Growth control: brassinosteroid activity gets context

Journal of Experimental Botany, Vol. 66, No. 4 pp. 1123–1132, 2015 doi:10.1093/jxb/erv026 

Review Paper

Growth control: brassinosteroid activity gets context Amar Pal Singh and Sigal Savaldi-Goldstein* Faculty of Biology, Technion-Israel Institute of Technology, Haifa 3200003, Israel *  To whom correspondence should be addressed. E-mail: [email protected]

Abstract Brassinosteroid activity controls plant growth and development, often in a seemingly opposing or complex manner. Differential impact of the hormone and its signalling components, acting both as promoters and inhibitors of organ growth, is exemplified by meristem differentiation and cell expansion in above- and below-ground organs. Complex brassinosteroid-based control of stomata count and lateral root development has also been demonstrated. Here, mechanisms underlying these phenotypic outputs are examined. Among these, studies uncovering core brassinosteroid signalling components, which integrate with distinct peptide, hormone, and environmental pathways, are reviewed. Finally, the differential spatiotemporal context of brassinosteroid activity within the organ, as an important determinant of controlled growth, is discussed. Key words:  Brassinosteroid signalling, growth coordination, meristem size, phytohormone, root growth, shoot growth.

Introduction Plant hormones control almost all aspects of plant growth and development. Their crucial role is reflected in the aberrant plant morphologies that arise upon suboptimal or supraoptimal endogenous plant hormone levels. Notably, the precise effect of a specific hormone on growth is not a result merely of its available levels, but also depends on the plant species, the organ, and even the tissue in which it acts. The activity of the brassinosteroid (BR) hormones triggers growth of aboveand below-ground organs, as apparent from the striking dwarf phenotype of BR-deficient mutants. This phenotype is a result of reduced cell proliferation and cell expansion, and provides unequivocal genetic evidence for the positive regulation of these two processes by BRs (Fridman and Savaldi-Goldstein, 2013). Recent studies have both shown a context-specific impact of BR activity on these generic growth stages, and supported a known concentration-dependent effect of the hormone (Mandava, 1988). In addition, downstream BR signalling factors have been shown to be shared by distinct signalling pathways, controlling different aspects of growth and development, including cell elongation, shoot branching, stomata formation, lateral root formation, and xylem differentiation. Such integrations can form either a temporary

interdependence between BR and additional hormonal pathways (e.g. hypocotyl cell elongation), opposing or complex effects (e.g. stomata formation, lateral root formation, and root meristem size), or act seemingly independent of BR (e.g. shoot branching, (Wang et al., 2013)). In this review, we examine and discuss the aforementioned complexities, where the BR signalling pathway or its downstream components trigger differential outcomes in various growth and developmental processes. Finally, we highlight recently uncovered differential BR signals acting in neighbouring cells and tissues, and their importance in the balance of organ growth.

The linear BR signalling pathway In Arabidopsis, BRs are perceived by the extracellular leucine-rich repeat (LRR) domain of three cell surface receptors kinases, BRI1 and its two homologues BRL1 and BRL3. BRI1 is the central receptor, with a broad expression pattern, while the expression of its two homologues has a more modest effect on growth and is limited to the vasculature (Clouse et al., 1996; Li and Chory, 1997; Friedrichsen et  al., 2000; Wang

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Received 27 November 2014; Revised 12 January 2015; Accepted 14 January 2015

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BRs and hypocotyl cell elongation: interdependence, but with the right timing Hypocotyl length is determined by the activity of distinct hormones and by environmental signals. For example, it is inhibited by light, through the photomorphogenesis programme, and is enhanced by elevated temperature. As such, how the promoting effect of the BR signalling integrates with these other pathways remains an open question. Recent studies have made tremendous progress, by demonstrating that light, temperature, and hormone signalling components modulate hypocotyl cell expansion via an interconnected battery of transcription factors, which can be roughly divided into two consecutive modules (Fig. 1A). The first module is composed of BES1/BZR1, that interacts with two key

transcription factors: a subset of the auxin response factor (ARF) family (i.e. ARF6 and ARF8) and the PHYTOCHROME INTERACTING FACTOR 4 (PIF4), probably forming a trimeric complex which shares common target genes, many of which encode cell wall enzymes, presumably involved in cell expansion (Bai et al., 2012b; Gallego-Bartolome et al., 2012; Li et al., 2012; Oh et  al., 2012, 2014a). The BES1/BZR1–ARF complex supports the longstanding reported synergistic interaction between auxin and BRs, implicated in both elevated gene expression and extended hypocotyl growth (Nemhauser et  al., 2004; Walcher and Nemhauser, 2012). Since the expression level of PIF is negatively regulated by light, positively regulated by elevated temperature, and oscillates with the circadian clock, its accumulation acts as a rheostat, which transduces environmental signals to growth decision-making factors (de Lucas and Prat, 2014). Intriguingly, the three interacting transcription factors BES1/BZR1, ARF6, and PIF4 are directly inhibited by DELLA, a negative regulator of the gibberellin (GA) pathway, which prevents their binding to DNA (Bai et al., 2012b; Gallego-Bartolome et al., 2012; Li et al., 2012; Oh et al., 2014a). In this model, high GA levels destabilize DELLA, promoting hypocotyl length via simultaneous activity of BES1/BZR1, ARF6, and PIF4 (Oh et al., 2014a), in line with reports of a synergistic interaction between BR and GA in elongation growth of light-grown Arabidopsis hypocotyls (Tanaka et al., 2003). In addition to genes encoding cell wall-remodelling enzymes, the first module directly promotes the expression levels of the helix–loop–helix PACLOBUTRAZOLRESISTANCE (PRE) family of transcription factors, comprising the second module (Fig. 1A) (Oh et al., 2012). In this module, the PRE family promotes growth via a series of antagonistic interactions with other helix–loop–helix proteins, including ILI1 BINDING bHLH 1 (AtIBH1) (Zhang et al., 2009), ATBS1 INTERACTING FACTORs (AIFs) (Wang et al., 2009; Ikeda et al., 2013), IBH1-LIKE1 (IBL1) (Zhiponova et al., 2014), and PHYTOCHROME RAPIDLY REGULATED1/2 (PAR1/2) (Hao et al., 2012). This inhibition enables the activation and binding of several basic helix–loop–helix (bHLH) factors, such as HOMOLOG OF BEE2 INTERACTING WITH IBH1 (HBI1) and ACTIVATORS FOR CELL ELONGATION (ACEs), to promoters of cell wall genes (Fig. 1A) (Bai et al., 2012a; Ikeda et al., 2012, 2013). Typical to complex signalling networks, the growth-promoting activity of these converged pathways involves various feedback loops, which control transcription factor accumulation and the hormone biosynthesis levels via positive, negative, and incoherent feed-forward loops (Oh et al., 2014a; Zhiponova et al., 2014). The hypocotyl growth rate is not constant over time. For example, under short-day conditions, hypocotyl elongation is maximal at dawn (Nozue et al., 2007). Under these same conditions, BR biosynthesis and signalling genes oscillate, with peak expression at or around dawn, a pattern that is important for setting the phase of maximal hypocotyl elongation (Michael et  al., 2008). How BR signalling integrates with time-of-day control of growth is not clearly defined. A recent study showed that BIN2 phosphorylates and destabilizes PIF4. Analysis of PIF4 protein accumulation at different times of the day, as compared with PIF4 with mutated BIN2 phosphorylation sites (PIF41A), suggested that BIN2 activity prevailed at

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et  al., 2001; Cano-Delgado et  al., 2004; Zhou et  al., 2004; Fabregas et al., 2013). The BR ligand is proposed to serve as a ‘glue’ that promotes the association of BRI1 LRR with that of its co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1) (Santiago et al., 2013; Sun et al., 2013). Activated BRI1 phosphorylates its negative regulator BRASSINSOSTEROID KINASE INHIBITOR1 (BKI1) (Wang and Chory, 2006; Jaillais et  al., 2011), which in turn leaves the plasma membrane, allowing BRI1 to complex with BAK1 (Li et al., 2002; Nam and Li, 2002; Clouse, 2011). As a result, both undergo reciprocal transphosphorylation, resulting in an enhanced signalling output. Activated BRI1 also phosphorylates the BR SIGNALLING KINASE 1 (BSK1) and CONSTITUTIVE DIFFERENTIAL GROWTH 1 (CDG1) kinases, which phosphorylate and activate the phosphatase bri1 SUPPRESSOR 1 (BSU1) (Kim et al., 2009). BSU1 (Mora-Garcia et al., 2004) dephosphorylates and inactivates the GSK3-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), the key inhibitor of the signalling cascade (Li et al., 2001; Kim et al., 2009). This enables the subsequent dephosphorylation (Tang et al., 2011) and activation of BRASSINAZOLE RESISTANT1 (BZR1) and its homologous transcription factor BRI1-EMSSUPPRESSOR1 (BES1)/BZR2 (hereafter called BES1), which regulate the expression of hundreds of genes (Sun et al., 2010; Yu et al., 2011). This regulation involves both gene activation and gene repression, depending on their interacting partners. Genetic analyses revealed different degrees of the effects of gene redundancy on distinct growth and developmental processes. For example, while the bin2 loss-of-function mutant has no apparent phenotype, higher order mutant combination of its homologues (Vert and Chory, 2006) and the use of a specific Arabidopsis GSK3-like kinase inhibitor drug (De Rybel et al., 2009) resulted in a high BR response-like phenotype. In contrast, overexpression of different BIN2 family members mimicked a BR-deficient phenotype (Kim et al., 2009; Rozhon et al., 2010). Ligand-dependent BRI1 activation has also been shown to trigger fast proton ATPase activity required for cell expansion (Caesar et  al., 2011) and cytosolic calcium, which may affect BR signalling (Frei dit Frey et al., 2012; Oh et al., 2012; Zhao et al., 2013). How these rapid responses integrate with the linear pathway is unknown. Key BR signalling components are also controlled by additional pathways, as will be discussed below.

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dawn, maintaining temporary low PIF levels (Fig. 1B). Plants expressing PIF41A (i.e. expressing PIF4 that ‘escapes’ BIN2 phosphorylation) had long hypocotyls, with arithmetic growth and maximal hypocotyl elongation occurring during the day. Hence, this study further supports the relevance of diurnal oscillations of BR pathway components for proper growth. A  future challenge lies in the direct quantification of BIN2 phosphorylation and BR levels at different times of the day. Another aspect affecting growth regulation relates to seedling age. A time-lapse imaging study revealed that the synergistic impact of BR and GA on hypocotyl length is limited to a specific time (also defined as a developmental stage) after germination (Fig. 1C) (Stewart Lilley et al., 2013). This analysis also showed a complex interaction between DELLA and BRs, where DELLA in fact enhances the growth-promoting effect of the latter. These results point to dynamic interactions between BR and GA, depending on the developmental stage of the seedling (Stewart Lilley et al., 2013). In addition to the aforementioned modules, other transcription factors and histone-modifying enzymes have been reported to bind BES1/BZR1, yielding co-operative gene expression control, leading to growth promotion (Li et  al., 2010; Ye et  al., 2012; Hao et  al., 2014; Oh et  al., 2014b; Wang et al., 2014; Zhang et al., 2014). Two of them, MYELOBLASTOSIS FAMILY TRANSCRIPTION FACTOR-LIKE 2 (MYBL2) (Ye et  al., 2012) and HOMEOBOX ARABIDOPSIS THALIANA 1 (HAT1), were reported to be stabilized by BIN2 phosphorylation,

suggesting an elegant means of buffering the impact of BR levels on growth (Ye et al., 2012; Zhang et al., 2014).

BRs differentially control stem cell activity, cell elongation, and organ growth in above-ground organs In the shoot apical meristem (SAM), transition (or differentiation) from meristematic cells to primordia (young leaf) cells is largely controlled by Class I knotted1-like homeobox (KNOX) transcription factors. KNOX expression in the SAM is crucial for maintenance of meristematic activity (Hake et  al., 2004). Among the direct and up-regulated targets of the rice KNOX gene OSH1 are three BR catabolic genes coding for CYP734A/ BAS1 (Tsuda et al., 2014). CYP734A/BAS1 is a carbon-26-hydroxylase of the two most active BRs, brassinolide and castasterone (Turk et al., 2003). Similar to the loss-of-function osh1 mutant, knockdown of these genes showed boundary defects in the SAM. The boundary is formed by limited growth that separates the emerging primordia from the meristem and neighbouring primordia from each other. Hence, local up-regulation of BAS1 by the boundary-localized protein LOB in Arabidopsis defines the boundary, presumably by down-regulation of BR levels that lead to attenuated cell proliferation (Fig.  2A) (Bell et  al., 2012). Low BR levels attenuate the accumulation of BES1/BZR1 in the nucleus of these cells, preventing them from repressing CUC genes (Gendron et al., 2012). As a result,

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Fig. 1.  BR signalling, time of day, and seedling age in the control of hypocotyl cell expansion. (A) BZR1, ARF6, and PIF4 proteins form a complex as part of a primary module which regulates common target genes, among them the PRE transcription factors (Bai et al., 2012b; Gallego-Bartolome et al., 2012; Li et al., 2012; Oh et al., 2012, 2014a). PREs regulate their target genes via a secondary module. BZR1, ARF6, and PIF4 are each directly inhibited by DELLA, thereby explaining a synergistic interaction between BR, auxin, and GA. The expression level of PIF is negatively regulated by light and it oscillates with the circadian clock. Hence, PIF accumulation acts as a rheostat, which transduces environmental signals to growth. (B) In addition to controlling PIF4 transcription, the clock is thought to regulate the time of BIN2 activity, which prevails at dawn, thereby maintaining temporary low PIF4 levels (Bernardo-Garcia et al., 2014). Shown is PIF4 and PIF1A protein accumulation during the day (PIF41A is PIF with mutated BIN2 phosphorylation sites). The dark period is marked by grey and light by white. This model is important to achieve diurnal phase of hypocotyl growth occurring at late night. (C) Interactions between BR and GA are dynamic and depend on the developmental stage of the seedling. Green boxes mark the stage where synergistic interaction has been detected (Stewart Lilley et al., 2013).

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elevated CUC genes slow cell proliferation, thus defining the boundary. Boundary definition is facilitated by negative feedback regulation, in which high levels of BES1/BZR1 promote LOB expression (Fig.  2A) (Bell et  al., 2012). However, OSH1 and CYP734As are expressed throughout the SAM, raising the question of how this module culminates into a defined boundary (Tsuda et al., 2014). As described above, the role of BRs, mediated by BES1/ BZR1, suggests that their accumulation promotes cell proliferation in the SAM. In contrast, high hormone activity (as inferred by the analysis of a CYP734As/BAS1 knockdown line) occasionally promotes vacuolization of the SAM and the primordia cells. This indicates a premature differentiation of these cells, resulting in arrest of leaf initiation (Fig. 2A). Hence, BR activity promotes two opposing processes, cell proliferation and cell differentiation, within the small region of the SAM. A complex impact of BR on organ growth was also reported in leaves, where the ratio between the number of cells and their size varied when BR signalling and BR

biosynthesis were stimulated, with the former promoting cell proliferation and the latter cell differentiation (Zhiponova et al., 2013). Hence, while high BRI1 activity led to increased numbers of dividing cells (Gonzalez et al., 2010; Oh et al., 2011; Zhiponova et  al., 2013), continuous exposure to the hormone resulted in a lower proportion of mitotic cells and to enhanced cell expansion (Fig. 2B) (Zhiponova et al., 2013). The mechanisms underlying these observations are unknown. In rice, BRs have been shown to have opposing effect on cell elongation in the leaf sheath (Tong et  al., 2014), which has been attributed to a complex interaction between BRs and GA. Physiological levels of BRs trigger cell elongation in the leaf sheath by elevating GA biosynthesis. In contrast, high BR levels, applied exogenously to the leaf sheath, inhibit cell elongation by triggering GA catabolism. Whether ‘supraoptimal’ BR concentrations occur in response to specific conditions during leaf sheath growth, such as after exogenous application of the hormone, remains unclear.

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Fig. 2.  Examples of differential BR effects. (A) High BR activity enhances both cell proliferation and cell differentiation in the SAM. Local elevation of the BR catabolic enzyme CYP34/BAS1 is necessary to slow cell proliferation at the boundary zone (Bell et al., 2012; Tsuda et al., 2014). In accordance with this, high BR causes organ boundary defects, presumably by promoting proliferation of these cells (Bell et al., 2012; Gendron et al., 2012; Tsuda et al., 2014). High BR activity also triggers cell differentiation in the SAM, outside the boundary region (Tsuda et al., 2014). SAM image is reprinted and modified from Shani et al., 2006. The role of hormones in shoot apical meristem function. Current Opinion in Plant Biology 9:484–489. Copyright 2006, with permission from Elsevier. (B) High BR signalling triggers cell proliferation in the leaves and high hormone levels trigger their early differentiation (Gonzalez et al., 2010; Oh et al., 2011; Zhiponova et al., 2013). How these two processes are coordinated and the molecular mechanism underlying these contrasting observations remain unknown. (C) The TDIF–TDR–BIN2 module inhibits xylem differentiation, counteracting the BR effect (Kondo et al., 2014). (D) In roots, the TDIF–TDR–BIN2 module promotes lateral root formation (Cho et al., 2014), while a similar promoting effect is triggered by BRI1, probably via enhanced auxin polar transport (PAT) (Bao et al., 2004), independent of BIN2. How these two models integrate is unknown. (E) BIN2 negatively regulates YODA, MKK4, and SPCH, thereby promoting and inhibiting stomata count, respectively (Gudesblat et al., 2012b; Kim et al., 2012; Khan et al., 2013). These contrasting effects are associated with specific organs, environmental conditions, and seedling age. SPCH regulates the transcription of BR biosynthesis and signalling components in a complex manner (Lau et al., 2014). SAM image is reprinted and modified from (Shani et al., 2006) . The role of hormones in shoot apical meristem function. Current Opinion in Plant Biology 9:484–489. Copyright (2006), with permission from Elsevier. Arabidopsis image courtesy of Yulia Fridman.

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The emerging repertoire of BIN2 interactions and the complexity of BR signalling

BRs impose contrasting tissue-dependent impacts on root growth Root meristem size Root length is determined by the number of cells in the meristem and by their final size. As seen with shoot growth, both processes are affected by BRs in an opposing manner, depending on the concentration of the hormone and the intensity of the signal. For example, BRs promote stem cell daughter divisions in the primary root meristem, as revealed by a comparison of the small meristem of BR-deficient mutants with that of the wild type (Gonzalez-Garcia et al., 2011; Hacham et al., 2011). In contrast, high BR levels promote meristem differentiation (Gonzalez-Garcia et al., 2011). How can BRs both promote and inhibit growth? One proposed mechanism relates to the hormone concentrations per se. In this scenario, different signalling components are differentially affected by such levels, as was recently suggested for salicylic acid effectors (Fu and Dong, 2013). Using mathematical modelling, it was proposed that sensitivity to BRs is primarily determined by the availability of the hormone, since only a small fraction of BRI1 molecules is occupied by the ligand under physiological conditions (van Esse et al., 2012, and examples therein). However, a plant line expressing BRI1–green fluorescent protein (GFP) under its natural promoter showed enhanced sensitivity in a root growth assay as compared with the wild type, contradicting this model (van Esse et al., 2012). While the molecular basis for this observation remains unknown, differential response to the hormone may be due to tissue-specific dynamics of signalling components, a critical aspect in interpretation of and response to a given stimulus. Recent models proposed that the spatial distribution of the BR receptors determine the opposing impact of BR on growth, as will be elaborated below (Fig. 3A, C) (Fridman et al., 2014; Vragovic et al., 2015). Similar to above-ground organs (Savaldi-Goldstein et al., 2007), targeted expression of BRI1 to the root epidermis (in its corresponding bri1 mutant background) is sufficient to promote stem cell daughter divisions (Hacham et al., 2011). In fact, these roots have enlarged meristems as compared with the wild type, a phenotype that could not be mimicked either by exogenous application of the hormone or by overexpression of the receptor. This observation suggested a

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BIN2 has recently been shown to be a signalling component shared by other LRR receptor kinases that control stomata formation, xylem differentiation, and lateral root development. While uncovering novel upstream and downstream BIN2 regulation, these studies also exposed new complexities associated with its function, as discussed below. BRs promote xylem differentiation via an unknown mechanism (Szekeres et al., 1996; Nagata et al., 2001; Cano-Delgado et  al., 2004). The LRR receptor kinase TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) RECEPTOR (TDR), also called PHLOEM INTERCALATED WITH XYLEM (PXY), suppresses xylem differentiation and promotes cambium proliferation (Fisher and Turner, 2007; Hirakawa et al., 2008, 2010). TDR is activated by its ligand TDIF, a peptide of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (CLE)related family. Activated TDR suppresses xylem differentiation by activating members of the GSK3 family, including BIN2, which leads to inactivation of BES1, thereby opposing the BR signalling pathway (Fig. 2C) (Kondo et al., 2014). Hence, open questions relating to how the TDR pathway prevails over the BR pathway in preventing xylem differentiation and how BR signalling counteracts this effect remain. A similar TDIF–TDR–BIN2 module has recently been shown to promote lateral root formation, involving an unexpected positive interaction with auxin (Cho et  al., 2014). Lateral roots are initiated in a subset of pericycle cells, and localized auxin activity is crucial for their development (reviewed in Lavenus et al., 2013). Auxin activates the transcription factors ARF7 and ARF19, by mediating the degradation of their inhibitor AUX/IAA14. Interestingly, in response to TDIF, activated BIN2 phosphorylates and activates ARF7 and ARF19, promoting their DNA binding activity (Fig.  2D) (Cho et  al., 2014). Thus, BIN2 phosphorylation activates and inhibits key auxin and BR transcription factors, respectively. Somewhat counterintuitively, BR signalling promotes lateral root formation (Bao et  al., 2004). How BIN2 is simultaneously activated and inactivated to promote lateral root development remains unclear. One model suggests that BRI1 enhances polar auxin transport (Bao et al., 2004) in a BIN2independent manner (Cho et al., 2014). How this bypass signalling occurs remains an open question. In a different developmental context, BIN2 triggers opposing impacts of BR activity during stomatal precursor stem cell activity (Fig. 2E). In this case, BR activity has a differential impact on stomata density and distribution (Gudesblat et  al., 2012b; Kim et  al., 2012; Khan et  al., 2013) with dependence on organ (hypocotyl, cotyledons, and leaves), environmental conditions (e.g. in vitro versus in vivo plant growth), and seedling age (Gudesblat et  al., 2012a). These distinct outcomes are in accordance with three points of interaction between BIN2 and components of the mitogenactivated protein kinase (MAPK) signalling cascade. The MAPK kinase kinase (MAPKKK) YODA (YDA), activated

by the ERECTA (ER) family of receptors, phosphorylates the MAPKKs MKK4/MKK5, which, in turn, activate the MAPKs MPK3/MPK6, thereby inhibiting the bHLH transcription factor SPEECHLESS (SPCH), which triggers the formation of stomatal precursor stem cells (Fig. 2E). BIN2 phosphorylates and inactivates both YDA and MKK4 and their downstream target SPCH, hence resulting in high and low stomata count, respectively. In addition, it has been reported recently that among the direct targets of SPCH in the stomata founder cells are genes encoding BR signalling and biosynthesis proteins (Lau et al., 2014). However, SPCH elevates and reduces the expression of BR signalling and biosynthesis components, potentially culminating in both a positive and negative effect on the pathway, thus adding another dimension of complexity to the BR effect.

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tissue-dependent function of the hormone signal. Indeed, using a series of genetic studies, it was recently shown that while the hormone triggers cell proliferation via epidermally localized BRI1, it promotes differentiation via the innermost, namely the stele-localized, receptors (Vragovic et al., 2015). BR activity in the stele buffers the epidermal effect, thereby co-ordinating the size of the meristem (Fig. 3A). The aforementioned model for spatially differential BR activity was further supported by translatome mapping of BR responses in roots, which uncovered a context-specific BR impact on gene expression. Genes down-regulated by BR are enriched in the apical meristem zone of the stele. In contrast, genes up-regulated by the hormone are mainly resident in the basal meristem zone of the epidermis. The precise mechanism through which BRs activate the cell cycle remain unknown (as elaborated in Fridman and Savaldi-Goldstein, 2013). However, local interaction between BR and auxin (which is known to activate the cell cycle) has recently been uncovered. In the translatome study, BR was shown to trigger genes enriched by those related to auxin, in the epidermis. Among them are auxin biosynthesis genes and

kinases, which promote the activity of the auxin efflux facilitator PIN2. These findings are in agreement with the observation that epidermal BRI1 triggers membrane accumulation of PIN2 (Hacham et al., 2012). Indeed, the enlarged meristem imposed by epidermal BRI1 has been shown to depend on the auxin biosynthesis gene TAA1 and PIN2 (Fig.  3A) (Vragovic et  al., 2015). Hence, auxin acts as a non-autonomous signal, triggered by BRI1 in the epidermis, and delays cell differentiation of stem cell daughters. In agreement with this, the region of high auxin concentrations in the root apical meristem, known to promote cell proliferation, is extended when BRI1 is active in the epidermis only. How does BR activity in the stele promote cell differentiation? This question was addressed when the translatome mapping was performed in the loss-of-function bri1, which harbours tissue-specific expression of the receptor. Genes in the inner cells were shown to be autonomously regulated by the hormone; that is, their modulation by BR, at least within hours of hormone application, depended on locally expressed receptor(s) (Vragovic et  al., 2015). Further analyses of the role of these genes will be required to understand the process.

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Fig. 3.  Contrasting tissue-dependent impact of BRs co-ordinate root growth. (A) BRI1, active in the epidermis, promotes auxin biosynthesis and its transport to the inner cells, thereby promoting daughter stem cell proliferation. In contrast, BRI1 and its two homologues, active in the stele, promote cell differentiation, buffering the epidermal effect. These tissue-dependent effects of BR activity are accompanied by context-specific modulation of gene expression (Vragovic et al., 2015). The orange arrow indicates the meristem transition zone. Epidermal tissue is marked in green. The apical meristem zone of the stele tissue is marked in purple. The QC is highlighted in orange and the stem cell niche is enclosed in a yellow line. (B) BR activity promotes QC cell divisions. BES1 and BRAVO interact in a non-linear fashion, forming a regulatory switch controlling QC cell quiescence (Vilarrasa-Blasi et al., 2014). BR also stimulates QC divisions by elevating the ERF115 transcriptional level (Heyman et al., 2013). (C) The relative expression level of BRI1 in hair (purple colour) and non-hair cells (green colour) determines the intensity of its downstream signalling and subsequent whole-root growth, via positive and negative effects on unidirectional cell expansion, respectively (Fridman et al., 2014).

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Root cell elongation The Arabidopsis root epidermis contains two types of cells, differing in their final length and subcellular organization, whose fates are determined by positional effects in the embryo, and which differentiate to root hair cells and nonhair cells upon completion of elongation, hence their names (Dolan et  al., 1993, 1994). The relative expression level of

BRI1 in hair and non-hair cells determines the outcome of its downstream signalling (Fig. 3C). Low relative BRI1 activity in hair cells leads to enhanced BR signalling in non-hair cells, without affecting the accumulation of BRI1 at the plasma membrane. This local high BR signalling in non-hair cells inhibits unidirectional cell expansion and whole root growth. In contrast, BRI1 expression in hair cells promotes unidirectional cell expansion, buffering the inhibitory effect of the receptor in non-hair cells. The discrepancy between intensified BR signalling in nonhair cells despite low BRI1 levels and how BRI1 signalling is buffered in neighbouring cells can be explained via cell wall homeostasis integrity pathways (Wolf et  al., 2012). Impaired cell wall composition triggers BR signalling via the receptorlike protein (RLP) 44 (Wolf et al., 2014), which may be triggered by lack of coordination between neighbouring expanding cells. Indeed, relatively high BRI1 levels in non-hair cells stimulates local deposition of crystalline cellulose, which, at least partially, limits unidirectional cell expansion (Fridman et al., 2014). This cell wall modification may reflect a compensatory mechanism that can account for impaired cell wall homeostasis. Enhanced BR signalling in non-hair cells also triggers the transcription of the ethylene biosynthesis genes ACS5 and ACS9, which enhance ethylene signalling necessary for the accumulation of crystalline cellulose and inhibition of unidirectional growth (Fig.  3C) (Fridman et  al., 2014). Taken together, differential growth rates between adjacent cells, whether imposed by stochastic differences in BR signalling strength or by other effectors, modulate hormonal signalling pathways, probably as a means of fine-tuning whole organ growth. In parallel, BR signalling modulates the specification of hair and non-hair cells (Kuppusamy et  al., 2009; Cheng et al., 2014). BR activity promotes non-hair cell identity by facilitating the formation of the known WER–GL3/EGL3– TTG1 transcriptional complex (Cheng et  al., 2014). This complex, which is subjected to sophisticated feedback regulatory events, induces the transcription of GL2, which inhibits hair cell fate (Schiefelbein et  al., 2014). Interestingly, some of these patterning genes are important in maintaining the differential sizes between hair and non-hair cells, and also impact whole-root growth (Lofke et al., 2013). Although very speculative, differential strengths of BR signalling may also regulate unidirectional cell expansion via these genes.

Perspective In this review, the context-specific effect of BR activity was shown to be an important determinant of plant growth and development. The mechanisms underlying specific BR effects on growth, including their dependence on age, time of day, environmental conditions, and the relative spatial distribution of the signalling components, were discussed. Open questions, shared by all presented examples, still remain: identification of the BR signalling components modulated by increasing concentrations of the hormone and how they are regulated, determinants of tissue-specific responses to the hormone, characterization of the dynamics of BR responses

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In the root meristem, the stem cells surround the quiescent centre (QC) which maintains their identity by an unknown signal, together forming the plant stem cell niche (Dolan et al., 1993; van den Berg et al., 1997). The QC cells have a slow proliferation rate as compared with their surrounding stem cells and serve as a source of cells that replenish the stem cells under stress conditions (Heidstra and Sabatini, 2014). Hence, under adverse conditions, such as DNA damage, the QC cells divide faster, enabling recovery of the stem cell niche. BRs enhance QC divisions via BES1, that down-regulates the transcription of the R2R3-MYB transcription factor BRASSINOSTEROIDS AT VASCULAR AND ORGANIZING CENTER (BRAVO) and physically interacts with the protein product, while accumulation of BRAVO further elevates its own transcription (Fig. 3B) (Vilarrasa-Blasi et al., 2014). These non-linear interprotein relationships triggered by changes in BR levels were interpreted, using mathematical modelling, to facilitate a robust switch between quiescence and cell proliferation. BRs also enhance QC divisions by elevating the transcription of ETHYLENE RESPONSE FACTOR 115 (ERF115) (Fig.  3B) (Heyman et  al., 2013). Elevation of ERF115 and ectopic expression of BRAVO promote and abolish stem cell niche recovery after DNA damage, respectively, highlighting the important role of BRs during stress response. Constant induction of QC cell proliferation exhausts the root meristem, causing root growth abnormalities, thus explaining, at least in part, the deleterious effect of high BR levels on the root meristem. The QC and the basal meristem zone have recently been shown to coordinate the size of the root meristem, via auxin (Moubayidin et  al., 2013). It will therefore be interesting to address whether the BES1–BRAVO module in the QC integrates with the spatial coordination imposed by BRs, between the epidermis and the stele tissues. While these growth processes are typically studied under normal conditions, the impact and integration of environmental signals remain largely unknown. In one example, low phosphate availability modulates root system architecture, and causes exhaustion of the meristem and inhibition of unidirectional cell expansion. It has recently been demonstrated that this developmental switch depends on reduced nuclear to cytoplasmic BES1/BZR1 ratios (Singh et al., 2014), but how this integrates with a tissue-dependent effect of BR signal on growth is unknown. Taken together, these studies have shown that the spatial distribution of BR components, rather than their absolute level, is an important determinant of controlled growth. This principle has recently been shown also to underlie the coordination between elongating cells (Fridman et  al., 2014), as discussed next.

1130  |  Singh and Savaldi-Goldstein

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Distinctive effects of BR activity on growth  |  1131

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