Translatome analyses capture of opposing tissue- specific brassinosteroid signals orchestrating root meristem differentiation Kristina Vragovica,1, Ayala Selaa,1, Lilach Friedlander-Shania, Yulia Fridmana, Yael Hachama, Neta Hollanda, Elizabeth Bartomb,c, Todd C. Mocklerd, and Sigal Savaldi-Goldsteina,2 aFaculty of Biology, Technion-Israel Institute of Technology, Haifa 3200003, Israel; bBioinformatics Knowledge Unit, Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering, Technion, Haifa 3200003, Israel; cCenter for Research Informatics, Biological Sciences Division, University of Chicago, Chicago, IL 60637; and dCrop Computational Biology Laboratory, Donald Danforth Plant Science Center, St. Louis, MO 63132 Edited by Mark Estelle, University of California at San Diego, La Jolla, CA, and approved December 4, 2014 (received for review September 19, 2014) The mechanisms ensuring balanced growth remain a critical question BRI1 phosphorylates its negative regulator Brassinsosteroid Ki- in developmental biology. In plants, this balance relies on spatiotem- nase Inhibitor 1 (BKI1) (4, 5), which enables BRI1 to form poral integration of hormonal signaling pathways, but the under- a complex with its coreceptor BRI1-Associated Kinase 1 (BAK1) standing of the precise contribution of each hormone is just beginning (6, 7). The activated receptor triggers transmission of the BR to take form. Brassinosteroid (BR) hormone is shown here to have signal to the nucleus, after various regulatory steps, including opposing effects on root meristem size, depending on its site of inhibition of GSK3-like kinase Brassinosteroid Insensitive 2 action. BR is demonstrated to both delay and promote onset of (BIN2), the key inhibitor of the signaling cascade. Consequently, stem cell daughter differentiation, when acting in the outer tissue Brassinazole Resistant 1 (BZR1) and its homologous transcrip- of the root meristem, the epidermis, and the innermost tissue, the tion factor BRI1-EMS-Suppressor1 (BES1)/BZR2, are activated stele, respectively. To understand the molecular basis of this phe- and regulate the expression of hundreds of genes (7, 8). nomenon, a comprehensive spatiotemporal translatome mapping of BRs have both promoting and inhibitory effects on root Arabidopsis roots was performed. Analyses of wild type and mutants growth, depending on the concentration of the hormone and the PLANT BIOLOGY featuring different distributions of BR revealed autonomous, tissue- intensity of the signaling pathway. BR-insensitive mutants (e.g., specific gene responses to BR, implying its contrasting tissue-dependent bri1) feature reduced meristem size and cell elongation (9, 10). impact on growth. BR-induced genes were primarily detected in epi- Conversely, BR-treated roots have reduced meristem size due to dermal cells of the basal meristem zone and were enriched by auxin- early differentiation (10). In addition, enhanced BR signaling related genes. In contrast, repressed BR genes prevailed in the stele of triggered by impaired spatial distribution of BRI1, limits cell the apical meristem zone. Furthermore, auxin was found to mediate elongation and whole root growth (11). BRs are also perceived the growth-promoting impact of BR signaling originating in the epi- by BRI1-Like 1 (BRL1) and BRI1-Like 3 (BRL3), two BRI1 dermis, whereas BR signaling in the stele buffered this effect. We homologs that are confined to the stem cell niche and the stele, propose that context-specific BR activity and responses are oppositely where they promote QC cell divisions (12–14). BRI1 acting in the interpreted at the organ level, ensuring coherent growth. epidermis promotes stem cell daughter divisions, stimulating root meristem size and whole root growth via an unknown signal (9). root meristem | brassinosteroids | auxin | intertissue communication | BRI1 However, its activity in the inner tissues, the endodermis/QC and stele, has no growth-promoting effect (9). Whether BR-mediated o ensure coherent organ growth, multicellular organisms Tmust develop mechanisms to integrate multiple cellular sig- Significance nals and to interpret them at the organ level. The Arabidopsis primary root provides a convenient system for deciphering how such spatiotemporal coordination is achieved (Fig. 1A). Newly Brassinosteroid (BR) differentially regulates the number of stem cell formed root cells originate from their initials (stem cells) at the daughters in the root meristem. How its activity coordinates and apical region of the root meristem. The stem cells surround the maintains the meristem size remains unknown. We show that BR quiescent center (QC), which maintains their identity, together signal coordinates root growth by evoking distinct and often op- forming the plant stem cell niche (1, 2). Stem cell daughters posing responses in specific tissues. Whereas epidermal BR signal contributing to the root length, repeatedly divide along the promotes stem cell daughter proliferation, the stele-derived BR apical–basal axis until they begin to differentiate, elongate, and signal induces their differentiation. Using a comprehensive tissue- maturate to exert specific functions. This longitudinal sequence specific translatome survey, we uncovered a context-specific effect of BR signaling on gene expression. Auxin genes, activated by is apparent along four consecutive zones of the growing root: epidermal BR perception, are necessary for induction of cell di- apical meristem, basal meristem or transition zone, elongation/ vision. Conversely, the stele BR perception, accompanied by gene differentiation zone, and maturation zone, which provide a si- repression, restrains the epidermal effect. Therefore, a site-specific multaneous view of the temporal events. The tissues comprising BR signal is essential for balanced organ growth. the root are organized in concentric layers around the stele and its constituent vasculature tissues, epidermis, cortex, and endo- Author contributions: K.V., A.S., and S.S.-G. designed research; K.V., A.S., L.F.-S., Y.F., Y.H., dermis, from outside to inside. The lateral root cap and colu- N.H., and T.C.M. performed research; K.V., A.S., L.F.-S., Y.F., E.B., and S.S.-G. analyzed data; mella surround the meristematic zones, thus protecting the stem K.V. generated the translatome data; A.S. performed genetic crosses and characterization cell niche from physical barriers. of the BR mutants; and K.V., A.S., E.B., and S.S.-G. wrote the paper. Phytohormones, including the brassinosteroid (BR) group of The authors declare no conflict of interest. hormones, play a pivotal role in the regulation of root growth This article is a PNAS Direct Submission. (3). BRs are perceived at the cell surface by Brassinosteroid 1K.V. and A.S. contributed equally to this work. Insensitive 1 (BRI1), a leucine-rich repeat (LRR)-receptor ki- 2To whom correspondence should be addressed. Email: [email protected]. nase, which is the central receptor controlling root growth, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with a broad expression pattern in the root meristem. Activated 1073/pnas.1417947112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1417947112 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 BR-repressed genes resided mainly in the stele of the apical meristem and were largely autonomously regulated by resident BRI1. By contrast, BR activity in the epidermis involved in- duction of genes in the basal meristem, among which auxin-related genes were overrepresented, uncovering auxin as a mediator of BRI1-promoted cell divisions. Taken together, this study shows that differential tissue-specific BR perception triggers opposing growth effects, together contributing to coordinated growth. Results Epidermal BRI1 Activity Delays Cell Differentiation, Whereas Stele- Localized Receptors Restrain This Effect. Roots expressing BRI1 in the epidermis only, in the bri1 background (e.g., in bri1; pGL2- BRI1), have a slightly enlarged meristem compared with wild type, a phenotype that could not be mimicked by exogenous application of the hormone (9). We therefore hypothesized that BR activity in the inner cells may counteract the epidermal effect on root meristem size. Thus, we assessed the impact of BRI1 activity in the epidermis on the size of the root meristem, when the BR activity is fully abrogated elsewhere, as in bri1, brl1, brl3; pGL2-BRI1. Remarkably, the enlarged meristem phenotype imposed by epidermal BRI1 activity in bri1, was enhanced in the triple mutant by about 30% compared with wild type (Fig. 1 B and C, Right). By contrast, the reduced meristem size observed in bri1 was not significantly different compared with bri1 brl1 brl3 (Fig. 1C, Left). Thus, BR signaling in the epidermis is required to promote the number of proliferating cells, whereas BR activity in the inner cells restrained this effect. Loss of function of both BRL1 and BRL3, in roots expressing pGL2-BRI1 or in wild-type roots, had no effect on meristem size compared with wild type (SI Appendix, Fig. S1 A and B). However, the impact of epi- dermal BRI1 activity on root meristem size was dramatically enhanced by the absence of active BRI1 elsewhere, as observed Fig. 1. Stem cell daughter differentiation is inversely controlled by BR ac- when combining its epidermis-targeted expression with loss of tivity in the epidermis and the stele. (A) Longitudinal and cross-sections of the Arabidopsis root. Each color depicts a tissue, also corresponding