Specific Brassinosteroid Signals Orchestrating Root Meristem Differentiation

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 induction 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 epidermal 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 to the function of either brl1 or brl3 (SI Appendix, Fig. S1C). expression pattern of the tagged ribosomal protein (see Fig. 2A). C, cortex; To characterize the cellular basis for the enlarged meristem, En, endodermis; Ep, epidermis; LR/C, lateral root cap and columella; and St, we performed a growth dynamic analysis of growing seedlings (Fig. stele. Yellow line marks the stem cell niche. Asterisks indicate QC cells. (B) 1D). In the bri1, brl1, brl3; pGL2-BRI1 mutant, the enlarged meri- Confocal microscopy image of the root meristem of the triple mutant bri1, stem was apparent early after germination and the meristem size brl1, brl3, Col-0 (wild type), and both single and triple mutants expressing increased until plateauing on day 5 at the growth rate observed pGL2-BRI1. The arrow marks the onset of cell elongation/differentiation. PI in wild type (Fig. 1D). Thus, the dynamics of stem cell daughter staining to show cell borders appears in white. (Scale bar, 50 μm.) (C) Cortical cell numbers of the wild type and mutants depicted in B.(D) Root-meristem proliferation and differentiation rate appeared normal in roots wild type and bri1, brl1, brl3; pGL2-BRI1 cell numbers, counted over time. DAG, expressing BRI1 in the epidermis only. In addition, the number of days after germination. Note the similar curve, reflecting balanced rate of cell cells in the S phase proportionally increased with meristem size (Fig. proliferation and cell differentiation. (E) Edu staining, applied to detect cells in 1E), as previously reported for the number of cells in the G2–M S phase, is shown as pink nuclei. Nuclei with DAPI staining appear purple. phase (9). Thus, the enlarged meristem size in roots expressing BRI1 Meristems correspond to the same genotypes shown in B.(F) Cortical cell in the epidermis only is an extended zone of actively dividing cells. number of wild type and bri1, brl1, brl3; pGL2-BRI1 in the absence and presence To determine which tissue counteracts the epidermal BRI1 of endodermal BRI1 (pSCR-BRI1). (G and H) Cortical cell number of wild type, bri1, brl1, brl3; pGL2-BRI1 in the absence and presence of stele BRI1 (pSHR-BRI1), effect, we combined the triple mutant expressing both epidermal and bri1, brl1, brl3 expressing pSHR-BRI1 (G), and in the absence and presence BRI1 and BRI1 targeted to either the endodermis or the stele, of BR (H). Mean ± SE; *P < 0.05 and **P < 0.01 with two-tailed t test. by crossing bri1, brl1, brl3; pGL2-BRI1 with either pSCR-BRI1 or pSHR-BRI1, respectively. Cellular analysis revealed that BRI1 activity in the stele buffered the effect of epidermal BRI1, inhibition of root meristem size is also tissue specific is unknown. resulting in wild-type–sized meristems (compare Fig. 1 F and G). Hence, experimental dissection of mechanisms underlying BR We then explored whether stele BRI1 expression leads to ability to both promote and restrict the size of the meristem is reduced meristem size in a BR-dependent manner and whether fundamental for understanding growth regulation. limiting BR receptor activity to the epidermis triggers further Here, we performed a comprehensive tissue-specific trans- growth. Interestingly, whereas wild-type roots responded to low latome survey, based on immunopurification of polysome-asso- BR levels, as manifested by reduced meristem size, bri1, brl1, ciated mRNA, following high BR treatment, in efforts to map brl3; pGL2-BRI1 roots were hormone insensitive and maintained BR effects on wild-type Arabidopsis root gene expression. In their enlarged meristem. However, when this mutant line also parallel, similar analyses were performed in bri1 with tissue- expressed BRI1 in the stele, wild-type–like sensitivity to the specific overexpression of BRI1, as a means of distinguishing hormone was observed (Fig. 1H). In summary, these results are between modulation of genes in the inner tissues by local BRI1 consistent with the observed impact of epidermal BR activity on activity (autonomous), versus modulation via BRI1 activity in the the duration of the stem cell daughter division phase and of stele epidermis (nonautonomous). These approaches revealed that BR activity promoting their differentiation.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1417947112 Vragovicetal. Downloaded by guest on September 25, 2021 Fig. 2. Tissue-specific sequence analysis of polyribosome-associated mRNA, in wild-type and mutant backgrounds, designed to capture context-specific BR responses. (A) Summary of all polysome-associated mRNA samples, subjected to RNA-seq analysis. (Upper) Outlines of samples collected from roots with wild-type distribution of BRI1. (Lower)Outlinesofsamplescol- lected from bri1, harboring targeted expression of the receptor. Colors correspond to the root tissues marked in Fig. 1. (B) Genes in the inner cells, e.g., the endodermis, respond to BR via epidermal BRI1 activity (nonautonomous) or via local BRI1 perception (autonomous). En, endodermis; Ep, epidermis; LR/C, lateral rootcapandcolumella;St,stele;andWR,wholeroot. (C) Principal-component analysis of all samples used in this study. Samples are categorized by the tissue to which the tagged ribosomal protein was targeted. (D) Normalized counts of endogenous genes corresponding to the promoter fragments used to drive the expression of the tagged ribosomal protein in roots. PLANT BIOLOGY

Precise Tissue-Specific Translatome Profiling in Roots Is Suitable for precise construction of a tissue-specific model, suitable for fur- Studying Context-Specific BR Effects. Our data thus far showed that ther analysis of BR impact on root growth. BR signaling has a contrasting impact on the size of the root meristem, depending on the location of its action. We therefore Opposing Response to BRs Between the Outer and the Inner Tissues. reasoned that gene responses to BR signaling may be tissue In efforts to characterize the spatiotemporal translatome map of specific. To test this hypothesis, we delineated a spatiotemporal Arabidopsis wild-type roots in response to BR, we conducted translatome map of wild-type and BR mutant Arabidopsis roots, a pairwise analysis and considered significantly modulated genes by establishing transgenic lines expressing a tagged ribosomal (SI Appendix, SI Material and Methods and Dataset S1). Re- protein in specific cells (15, 16). markably, about 10-fold more genes were modulated in the outer The tagged protein was expressed under select tissue-specific tissues, LR/C, and epidermis, compared with the inner, endo- promoters that were previously used to analyze the effects of dermis, and stele tissues, in the different genetic backgrounds targeted BRI1 expression (9). The promoters were specific for tested at both time points (Fig. 3A). the epidermis, endodermis (and QC), stele, and the lateral root Next, a series of hierarchal clustering analyses was performed cap and differentiated cells of the columella (hereafter called on genes significantly up-regulated or down-regulated in re- LR/C) (17) (Figs. 1A and 2A, Upper). A line expressing ribo- sponse to BR treatment (Fig. 3A and Dataset S1) by comparing somal protein under the 35S promoter served as a control for the their relative expression over all samples in the dataset (i.e., 24 whole-root response (15). In addition, to distinguish between samples; Dataset S2 and SI Appendix, SI Material and Methods). genes potentially autonomously or nonautonomously responsive Patterns obtained for whole-root up-regulated and down-regu- to BR in the inner cells (Fig. 2B), we crossed selected lines lated genes, resembled those observed in the epidermal tissue. In expressing tagged ribosomal protein in these tissues, to bri1, addition, a positive correlation was observed between induced expressing targeted BRI1, as outlined in Fig. 2A, Lower (9, 11). genes and their enriched localization in the epidermis, at both Samples of polyribosome-associated mRNA were collected and time points (Fig. 3 B and C). Conversely, genes repressed in the subjected to RNA-sequencing (RNA-seq) analysis after up to 8 h whole root or epidermis (note Z < 0) tended to show increased of BR treatment (Fig. 2A and SI Appendix, Fig. S2). basal expression in the endodermis and the stele (Fig. 3 D and E). Principal-component analysis showed tissue-specific clustering In efforts to characterize the distribution of BR-regulated of transcripts (Fig. 2C). Samples corresponding to whole root gene expression along the root zones, we used predefined clustered at the center, at similar distances from the tissue-type dominant expression patterns in 12 consecutive regions along the clusters (Fig. 2C). The tissue specificity of the isolated transcripts longitudinal axis of the root (Fig. 4A) (18). Each predefined was further confirmed by endogenous tissue-specific genes that longitudinal pattern (LP) was filtered to highlight BR-responsive were highly enriched in their corresponding tissues (Fig. 2D and SI genes in at least one of our samples. These genes were plotted Appendix,Fig.S3). Transcripts showed no clustering based on their against our entire dataset (Fig. 4, Dataset S3, and SI Appendix, SI response to the BR treatment (SI Appendix,Fig.S4). The fluo- Material and Methods). As an example, 263 BR-regulated genes rescently tagged BKI1 was used to verify that sufficiently high BR were highlighted for the apical meristem (as in LP 25) and 97 levels accumulated in both the outer and the inner tissues (4, 5) (SI BR-regulated genes were described for the basal meristem (as in Appendix,Fig.S5A). Response to the hormone was also confirmed LP 29). When these gene sets were compared with the trans- by the behavior of known BR targets (SI Appendix,Fig.S5B). latome, significant enrichment of apical meristem-expressed Thus, tissue identity accounts for the majority of the variation genes was noted in the samples originating from the stele, 95% between samples. In addition, our analysis shows a reliable and of which were reduced by the hormone (Fig. 4B, yellow area). In

Vragovicetal. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 21). Hence, many genes repressed by BR in the endodermis behave in a cell-autonomous fashion. To determine whether the activity of BR in the stele induces similar autonomous gene repression, we established a bri1, brl1, brl3; pGL2-BRI1 line expressing the ribosomal protein in the stele. Real-time analysis of the immunopurified polyribosome- associated mRNA revealed BR-mediated repression of PLT1, PLT2, and CPD in the wild-type tissue (Fig. 5C and SI Appendix, Fig. S6D). Furthermore, the tested genes showed high constitu- tive basal expression in bri1, brl1, brl3; pGL2-BRI1 stele, that was not significantly repressed by the hormone (Fig. 5C and SI Ap- pendix, Fig. S6D). Hence, a pattern of misregulation of autonomously controlled BR-repressed genes is shared between the endodermis and the stele. Taken together, the root translatome reveals distinct features between the outer and the inner tissues and demonstrates autonomous regulation of BR-responsive genes in the inner cells, indicative of a unique function.

BRI1 Activity in the Epidermis Delays Timing of Cell Differentiation via Auxin. To further characterize the tissue-specific impact of BR signaling, we subjected the significantly modulated genes to a functional annotation analysis (http://david.abcc.ncifcrf.gov/, SI Appendix,TableS1). Only a few significantly enriched categories were defined in the inner tissues, whereas the outer tissues, LR/C and epidermis, contained more categories. Up-regulated genes responding to hormones prevailed in the epidermis, among them ethylene- and auxin-related genes and kinases enhancing the efflux activity of PIN2 in the epidermis (22–26) (Fig. 6A). Next, we analyzed the functional categories enriched in steady-state bri1 roots overexpressing BRI1 in the epidermis only (i.e., in bri1; pGL2- BRI1) or in endodermis only (i.e., in bri1; pSCR-BRI1), compared Fig. 3. Gene response to BR in the outer tissues is robust, where induced with their respective tissues in wild type (SI Appendix,Fig.S7). BRI1 genes prevail, whereas BR-repressed genes are enriched in the stele. (A) The overexpression in the epidermis led to further enrichment of auxin- number of significantly differentially expressed genes in response to BR ≥ ≤ related genes and a more robust response to BR (Figs. 3D and 6A treatment ( 1.5 fold change, 0.1 false discovery rate) in both wild-type and and SI Appendix,Fig.S7), whereas the unique steady-state response mutant backgrounds. Hierarchical clustering of genes induced (B and C) and repressed (D and E) in whole root (B and D), in wild-type epidermis (C and E) to high endodermal BRI1 largely involved genes of the translational (and in epidermis of bri1, expressing BRI1 in the epidermis only; Dataset S2), machinery (SI Appendix, Fig. S7). Cell wall- and hormone-related upon 3 h of treatment with the hormone. Each gene identified as differ- gene expression was enriched in both the epidermis and the entially expressed in each of these categories has its expression levels across the entire dataset clustered and plotted. The color scale is adjusted for each gene independently, depending on its distribution of expression levels.

contrast, the basal meristem profile overlapped with epidermal- enriched translatome, with 82% of the genes induced by the hormone (Fig. 4C). Taken together, genes activated and repressed by BRs were primarily observed in the basal domain of the outer and the apical domain of the inner tissues, respectively.

The Translatome Reveals Autonomous BR-Driven Regulation in the Inner Cells. To explore the extent to which BR-responsive inner cell genes are controlled by epidermal versus local BRI1 (Fig. 2B and SI Appendix, Fig. S6), all genes significantly repressed in wild-type endodermis upon BR treatment (3 and 8 h, Dataset S1) were subjected to a pattern similarity analysis across all endodermal datasets. The most prominent expression pattern included 40% of the genes and showed a pattern as depicted in Fig. 5A, reaching repression in the endodermis within 3 h after treatment in wild-type tissue (Fig. 5A, Left). When BRI1 expression was limited to the endodermis, the corresponding gene set was constitutively repressed (Fig. 5A, Middle). Conversely, in a line expressing BRI1 in epidermis, the expression of the gene Fig. 4. BR-repressed genes are enriched in the apical meristem zone, set was constitutively up-regulated (Fig. 5A, Right), suggesting whereas BR-induced genes are enriched in the consecutive basal meristem loss of responsiveness in the absence of local BRI1. Genes in this zone. (A) Confocal image of a wild-type root depicting its different longitudinal zones. Numbers indicate the corresponding zones taken to generate gene set included known transcription factors regulating root publically available longitudinal transcriptional datasets (18). Hierarchical development and root zonation [e.g., Plethora (PLT2, PLT3, and clustering of BR-regulated genes, identified in representative dominant ex- BBM), HD-ZIP II (HAT1 and HAT4), and Wuschel-Related pression patterns along the longitudinal axis of the root called longitudinal Homeobox (WOX12); Fig. 5B and SI Appendix, Fig. S6C] (19– patterns 25 (B) and 29 (C) (18), plotted against the entire dataset, as in Fig. 3.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1417947112 Vragovicetal. Downloaded by guest on September 25, 2021 even when active in a subset of cells (29). However, how these pathways integrate to maintain coherent growth is largely unknown. Our study reveals opposing responses to the same hormone ligand in time and space as a mechanism for determination of meristem size (Fig. 6F). The outer tissues exhibit a robust genomic response to BR, largely characterized by gene activation, particularly related to auxin signaling. We thus propose that BR-triggered auxin biosynthesis and transport in the outer tissue results in translocation of auxin required to drive stem cell daughter divisions and delay the onset of cell differentiation. Hence, auxin acts as an epidermis-derived nonautonomous signaling component, promoting cell proliferation in the meristem. The role of auxin, acting as a nonautonomous signal coordinating the rate of stem cell division with the rate of stem cell daughter differentiation, has been recently described (30). The response to BR in the inner tissues was surprisingly mild and accompanied by autonomous repression of gene expression. The absence of obvious nonautonomous patterns of gene regulation up to 8 h after BR treatment suggests that these gene targets might be affected at later time points (e.g., AGL42, SI Ap- pendix,Fig.S9) (9). Alternatively, some targets could be controlled posttranslationally and undetected by our translatome system. BR perception in the stele triggered early cell differentiation, Fig. 5. Delineating the autonomous mode of gene regulation in the inner restraining the epidermal impact of the hormone. A combination cells. (A) Representative expression pattern describing 40% of genes significantly repressed by BR, indicating autonomous regulation. This analysis of epidermal BRI1 activity with combined loss of function of the compares the expression of genes in the endodermis of wild type, bri1 expressing BRI1 in the endodermis only, and bri1 expressing BRI1 in the

epidermis only (PLT3-like pattern is shown). (B) Gene list heatmap of a rela- PLANT BIOLOGY tive expression pattern showing an autonomous expression pattern similar to that of PLT3 (Pearson correlation of 0.9). (C) Autonomous response observed in the stele. Real-time analysis of selected genes performed on polyribosome-associated mRNA isolated from the stele of wild type and of a line expressing BRI1 in the epidermis of the triple mutant bri1, brl1, brl3.

endodermis in response to high local BRI1 levels (SI Appendix,Fig. S7). Hence, both short-lived and sustained high BR activity in the epidermis triggers activation of hundreds of genes, many of which relate to hormone signaling in general, and to auxin in particular. To address the possibility that BRI1 activity in the epidermis promotes cell proliferation via auxin, we crossed bri1; pGL2-BRI1 and bri1, brl1, brl3; pGL2-BRI1 with the loss-of-function mutant taa1. TAA1 is an essential auxin biosynthetic gene, modulated by BR in the epidermis only (Fig. 6 A and B)(25,26).Remarkably, loss of TAA1 suppressed the enlarged meristem size of the progeny of both genetic crosses, yielding a wild-type meristem phenotype (Fig. 6B). In agreement, it also suppressed the extended zone of cells present in S phase (SI Appendix,Fig.S8A). To characterize the distribution of auxin activity, bri1; pGL2-BRI1 was then crossed with a line expressing the auxin sensor DII-Venus, the expression of which negatively correlates with auxin levels (27). Homozygous progeny of this cross showed less DII-Venus activity than in the parental line, manifested by an extendedregionintheapicalmer- istem with high auxin levels. These observations suggest the pres- Fig. 6. Epidermal BRI1 activity delays onset of cell differentiation via auxin. ence of an extended auxin gradient, in agreement with the increased (A) Expression level of the auxin biosynthesis genes (gray bars) and auxin-related meristem size (Fig. 6 C and D and SI Appendix,Fig.S8B). PIN kinase genes (green bars), as captured in the translatome analysis. (B) Cortical The extended auxin gradient zone could also be a result of en- cell numbers in wild type, taa1, bri1,andbri1, brl1, brl3 expressing BRI1 in the epidermis only, and in the background of wild-type TAA1 and taa1 mutant. (C) hanced auxin transport (Fig. 6A) (28). To reveal whether PIN2- Confocal images of the auxin sensor DII-Venus in wild type and in the bri1; pGL2- mediated auxin transport is important for driving the epidermal BRI1 mutant (apical meristem is shown, see also SI Appendix,Fig.S8B). The green effect, bri1; pGL2-BRI1 was crossed with the loss-of-function mu- color in the epidermal membrane of bri1; pGL2-BRI1 corresponds to BRI1-GFP. tant pin2, which yielded progeny with a normal meristem size (Fig. (D) Quantification of the length of the zone with no DII signal, from the QC 6E and SI Appendix,Fig.S8A). Thus, high auxin concentrations, (contained within the yellow oval region in C). (E) Cortical cell numbers in wild likely synthesized in the epidermis, delay stem cell daughter dif- type, pin2, bri1; pGL2-BRI1, and the double mutant pin2; bri1; pGL2-BRI1.(F) ferentiation, whereas BR activity in the stele counteracts this effect. Model for BR regulation of root meristem size. BRI1 activity in the epidermis promotes cell proliferation by elevating auxin levels, whereas the activity of BRI1, Discussion BRL1, and BRL3 in the stele buffers this effect, promoting differentiation. The differential spatiotemporal pattern of BR-responsive genes is evidence of the In the past years, accumulating research in plants revealed an un- context-specific effect of the hormone. Mean ± SE; *P < 0.05, **P < 0.01, and expected ability of hormonal signaling pathways to control growth ***P < 0.001 with two-tailed t test. (Scale bar, 50 μM.)

Vragovicetal. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 three BR receptors (12), revealed unequal redundancy between (9), and under the pBRN2 promoter, were established. Seeds expressing the these receptors. Stele-localized BRI1 had the most significant ribosomal protein under p35S (35S:HF-RPL18) (15), DII-Venus (27), and impact on restraining the extent of stem cell daughter pro- BKI1-mCherry (5) were as previously described. Seeds of the T-DNA insertion liferation. The emerging gene list autonomously regulated by BR mutants pin2 (eir1-4, SALK_091142), taa1 (wei8-4, SALK_022743), brl1 includes BR biosynthesis genes and those encoding proteins (SALK_005982), and brl3 (SALK_006024) were obtained from Nottingham previously reported to impact root meristem zonation and the Arabidopsis Stock Centre. Primers used to confirm T-DNA insertion sites stem cell niche (e.g., PLTs). BR stimulates cell divisions of the and to identify homozygous lines are listed in SI Appendix, Table S2.The homozygous bri1-116 background was verified using cleaved amplified QC via repression of one of these genes, Bravo (31). Whether polymorphic sequences (CAPS) marker digested with PmeI. Growth conditions this activity integrates with the intertissue coordination of root and growth media were as previously described (11). For the translatome meristem size is currently unknown. We speculate that many genes treatments, 100 nM brassinolide (BL) was added, and for physiological are involved in transducing the contrasting impact of BR activity, experiments, as indicated in the text. forming a complex regulatory network. Some, like PLTs and BR biosynthesis genes (32, 33), are positively regulated by auxin and, as RNA Extraction and Real-Time Analysis. Total RNA extraction and quantitative such, could reflect a potential convergence point for the context- real-time PCR assays were performed as previously described (9), with the specific opposing effect of BR. In this scenario, the increased auxin exception of the At5g62690 gene, which served as an endogenous control. concentrations will trigger stem cell daughter proliferation but also Immunopurification of FLAG-tagged polysomes from root cells was increase their differentiation, stimulating internal tissue BR performed as described (16), with few modifications. biosynthesis, culminating in controlled growth. The strong contrast in the magnitude and the nature of the outer Confocal Microscopy. Fluorescence signals were detected using an LSM 510 and the inner cell hormonal responses could be explained by the META confocal laser-scanning microscope (Zeiss) equipped with a 25× water existence of intrinsic variability in the expression of signaling pro- immersion objective lens (N.A. 0.8). Roots were imaged in water supple- teins and their dynamics, as reported for animal signaling systems mented with propidium iodide (PI, 10 μg/mL). PI and green fluorescent (34). Here, the same receptor expressed in adjacent cells, stimulates protein (GFP) were viewed at excitation wavelengths of 488 nm and 561 nm, different developmental programs. This may be achieved by a respectively. Fluorescence emission was collected at 575 nm for PI and be- combination of altered coupling of BR signal transduction path- tween 500 and 530 nm band pass for GFP. Venus was viewed at an excitation wavelength of 488 nm and emission was collected at 518 nm. DAPI and ways in each cell type and a complex interplay of superimposed Alexa Fluor 555 were viewed at a excitation wavelength of 750 and emission hormonal gradients. The combined genetics and translatome-based was collected at 738 nm and 710 nm, respectively. mapping of BR responses provides solid evidence for the context- specific impact of BR and shows that its differential activity across ACKNOWLEDGMENTS. We thank Y. Eshed, R. Fluhr, and B. Podbilewicz for tissues is essential to maintain balanced growth. helpful comments on the manuscript; L. Elkouby for contributing at the early stages of this project; R. Blatt, N. Henig (Lorry I. Lokey Interdisciplinary Materials and Methods Center for Life Sciences and Engineering Infrastructure Unit), and M. Levin (I. Yanai laboratory) for their technical assistance; J. Bailey-Serres (University Plant Material, Growth Conditions, and Chemical Treatments. All Arabidopsis of California at Riverside) for her support and for sharing published materials; thaliana lines used in the study were in the Columbia (Col-0) background. To and the Russell Barrie Nanotechnology Institute at Technion. This research was generate the translatome data, transgenic lines expressing the ribosomal supported by grants from FP7-PEOPLE-International Reintegration Grant-2008, protein RPL18 fused through its N terminus to His6-FLAG dual epitope tag United States - Israel Binational Agricultural Research and Development Fund (IS- (16) under the same promoters and vectors previously used to express BRI1 4246–09), and Israel Science Foundation (1990/14).

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