed (Fig. 3B). of QC cells (Vilarrasa-Blasi et al., 2014). This constitutes a However, it is still unknown where the signals that activate the BR regulatory circuit that controls QC division via interactions at pathway originate from, i.e. if they come from external tissues or if both the transcriptional and protein levels. Another transcription this process is carried out in a cell-autonomous way. Nonetheless, a A Control conditions BRI1 BAK1 QC cell BES1 BRAVO QC divisions B Upon DNA damage BR QC cell BES1 BES1 BES1 ERF115 DEVELOPMENT BES1 BRAVO QC divisions Fig. 3. Functional role of BRs in stem cell regeneration. (A) In normal (‘control’) conditions, BR signaling in the QC is not active. This maintains BES1 in a phosphorylated and inactive state, permitting BRAVO to act and repress QC divisions. (B) In the presence of DNA damaging agents, vascular cells die and produce a local pool of BR that is sensed by BRI1/BRLs in a paracrine way in the QC. This leads to the dephosphorylation of BES1, the inactivation of BRAVO (both transcriptionally and via BRAVO heterodimerization with BES1) and the induction of ERF115 expression, which together promote the division of QC cells to replenish the dying cells. 4 REVIEW Development (2019) 146, dev151894. doi:10.1242/dev.151894 recent study has shed some light on this matter, revealing that QC cell division is an autonomous process that needs BRI1 action within the stem cell niche (Lozano-Elena et al., 2018). This study also suggested BR that a paracrine signal leads to the activation of BES1 in QC cells in order to promote their division when needed. Thus, when the root suffers damage and stem cells undergo programmed cell death, the ABA BRI1 BAK1 plant detects this scenario and starts promoting QC cell divisions to replenish damaged cells and to assure its survival. Although the mechanism underlying this response remains to be elucidated, it appears to involve a steroid paracrine signal from dead cells to the QC and that is perceived by BRI1 and transduced by BES1 (Lozano- Elena et al., 2018). We hypothesize that one such mobile signal could be the BR molecule itself, and that the increase in BR concentration in the stem cell niche could be due to a possible increase in BR PYR/PYL/RCAR PYR/PYL/R CAR biosynthetic genes, such as those encoding CPD and DWF4 (DWARF 4) (Lozano-Elena et al., 2018). However, further studies BIN2 PP2C of BR synthesis and mobility are required to shed light on this matter SnRKs (Vukasinovic and Russinova, 2018). BRs also promote the differentiation of columella stem cells ABI5 (CSCs), cells which are located distally to the QC. This occurs in a BZR1/ BZR1/ dose-dependent manner (González-García et al., 2011; Lee et al., BES1 BES1 Degradation 2015) via the transcription factor WOX5 (WUSCHEL-RELATED HOMEOBOX 5). WOX5 is a homolog of WUSCHEL, a ABI5 transcription factor that maintains the identity of stem cells in the shoot (Mayer et al., 1998). In the root, WOX5 is required to ABI5 maintain the identity of stem cells (Sarkar et al., 2007), and its transcript expression is restricted to the QC through external signals ABI5 (Ding and Friml, 2010; Zhang et al., 2015) where it represses CYCD activity to establish quiescence (Forzani et al., 2014). wox5 Stress-responsive genes mutants show increased QC divisions and differentiated CSCs in the root apex (Sarkar et al., 2007). Importantly, the expression of WOX5 is regulated by BR; WOX5 expression decreases in bri1-116 Fig. 4. BR-ABA crosstalk during the regulation of stress responses. mutants (lacking the BRI1 receptor) and in plants treated with Schematic of the crosstalk between the BR and ABA pathways. ABA is brassinazole (an inhibitor of BR biosynthesis). In contrast, WOX5 perceived by PYR/PYL/RCAR receptors and promotes the phosphorylation expression increases in plants treated with brassinolide (a bioactive and activation of SnRKs, thereby relieving them from PP2C-mediated form of BR) and in bes1-D or BRI1 overexpressor mutants repression. SnRKs, in turn, phosphorylate downstream transcription factors such as ABI5 that regulate the transcription of various stress-responsive (González-García et al., 2011). genes. BIN2, which is a negative regulator of BR signaling, can also directly In summary, BR levels are essential for regulating both cellular phosphorylate and activate SnRKs and ABI5, while PP2C is able to inactivate quiescence and the differentiation of stem cells in the root apex. BIN2. ABI5 is also a direct target of BZR1, which represses its transcription Further studies, including cell-specific ‘omics’ approaches, will be to negatively regulate stress-responsive gene expression. key to decipher, for example, BRAVO partners and targets in the stem cell niche. It will also be interesting to decipher which receptors are Lobato, 2017; Tunc-Ozdemir and Jones, 2017; Xia et al., 2009; Zou involved in this context, and if the promotion of QC divisions is et al., 2018); and (3) promoting the production of osmoprotectants mediated primarily by BRI1 or if BRL1/3 play a major role as a (Fàbregas et al., 2018). As we discuss below, these various consequence of their expression pattern throughout the root. mechanisms contribute to BR-mediated adaptation to drought, cold, heat and salinity. Brassinosteroid signaling in adaptation to environmental BRs and ABA perform mostly antagonistic physiological stress functions, converging at the level of BIN2 and BZR1 (Cai et al., The ability of a plant to tolerate stress, such as changes in water 2014; Hu and Yu, 2014). Whereas BIN2 acts a repressor of BR availability, temperature or soil salinity, depends on its ability to signaling (as discussed above), it enhances ABA-mediated stress switch between growth activation and repression in unfavorable responses by phosphorylating SnRK2 (SNF1-RELATED PROTEIN conditions (Bechtold and Field, 2018; Feng et al., 2016). A key KINASE 2), leading to ABA-responsive gene expression (Cai et al., pathway that controls responses to environmental stresses is the 2014). In addition, exogenous BR treatment inhibits the ABA- DEVELOPMENT abscisic acid (ABA) signaling pathway (Yoshida et al., 2014; Zhu mediated induction of RD26 (RESPONSIVE TO DESICCATION 26), et al., 2017). However, compelling evidence indicates that BRs also a gene encoding a transcriptional activator of stress-inducible gene play a prominent role in controlling the balance between normal expression (Chung et al., 2014). This reciprocal antagonism between growth and resistance against environmental assaults, acting either BR signaling and ABA-responsive transcription factors is key for via crosstalk with the ABA pathway or independently (Fig. 4). coordinating plant growth and drought tolerance in Arabidopsis Several mechanisms have been proposed to explain how BR (Fig. 4). Indeed, it has been shown that RD26 is also a direct target of signaling mediates adaptation to stress. These include: (1) fine- BES1 and is repressed by BR under drought conditions; reciprocally, tuning stress-responsive transcript machineries (Ye et al., 2017); (2) RD26 modulates the transcription of BES1-regulated genes to inhibit activating antioxidant machineries (Kim et al., 2012; Lima and BR function (Ye et al., 2017). The transcription factors WRKY46, 5 REVIEW Development (2019) 146, dev151894. doi:10.1242/dev.151894 54 and 70 also interact with BES1 directly to promote BR-regulated production, and hence signaling, by enhancing the activity of plant growth while repressing drought-inducible global transcripts 1-aminocyclopropane-1-carboxylate synthase (ACS), an ethylene to inhibit drought tolerance (Chen and Yin, 2017). BIN2 synthesis enzyme (Tao et al., 2015; Zhu et al., 2016). Conversely, phosphorylates and destabilizes WRKY54 to negatively regulate its blocking ethylene production and/or signaling components inhibits effect on the BES1-mediated BR response (Chen et al., 2017). BR-induced antioxidant enzyme activities and salt tolerance (Tao Recently, it was revealed that BR signaling via BIN2 interacts with et al., 2015; Zhu et al., 2016). The role for BR signaling in autophagy pathways to coordinate plant growth and survival under regulating salt stress tolerance may be mediated by BRI1; inhibiting drought stress and starvation (Nolan et al., 2017b) (Fig. 5A). In this the endoplasmic reticulum-associated protein degradation system is context, BIN2 phosphorylates and activates the ubiquitin receptor able to partially rescue the salt hypersensitivity of bri1-9 mutants, protein DSK2, which further interacts with BES1 and targets it for providing evidence for the involvement of a membrane-bound BRI1 degradation via autophagy (Nolan et al., 2017b). Together, these signaling complex in the salinity response (Cui et al., 2012). In findings highlight the complexity of BR-mediated responses to contrast, bin2-1 mutants are hypersensitive to salinity stress, and this drought. Future investigations are clearly needed to unravel the roles correlates with inhibited induction of stress-responsive genes (Zeng of individual BR signaling components and to understand how they et al., 2010). High salinity also causes growth quiescence in roots by switch the balance between normal versus drought-adapted growth suppressing nuclear accumulation of BZR1 and subsequent BR and development. signaling functions (Geng et al., 2013). It is evident from the above- BR signaling also modulates plant adaptation to different mentioned reports that exogenous application of BR helps plants to temperature stresses (Fig. 4B,C). The BR-regulated basic helix-loop- cope better under high salinity conditions by modulating both BR helix (bHLH) transcription factor CESTA activates the expression and ethylene signal outputs. of C-REPEAT/DEHYDRATION-RESPONSIVE ELEMENT In addition to the crosstalk and mechanisms discussed above, the BINDING FACTOR (CBF) transcriptional regulators, which interplay between BR signaling and redox signaling appears to be control the transcription of core cold responsive (COR) genes crucial for plant development under stress (Fig. 5A). It is known that (Eremina et al., 2016). Another BR-regulated transcription factor, BR induces the antioxidant system during abiotic stress tolerance BR-ENHANCED EXPRESSION 1 (BEE1), promotes cold (Jiang et al., 2012; Zhou et al., 2014). BR has also been reported to acclimation by indirectly influencing the transcription of MYB- utilize hydrogen peroxide (H2O2)- and nitric oxide (NO)-mediated bHLH-WD40 complex components (Petridis et al., 2016) (Fig. 5B). mechanisms to provide stress tolerance (Cui et al., 2012; Xia et al., In line with these findings, BIN2 overexpression has been shown to 2009). For example, during oxidative stress, BR increases ABA cause hypersensitivity to freezing stress under both non-acclimated production through NO-mediated machinery (Zhang et al., 2011). and acclimated conditions, whereas bin2-3 bil1 bil2 triple mutants, BR-mediated transient H2O2 production via NADPH oxidase also as well as the gain-of-function bzr1-1D and bes1-D mutants, have triggers ABA biosynthesis, which, along with enhanced H2O2 enhanced tolerance for freezing stress (Li et al., 2017). BZR1 production, acts as a positive-feedback mechanism for prolonged dephosphorylation is also induced upon cold treatment and can heat and oxidative stress tolerance (Zhou et al., 2014). The over- regulate COR genes, either directly or indirectly by binding to CBF1 accumulation of superoxide anions (O− 2 ) in the BR biosynthesis- and CBF2, and thereby affect the transcription of their downstream defective mutant det2-9 highlights yet another node of crosstalk targets (Li et al., 2017) (Fig. 5B). BR signaling is also involved in between the BR and reactive oxygen species (ROS) pathways that is regulating plant growth under high temperature stress (Fig. 5C). implicated in controlling root growth and development (Lv et al., Upon elevated temperature, BZR1 accumulates in the nucleus and 2018). Interestingly, this BR-mediated control of O− 2 accumulation induces the expression of growth-promoting genes, either directly or was found to occur through the peroxidase pathway rather than the via binding to the promoter of PHYTOCHROME INTERACTING NADPH oxidase pathway (Lv et al., 2018). H2O2-mediated FACTOR 4 (PIF4) to regulate thermomorphogenesis (Ibañez et al., oxidative modifications enhance the transcriptional activity of 2018; Oh et al., 2012). Elevated temperature has been shown to BZR1 and promote its interaction with ARF6 and PIF4. In contrast, increase the accumulation of active PIF4, thereby shifting the the thioredoxin TRXh5 interacts with BZR1 and catalyzes its balance of nuclear protein complexes towards BES1-PIF reduction (Tian et al., 2018) (Fig. 5A). Exogenous BR application heterodimers instead of BES1 homodimers (Martinez et al., also increases H2O2 production in the root stem cell niche, 2018). The subsequent reduced availability of active BES1 contributing to BR-induced QC division and cell elongation (Tian homodimers causes de-repression of BR biosynthesis and et al., 2018). feedback inhibition of BR signaling output. In contrast, abundant Nutrient availability in the soil microenvironment is another levels of BES1-PIF4 complexes activate the genes involved in limiting factor for optimal root growth. BR signaling components thermomorphogenesis (Martinez et al., 2018). Elevated ambient were recently shown to regulate root growth behavior under low iron temperatures can also reduce BRI1 levels and affect primary root or phosphate levels (Singh et al., 2018). Specifically, it was found elongation growth (Martins et al., 2017). Recently, the kinase- that BR signaling becomes activated upon iron deficiency and defective BRI1 protein from bri1-301 mutants was found to show promotes root growth, and similarly that perturbed BR signaling less stability and biochemical activity under elevated temperature affects iron distribution in Arabidopsis roots. In contrast, low DEVELOPMENT (29°C). A mutated version of this protein undergoes temperature- phosphate levels cause enhanced iron accumulation, inhibiting BR enhanced protein misfolding and degradation via an as-yet- signaling activation and subsequent root growth acceleration. The unknown mechanism (Zhang et al., 2018). Together, these studies BRI1 negative regulator BKI1 was found to be the center point of highlight a clear involvement of both BR receptors and downstream this signal interplay, with BZR1/BES1, along with their direct target signaling components in regulating growth responses under LPR1, which is a ferroxidase, acting at more downstream steps in fluctuating temperatures. this response (Singh et al., 2018). Moving forward, obtaining a BR signaling is also able to mediate salt tolerance. It does so via more comprehensive understanding of the complex interplay the regulation of ethylene biosynthesis and signaling (Fig. 5D). between BR signaling, cellular redox status and the surrounding Under salinity stress conditions, BR pre-treatment induces ethylene microenvironment will undoubtedly prove beneficial for 6 REVIEW Development (2019) 146, dev151894. doi:10.1242/dev.151894 A Drought B Cold NADPH oxidase Drought BR BR Low temperature NO ROS BRI1 BAK1 BRI1 BAK1 H2O2 BIN2 BIN2 SINAT2 DSK2 BZR1/ BES1/ BZR1 BZR1 BES1 Degradation BES1 BZR1/ BES1 CBFs Other TFs Other BZR1/ BZR1 BZR1 TFs BES1 Drought-responsive genes CBF-dependent CBF-independent COR genes COR genes Drought stress response Cold stress response C Heat D Salinity BR BR High ACS temperature BRI1 BAK1 BRI1 BAK1 ET ETR1/ETR2/EIN4 CTR1 BIN2 BIN2 EIN2 BZR1 BZR1 BZR1 BZR1 EIN3 BZR1 ERF1 BZR1 BR-responsive genes DRE Box genes BR-responsive genes DEVELOPMENT Growth and development Salt tolerance Growth and development Fig. 5. BR signaling controls the switch between growth and abiotic stress responses. (A-D) Schematics of cellular BR actions and crosstalk under conditions of drought (A), cold (B), heat (C) and high salinity (D). Notably, BRs act to control the balance between plant growth and stress responses. The BR and stress signaling pathways show multi-level crosstalk via their receptors, via the downstream kinase BIN2 and/or via transcription factors such as BZR1/BES1, depending on external as well as cellular environments. ET, ethylene. 7 REVIEW Development (2019) 146, dev151894. doi:10.1242/dev.151894 understanding the mechanisms of plant survival and growth BR signal in specific tissues, making, for example, plants that are adaptation in suboptimal growth conditions. resistant to drought without altering their growth. Such an approach Many of the BR-regulated stress adaptation responses discussed will be important to meet the food demands of an exponentially above have been described at the whole-plant survival level. growing world population, especially when increasing plant yield in However, recent technological advances are now allowing us to environmentally challenging conditions becomes essential (Food & deconstruct the complexity of stress traits in a more spatiotemporal Agriculture Organization, 2017). fashion. This approach has been instrumental in identifying the Excitingly, studies have indicated that the local activities of the spatiotemporal roles of other phytohormones during stress different BR receptors – BRI1 and the BRLs – and their effects on responses in plants (Dinneny and Benfey, 2008; Geng et al., root development vary (Kang et al., 2017; Vragović et al., 2015); 2013; Iyer-Pascuzzi et al., 2011). Recently, a role for the vascular this could be one of the mechanisms through which BRs execute cell-specific activation of BR signaling in regulating drought their pleiotropic effects on growth and stress adaptation. We propose adaptation in different developmental stages of root and shoot a scenario (Fig. 6) in which, under normal conditions, BRI1- organs was uncovered using a multi-omics approach (Fàbregas mediated signals drive the growth and development of roots and, et al., 2018). This study revealed that the quadruple BR receptor subsequently, of the whole plant. This idea is supported by the mutant (bri1 brl1 brl3 bak1) exhibits enhanced drought tolerance at finding that the lack of this receptor produces dwarf and sterile the expense of overall growth. However, the overexpression of plants with shorter roots (Li and Chory, 1997). In contrast, the vascular-localized BRL3 receptors significantly improves drought BRL1/BRL3 receptors seem to have little impact on these tolerance without penalizing growth. In this case, BRL3 receptor physiological processes, as mutants of both receptors (brl1 brl3 accumulation in vascular tissues triggers the transcription of mutants) do not show any visible phenotype (Caño-Delgado et al., canonical water stress-response genes and osmoprotectant 2004). However, these BR receptors, which exhibit tissue/cell- metabolism genes under both normal as well as water-deprived specific expression patterns, could be more involved during stress conditions. Metabolomic analyses confirmed that BRL3- responses and adaptation. For example, vascular BRL3 expression overexpressing roots are enriched in osmoprotectant sugars and is able to confer drought resistance, driving the accumulation of amino acids, and analysis of the transcriptome showed that it is osmoprotectant metabolites in the root by promoting the activity of enriched in genes involved in abiotic stress responses (Fàbregas genes involved in their production. Moreover, it is known that brl1 et al., 2018). Altogether, these changes indicate that BRL3- brl3 and brl1 brl3 bak1 mutants have phenotypes associated with overexpressing plants are better prepared for any upcoming stress, which in this case is drought (Fàbregas et al., 2018). This finding is corroborated by previous results reporting that BRs regulate metabolic flux, flavonol accumulation and anthocyanin synthesis during cold acclimation (Petridis et al., 2016), and other studies showing that BR application and BZR1 overexpression promote carotenoid, soluble sugar and ascorbic acid accumulation (Liu et al., 2014). Another example of spatiotemporal compartmentalization of BR signaling has recently been reported (Lozano-Elena et al., 2018). This study highlighted that paracrine BR signals from damaged cells can activate QC division and stem cell replenishment to compensate for root growth arrest upon genotoxic stress. Given the tissue-specific localization and regulation of different BR signaling components, combined with the complexity and diversity of stress-responsive mechanisms, it is likely that decisions of growth versus adaptation are made by signal activation/suppression on BRI1 spatiotemporal scales. Understanding how these spatiotemporal BRI1-mediated BR signaling variations in the activity of BRs control growth and plant adaptation from outer cell layers drives to various environmental stresses is essential for understanding the normal growth and development mechanism by which plants balance growth with adaptation to ensure survival. BRLs Conclusions and perspectives BRL-mediated BR signaling BRs are key for maintaining proper plant growth, both under normal from innermost cell layers conditions and in response to environmental stress, and ample acts during stress adaptation evidence now supports the idea that modifying the BR response pathway can be a powerful strategy for designing better-adapted DEVELOPMENT crops. However, our understanding of the main functions of BR signaling during stress is only generic, and the investigation of precise spatiotemporal- and context-specific regulatory mechanisms Fig. 6. Model depicting the tissue-specific actions of BR receptors during has only just begun (Kang et al., 2017; Lozano-Durán and Zipfel, growth and stress responses. Schematic of a scenario in which BRI1-based BR signaling from outer tissues (green) primarily regulates normal growth 2015; Lozano-Elena et al., 2018; Vragović et al., 2015). Further and development, whereas the signaling mediated by BRLs situated in the studies are clearly required to obtain a more mechanistic innermost cell layers (e.g. in the QC, the stem cell niche and vascular tissues; understanding of the global and local actions of the BR pathway. blue) controls stress adaptation responses. BRLs might also be involved in With such knowledge, we could improve both the growth rates of facilitating the mobilization of metabolic signals (orange arrow) from the shoot plants and their adaptation to the environment by only changing the to root to provide stress tolerance. 8 REVIEW Development (2019) 146, dev151894. doi:10.1242/dev.151894 hydrotropism that are independent of the BRI1 pathway (Fàbregas Chen, J., Nolan, T. M., Ye, H., Zhang, M., Tong, H., Xin, P., Chu, J., Chu, C., Li, Z. and Yin, Y. (2017). Arabidopsis WRKY46, WRKY54, and WRKY70 transcription et al., 2018), suggesting that the response to different stresses might factors are involved in brassinosteroid-regulated plant growth and drought be driven by BR receptors in specific cell types, such as stem cells responses. Plant Cell 29, 1425-1439. and vascular tissues (Fig. 6). The identification of these BR Choe, S., Noguchi, T., Fujioka, S., Takatsuto, S., Tissier, C. P., Gregory, B. D., receptor-driven differential signals will not only illustrate how Ross, A. S., Tanaka, A., Yoshida, S., Tax, F. E. et al. (1999). The Arabidopsis dwf7/ste1 mutant is defective in the delta7 sterol C-5 desaturation step leading to different tissues coordinate their organ growth, but may prove to be brassinosteroid biosynthesis. Plant Cell 11, 207-221. useful for engineering new plants that have improved adaptation to Chung, Y., Kwon, S. I. and Choe, S. (2014). Antagonistic regulation of Arabidopsis the environment without modified growth. growth by brassinosteroids and abiotic stresses. Mol. Cells 37, 795-803. Clouse, S. D. and Sasse, J. M. (1998). Brassinosteroids: essential regulators of Finally, it will also be important to capture the canonical as well as plant growth and development. Annu. Rev. Plant Phys. 49, 427-451. non-canonical signaling dynamics that function downstream of Cui, F., Liu, L., Zhao, Q., Zhang, Z., Li, Q., Lin, B., Wu, Y., Tang, S. and Xie, Q. different BR receptor complexes. Examining these over different (2012). Arabidopsis ubiquitin conjugase UBC32 is an ERAD component that functions in brassinosteroid-mediated salt stress tolerance. Plant Cell 24, time and spatial scales may enable the identification of novel candidates 233-244. that are relevant for adaptation upon stress-induced damage. A more De Bruyne, L., Hö fte, M. and De Vleesschauwer, D. (2014). Connecting growth precise and quantitative visualization of BRI1- and/or BRL-mediated and defense: the emerging roles of brassinosteroids and gibberellins in plant cellular responses, such as ROS and NO production, stress-responsive innate immunity. Mol. Plant 7, 943-959. Ding, Z. and Friml, J. (2010). Auxin regulates distal stem cell differentiation in transcription factor activation and downstream transcript regulation in Arabidopsis roots. Proc. Natl. Acad. Sci. USA 107, 12046-12051. different root tissues, will also help establish how BR executes stress Dinneny, J. R. and Benfey, P. N. (2008). Plant stem cell niches: standing the test of protection and subsequent growth recovery. Overall, studies of the time. Cell 132, 553-557. Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts, K. and mechanisms underlying BR-regulated growth, in both optimal and Scheres, B. (1993). Cellular organisation of the Arabidopsis thaliana root. stress conditions, will bring us closer to understanding the trade-off Development 119, 71-84. between growth and adaptation, and will help us strategize new Eremina, M., Unterholzner, S. J., Rathnayake, A. I., Castellanos, M., Khan, M., approaches for creating smart root systems with efficient water and Kugler, K. G., May, S. T., Mayer, K. F. X., Rozhon, W. and Poppenberger, B. (2016). Brassinosteroids participate in the control of basal and acquired freezing nutrient uptake abilities that can sustain crop biomass and yield. tolerance of plants. Proc. Natl. Acad. Sci. USA 113, E5982-E5991. Espinosa-Ruiz, A., Martı́nez, C., de Lucas, M., Fàbregas, N., Bosch, N., Cano- ̃ Acknowledgements Delgado, A. I. and Prat, S. (2017). TOPLESS mediates brassinosteroid control of We thank Cano-Delgado ̃ lab members for comments on the manuscript. shoot boundaries and root meristem development in Arabidopsis thaliana. Development 144, 1619-1628. Competing interests Fàbregas, N., Li, N., Boeren, S., Nash, T. E., Goshe, M. B., Clouse, S. D., de The authors declare no competing or financial interests. Vries, S. and Cano-Delgado, A. I. (2013). The brassinosteroid insensitive1-like3 signalosome complex regulates Arabidopsis root development. Plant Cell 25, 3377-3388. Funding Fàbregas, N., Lozano-Elena, F., Blasco-Escá mez, D., Tohge, T., Martı́nez- A.P.-R. is recipient of a PhD fellowship from the “Severo Ochoa Programme for Andú jar, C., Albacete, A., Osorio, S., Bustamante, M., Riechmann, J. L., Centers of Excellence in R&D” 2016-2019 from the Ministerio de Ciencia e Nomura, T. et al. (2018). Overexpression of the vascular brassinosteroid receptor Innovació n (SEV-2015-0533). A.G. is a recipient of a post-doctoral fellowship from BRL3 confers drought resistance without penalizing plant growth. Nat. Commun. the “Severo Ochoa Programme for Centers of Excellence in R&D” 2016-2019 from 9, 4680. the Ministerio de Ciencia e Innovació n (SEV-2015-0533). I.B.-P. is funded by a grant Food & Agriculture Organization. (2017). The Future of Food and Agriculture – from the Ministerio de Educació n, Cultura y Deporte (FPU15/02822). N.B. is funded Trends and Challenges. Food & Agriculture Organization of the United Nations. by a grant from the Agència de Gestió d’Ajuts Universitaris i de Recerca, Generalitat Feng, W., Lindner, H., Robbins, N. E., II and Dinneny, J. R. (2016). Growing out of de Catalunya (FI-DGR 2016FI_B 00472). M.I. acknowledges support from the stress: the role of cell- and organ-scale growth control in plant water-stress Ministerio de Economı́a y Competitividad (Spain) and FEDER (European Regional responses. Plant Cell. 28, 1769-1782. Development Fund of the European Union) (FIS2015-66503-C3-3-P) and from the Forzani, C., Aichinger, E., Sornay, E., Willemsen, V., Laux, T., Dewitte, W. and Generalitat de Catalunya through Grup de Recerca Consolidat (2017 SGR 1061). Murray, J. A. H. (2014). WOX5 suppresses CYCLIN D activity to establish A.I.C.-D. is recipient of a grant from the Ministerio de Economı́a y Competitividad quiescence at the center of the root stem cell niche. Curr. Biol. 24, 1939-1944. (BIO2013-43873) and of an ERC Consolidator Grant from the European Research Fridman, Y., Elkouby, L., Holland, N., Vragović , K., Elbaum, R. and Savaldi- Council (ERC-2015-CoG – 683163). Goldstein, S. (2014). Root growth is modulated by differential hormonal sensitivity in neighboring cells. Genes Dev. 28, 912-920. Friedrichsen, D. and Chory, J. (2001). Steroid signaling in plants: from the cell surface to the nucleus. BioEssays 23, 1028-1036. References Gampala, S. S., Kim, T.-W., He, J.-X., Tang, W., Deng, Z., Bai, M.-Y., Guan, S., Bechtold, U. and Field, B. (2018). Molecular mechanisms controlling plant growth Lalonde, S., Sun, Y., Gendron, J. M. et al. (2007). An essential role for 14-3-3 during abiotic stress. J. Exp. Bot. 69, 2753-2758. proteins in brassinosteroid signal transduction in Arabidopsis. Dev. Cell 13, Belkhadir, Y. and Jaillais, Y. (2015). The molecular circuitry of brassinosteroid 177-189. signaling. New Phytol. 206, 522-540. Geng, Y., Wu, R., Wee, C. W., Xie, F., Wei, X., Chan, P. M. Y., Tham, C., Duan, L. Brady, S. M., Orlando, D. A., Lee, J.-Y., Wang, J. Y., Koch, J., Dinneny, J. R., Mace, and Dinneny, J. R. (2013). A spatio-temporal understanding of growth regulation D., Ohler, U. and Benfey, P. N. (2007). A high-resolution root spatiotemporal map during the salt stress response in Arabidopsis. Plant Cell 25, 2132-2154. reveals dominant expression patterns. Science 318, 801-806. Gonzá lez-Garcı́a, M.-P., Vilarrasa-Blasi, J., Zhiponova, M., Divol, F., Mora- Cai, Z., Liu, J., Wang, H., Yang, C., Chen, Y., Li, Y., Pan, S., Dong, R., Tang, G., Garcia, S., Russinova, E. and Cano-Delgado, A. I. (2011). Brassinosteroids Barajas-Lopez, J. D. et al. (2014). GSK3-like kinases positively modulate control meristem size by promoting cell cycle progression in Arabidopsis roots. abscisic acid signaling through phosphorylating subgroup III SnRK2s in Development 138, 849-859. Arabidopsis. Proc. Natl. Acad. Sci. USA 111, 9651-9656. Hacham, Y., Holland, N., Butterfield, C., Ubeda-Tomas, S., Bennett, M. J., Caño-Delgado, A. I. and Blá zquez, M. A. (2013). Spatial control of plant steroid Chory, J. and Savaldi-Goldstein, S. (2011). Brassinosteroid perception in the DEVELOPMENT signaling. Trends Plant Sci. 18, 235-236. epidermis controls root meristem size. Development 138, 839-848. Caño-Delgado, A., Yin, Y., Yu, C., Vafeados, D., Mora-Garcia, S., Cheng, J. C., He, J.-X., Gendron, J. M., Yang, Y., Li, J. and Wang, Z.-Y. (2002). The GSK3-like Nam, K. H., Li, J. and Chory, J. (2004). BRL1 and BRL3 are novel kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid receptors that function in vascular differentiation in Arabidopsis. brassinosteroid signaling pathway in Arabidopsis. Proc. Natl. Acad. Sci. USA 99, Development 131, 5341-5351. 10185-10190. Chaiwanon, J. and Wang, Z.-Y. (2015). Spatiotemporal brassinosteroid signaling Heyman, J., Cools, T., Vandenbussche, F., Heyndrickx, K. S., Van Leene, J., and antagonism with auxin pattern stem cell dynamics in Arabidopsis roots. Curr. Vercauteren, I., Vanderauwera, S., Vandepoele, K., De Jaeger, G., Van Der Biol. 25, 1031-1042. Straeten, D. et al. (2013). ERF115 controls root quiescent center cell division and Chen, J. and Yin, Y. (2017). WRKY transcription factors are involved in stem cell replenishment. Science 342, 860-863. brassinosteroid signaling and mediate the crosstalk between plant growth and Heyman, J., Cools, T., Canher, B., Shavialenka, S., Traas, J., Vercauteren, I., drought tolerance. Plant Signal. Behav. 12, e1365212. Van den Daele, H., Persiau, G., De Jaeger, G., Sugimoto, K. et al. (2016). The 9 REVIEW Development (2019) 146, dev151894. doi:10.1242/dev.151894 heterodimeric transcription factor complex ERF115-PAT1 grants regeneration Mussig, C., Fischer, S. and Altmann, T. (2002). Brassinosteroid-regulated gene competence. Nat. Plants 2, 16165. expression. Plant Physiol. 129, 1241-1251. Hothorn, M., Belkhadir, Y., Dreux, M., Dabi, T., Noel, J. P., Wilson, I. A. and Mussig, C., Shin, G. H. and Altmann, T. (2003). Brassinosteroids promote root Chory, J. (2011). Structural basis of steroid hormone perception by the receptor growth in Arabidopsis. Plant Physiol. 133, 1261-1271. kinase BRI1. Nature 474, 467-471. Nolan, T., Chen, J. and Yin, Y. (2017a). Cross-talk of Brassinosteroid signaling in Hu, Y. and Yu, D. (2014). BRASSINOSTEROID INSENSITIVE2 interacts with controlling growth and stress responses. Biochem. J. 474, 2641-2661. ABSCISIC ACID INSENSITIVE5 to mediate the antagonism of brassinosteroids to Nolan, T. M., Brennan, B., Yang, M., Chen, J., Zhang, M., Li, Z., Wang, X., abscisic acid during seed germination in Arabidopsis. Plant Cell 26, 4394-4408. Bassham, D. C., Walley, J. and Yin, Y. (2017b). Selective autophagy of BES1 Ibanes, M., Fabregas, N., Chory, J. and Cano-Delgado, A. I. (2009). mediated by DSK2 balances plant growth and survival. Dev. Cell 41, 33-46.e7. Brassinosteroid signaling and auxin transport are required to establish the Oh, E., Zhu, J.-Y. and Wang, Z.-Y. (2012). Interaction between BZR1 and PIF4 periodic pattern of Arabidopsis shoot vascular bundles. Proc. Natl. Acad. Sci. USA integrates brassinosteroid and environmental responses. Nat. Cell Biol. 14, 106, 13630-13635. 802-809. Ibañez, C., Delker, C., Martinez, C., Bü rstenbinder, K., Janitza, P., Lippmann, Pavelescu, I., Vilarrasa-Blasi, J., Planas-Riverola, A., Gonzá lez-Garcı́a, M. P., R., Ludwig, W., Sun, H., James, G. V., Klecker, M. et al. (2018). Caño-Delgado, A. I. and Ibañes, M. (2018). A Sizer model for cell differentiation Brassinosteroids dominate hormonal regulation of plant thermomorphogenesis in Arabidopsis thaliana root growth. Mol. Syst. Biol. 14, e7687. via BZR1. Curr. Biol. 28, 303-310.e303. Peng, P., Yan, Z., Zhu, Y. and Li, J. (2008). Regulation of the Arabidopsis GSK3-like Iyer-Pascuzzi, A. S., Jackson, T., Cui, H., Petricka, J. J., Busch, W., Tsukagoshi, kinase BRASSINOSTEROID-INSENSITIVE 2 through proteasome-mediated H. and Benfey, P. N. (2011). Cell identity regulators link development and stress protein degradation. Mol. Plant 1, 338-346. responses in the Arabidopsis root. Dev. Cell 21, 770-782. Petridis, A., Dö ll, S., Nichelmann, L., Bilger, W. and Mock, H.-P. (2016). Jiang, Y.-P., Cheng, F., Zhou, Y.-H., Xia, X.-J., Mao, W.-H., Shi, K., Chen, Z.-X. Arabidopsis thaliana G2-LIKE FLAVONOID REGULATOR and and Yu, J.-Q. (2012). Hydrogen peroxide functions as a secondary messenger for BRASSINOSTEROID ENHANCED EXPRESSION1 are low-temperature brassinosteroids-induced CO2 assimilation and carbohydrate metabolism in regulators of flavonoid accumulation. New Phytol. 211, 912-925. Cucumis sativus. J. Zhejiang Univ. Sci. B 13, 811-823. Russinova, E., Borst, J. W., Kwaaitaal, M., Cano-Delgado, A., Yin, Y., Chory, J. Kang, Y. H., Breda, A. and Hardtke, C. S. (2017). Brassinosteroid signaling directs and de Vries, S. C. (2004). Heterodimerization and endocytosis of Arabidopsis formative cell divisions and protophloem differentiation in Arabidopsis root brassinosteroid receptors BRI1 and AtSERK3 (BAK1). Plant Cell 16, 3216-3229. meristems. Development 144, 272-280. Sabatini, S., Heidstra, R., Wildwater, M. and Scheres, B. (2003). SCARECROW is Kim, B. H., Kim, S. Y. and Nam, K. H. (2012). Genes encoding plant-specific class involved in positioning the stem cell niche in the Arabidopsis root meristem. Genes III peroxidases are responsible for increased cold tolerance of the brassinosteroid- Dev. 17, 354-358. insensitive 1 mutant. Mol. Cells 34, 539-548. Salazar-Henao, J. E., Lehner, R., Betegó n-Putze, I., Vilarrasa-Blasi, J. and Kinoshita, T., Cano-Delgado, ̃ A., Seto, H., Hiranuma, S., Fujioka, S., Yoshida, S. Cano-Delgado, ̃ A. I. (2016). BES1 regulates the localization of the and Chory, J. (2005). Binding of brassinosteroids to the extracellular domain of brassinosteroid receptor BRL3 within the provascular tissue of the Arabidopsis plant receptor kinase BRI1. Nature 433, 167-171. primary root. J. Exp. Bot. 67, 4951-4961. Lee, H.-S., Kim, Y., Pham, G., Kim, J. W., Song, J.-H., Lee, Y., Hwang, Y.-S., Sarkar, A. K., Luijten, M., Miyashima, S., Lenhard, M., Hashimoto, T., Nakajima, Roux, S. J. and Kim, S.-H. (2015). Brassinazole resistant 1 (BZR1)-dependent K., Scheres, B., Heidstra, R. and Laux, T. (2007). Conserved factors regulate brassinosteroid signalling pathway leads to ectopic activation of quiescent cell signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature 446, division and suppresses columella stem cell differentiation. J. Exp. Bot. 66, 811-814. 4835-4849. Scheres, B., Wolkenfelt, H., Willemsen, V., Terlouw, M., Lawson, E., Dean, C. Li, J. and Chory, J. (1997). A putative leucine-rich repeat receptor kinase involved in and Weisbeek, P. (1994). Embryonic origin of the arabidopsis primary root and brassinosteroid signal transduction. Cell 90, 929-938. root-meristem initials. Development 120, 2475-2487. Li, J. and Nam, K. H. (2002). Regulation of brassinosteroid signaling by a GSK3/ Singh, A. P. and Savaldi-Goldstein, S. (2015). Growth control: brassinosteroid SHAGGY-like kinase. Science 295, 1299-1301. activity gets context. J. Exp. Bot. 66, 1123-1132. Li, H., Ye, K., Shi, Y., Cheng, J., Zhang, X. and Yang, S. (2017). BZR1 positively Singh, A. P., Fridman, Y., Holland, N., Ackerman-Lavert, M., Zananiri, R., regulates freezing tolerance via CBF-dependent and CBF-independent pathways Jaillais, Y., Henn, A. and Savaldi-Goldstein, S. (2018). Interdependent nutrient in arabidopsis. Mol. Plant 10, 545-559. availability and steroid hormone signals facilitate root growth plasticity. Dev. Cell Lima, J. V. and Lobato, A. K. S. (2017). Brassinosteroids improve photosystem II 46, 59-72.e4. efficiency, gas exchange, antioxidant enzymes and growth of cowpea plants Sun, Y., Fan, X.-Y., Cao, D.-M., Tang, W., He, K., Zhu, J.-Y., He, J.-X., Bai, M.-Y., exposed to water deficit. Physiol. Mol. Biol. Plants 23, 59-72. Zhu, S., Oh, E. et al. (2010). Integration of brassinosteroid signal transduction Liu, L., Jia, C., Zhang, M., Chen, D., Chen, S., Guo, R., Guo, D. and Wang, Q. with the transcription network for plant growth regulation in Arabidopsis. Dev. Cell (2014). Ectopic expression of a BZR1-1D transcription factor in brassinosteroid 19, 765-777. signalling enhances carotenoid accumulation and fruit quality attributes in tomato. Sun, Y., Han, Z., Tang, J., Hu, Z., Chai, C., Zhou, B. and Chai, J. (2013). Structure Plant Biotechnol. J. 12, 105-115. reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide. Cell Long, Y., Stahl, Y., Weidtkamp-Peters, S., Postma, M., Zhou, W., Goedhart, J., Res. 23, 1326-1329. Sá nchez-Pé rez, M.-I., Gadella, T. W. J., Simon, R., Scheres, B. et al. (2017). In Szekeres, M., Né meth, K., Koncz-Ká lmá n, Z., Mathur, J., Kauschmann, A., vivo FRET-FLIM reveals cell-type-specific protein interactions in Arabidopsis Altmann, T., Ré dei, G. P., Nagy, F., Schell, J. and Koncz, C. (1996). roots. Nature 548, 97-102. Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, Lozano-Durá n, R. and Zipfel, C. (2015). Trade-off between growth and immunity: controlling cell elongation and de-etiolation in Arabidopsis. Cell 85, 171-182. role of brassinosteroids. Trends Plant Sci. 20, 12-19. Tao, J. J., Chen, H. W., Ma, B., Zhang, W. K., Chen, S. Y. and Zhang, J. S. (2015). Lozano-Elena, F., Planas-Riverola, A., Vilarrasa-Blasi, J., Schwab, R. and The role of ethylene in plants under salinity stress. Front. Plant Sci. 6, 1059. Caño-Delgado, A. I. (2018). Paracrine brassinosteroid signaling at the stem cell Tian, Y., Fan, M., Qin, Z., Lv, H., Wang, M., Zhang, Z., Zhou, W., Zhao, N., Li, X., niche controls cellular regeneration. J. Cell Sci. 131, jcs204065. Han, C. et al. (2018). Hydrogen peroxide positively regulates brassinosteroid Lv, B., Tian, H., Zhang, F., Liu, J., Lu, S., Bai, M., Li, C. and Ding, Z. (2018). signaling through oxidation of the BRASSINAZOLE-RESISTANT1 transcription Brassinosteroids regulate root growth by controlling reactive oxygen species factor. Nat. Commun. 9, 1063. homeostasis and dual effect on ethylene synthesis in Arabidopsis. PLoS Genet. Tunc-Ozdemir, M. and Jones, A. M. (2017). BRL3 and AtRGS1 cooperate to fine 14, e1007144. tune growth inhibition and ROS activation. PLoS ONE 12, e0177400. Marquès-Bueno, M. M., Morao, A. K., Cayrel, A., Platre, M. P., Barberon, M., van den Berg, C., Willemsen, V., Hendriks, G., Weisbeek, P. and Scheres, B. Caillieux, E., Colot, V., Jaillais, Y., Roudier, F. and Vert, G. (2016). A versatile (1997). Short-range control of cell differentiation in the Arabidopsis root meristem. Multisite Gateway-compatible promoter and transgenic line collection for cell type- Nature 390, 287-289. specific functional genomics in Arabidopsis. Plant J. 85, 320-333. Vert, G., Nemhauser, J. L., Geldner, N., Hong, F. and Chory, J. (2005). Molecular Martinez, C., Espinosa-Ruiz, A., de Lucas, M., Bernardo-Garcia, S., Franco- mechanisms of steroid hormone signaling in plants. Annu. Rev. Cell Dev. Biol. 21, DEVELOPMENT Zorrilla, J. M. and Prat, S. (2018). PIF4-induced BR synthesis is critical to diurnal 177-201. and thermomorphogenic growth. EMBO J. 37, e99552. Vilarrasa-Blasi, J., Gonzá lez-Garcı́a, M.-P., Frigola, D., Fàbregas, N., Alexiou, Martins, S., Montiel-Jorda, A., Cayrel, A., Huguet, S., Roux, C. P.-L., Ljung, K. K. G., Ló pez-Bigas, N., Rivas, S., Jauneau, A., Lohmann, J. U., Benfey, P. N. and Vert, G. (2017). Brassinosteroid signaling-dependent root responses to et al. (2014). Regulation of plant stem cell quiescence by a brassinosteroid prolonged elevated ambient temperature. Nat. Commun. 8, 309. signaling module. Dev. Cell 30, 36-47. Mayer, K. F., Schoof, H., Haecker, A., Lenhard, M., Jü rgens, G. and Laux, T. Vragović , K., Sela, A., Friedlander-Shani, L., Fridman, Y., Hacham, Y., Holland, (1998). Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot N., Bartom, E., Mockler, T. C. and Savaldi-Goldstein, S. (2015). Translatome meristem. Cell 95, 805-815. analyses capture of opposing tissue-specific brassinosteroid signals orchestrating Mitchell, J. W., Mandava, N., Worley, J. F., Plimmer, J. R. and Smith, M. V. (1970). root meristem differentiation. Proc. Natl. Acad. Sci. USA 112, 923-928. Brassins–a new family of plant hormones from rape pollen. Nature 225, Vukasinovic, N. and Russinova, E. (2018). BRexit: possible brassinosteroid export 1065-1066. and transport routes. Trends Plant Sci. 23, 285-292. 10 REVIEW Development (2019) 146, dev151894. doi:10.1242/dev.151894 Wang, Z.-Y., Seto, H., Fujioka, S., Yoshida, S. and Chory, J. (2001). BRI1 is a Zeng, H., Tang, Q. and Hua, X. (2010). Arabidopsis brassinosteroid mutants det2-1 critical component of a plasma-membrane receptor for plant steroids. Nature 410, and bin2-1 display altered salt tolerance. J. Plant Growth Regul. 29, 44-52. 380-383. Zhang, A., Zhang, J., Zhang, J., Ye, N., Zhang, H., Tan, M. and Jiang, M. Wang, Z.-Y., Nakano, T., Gendron, J., He, J., Chen, M., Vafeados, D., Yang, Y., (2011). Nitric oxide mediates brassinosteroid-induced ABA biosynthesis Fujioka, S., Yoshida, S., Asami, T. et al. (2002). Nuclear-localized BZR1 involved in oxidative stress tolerance in maize leaves. Plant Cell Physiol. 52, mediates brassinosteroid-induced growth and feedback suppression of 181-192. brassinosteroid biosynthesis. Dev. Cell 2, 505-513. Zhang, Y., Jiao, Y., Liu, Z. and Zhu, Y.-X. (2015). ROW1 maintains quiescent Xia, X.- J., Huang, L.-F., Zhou, Y.-H., Mao, W.-H., Shi, K., Wu, J.-X., Asami, T., centre identity by confining WOX5 expression to specific cells. Nat. Commun. Chen, Z. and Yu, J.-Q. (2009). Brassinosteroids promote photosynthesis and 6, 6003. growth by enhancing activation of Rubisco and expression of photosynthetic Zhang, X., Zhou, L., Qin, Y., Chen, Y., Liu, X., Wang, M., Mao, J., Zhang, J., genes in Cucumis sativus. Planta 230, 1185-1196. He, Z., Liu, L. et al. (2018). A temperature-sensitive misfolded bri1-301 receptor Yamaguchi, M., Goue, N., Igarashi, H., Ohtani, M., Nakano, Y., Mortimer, J. C., requires its kinase activity to promote growth. Plant Physiol. 178, 1704-1719. Zhao, B. and Li, J. (2012). Regulation of brassinosteroid biosynthesis and Nishikubo, N., Kubo, M., Katayama, Y., Kakegawa, K. et al. (2010). Vascular- inactivation. J. Integr. Plant Biol. 54, 746-759. related NAC-Domain6 and vascular-related NAC-Domain7 effectively induce Zhou, J., Wang, J., Li, X., Xia, X.-J., Zhou, Y.-H., Shi, K., Chen, Z. and Yu, J.-Q. transdifferentiation into xylem vessel elements under control of an induction (2014). H2O2 mediates the crosstalk of brassinosteroid and abscisic acid in system. Plant Physiol. 153, 906-914. tomato responses to heat and oxidative stresses. J. Exp. Bot. 65, 4371-4383. Yamamoto, R., Fujioka, S., Demura, T., Takatsuto, S., Yoshida, S. and Fukuda, Zhu, J.-Y., Sae-Seaw, J. and Wang, Z.-Y. (2013). Brassinosteroid signalling. H. (2001). Brassinosteroid levels increase drastically prior to morphogenesis of Development 140, 1615-1620. tracheary elements. Plant Physiol. 125, 556-563. Zhu, T., Deng, X., Zhou, X., Zhu, L., Zou, L., Li, P., Zhang, D. and Lin, H. (2016). Ye, H., Liu, S., Tang, B., Chen, J., Xie, Z., Nolan, T. M., Jiang, H., Guo, H., Lin, Ethylene and hydrogen peroxide are involved in brassinosteroid-induced salt H.-Y., Li, L. et al. (2017). RD26 mediates crosstalk between drought and tolerance in tomato. Sci. Rep. 6, 35392. brassinosteroid signalling pathways. Nat. Commun. 8, 14573. Zhu, Y., Wang, B., Tang, K., Hsu, C.-C., Xie, S., Du, H., Yang, Y., Tao, W. A. and Yin, Y., Wang, Z.-Y., Mora-Garcia, S., Li, J., Yoshida, S., Asami, T. and Chory, J. Zhu, J.-K. (2017). An Arabidopsis Nucleoporin NUP85 modulates plant (2002). BES1 accumulates in the nucleus in response to brassinosteroids to responses to ABA and salt stress. PLoS Genet. 13, e1007124. regulate gene expression and promote stem elongation. Cell 109, 181-191. Zou, L.-J., Deng, X.-G., Zhang, L.-E., Zhu, T., Tan, W.-R., Muhammad, A., Zhu, Yoshida, T., Mogami, J. and Yamaguchi-Shinozaki, K. (2014). ABA-dependent L.-J., Zhang, C., Zhang, D.-W. and Lin, H.-H. (2018). Nitric oxide as a signaling and ABA-independent signaling in response to osmotic stress in plants. Curr. molecule in brassinosteroid-mediated virus resistance to Cucumber mosaic virus Opin. Plant Biol. 21, 133-139. in Arabidopsis thaliana. Physiol. Plant 163, 196-210. DEVELOPMENT 11