An Auxin-Mediated Shift toward Growth Isotropy Promotes Organ Formation at the Shoot Meristem in Arabidopsis

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1 Current Biology, Volume 24 Supplemental Information An Auxin-Mediated Shift toward Growth Isotropy Promotes Organ Formation at the Shoot Meristem in Arabidopsis Massimiliano Sassi, Olivier Ali, Frédéric Boudon, Gladys Cloarec, Ursula Abad, Coralie Cellier, Xu Chen, Benjamin Gilles, Pascale Milan, Jiri Friml, Teva Vernoux, Christophe Godin, Olivier Hamant, and Jan Traas

2 Supplemental Information Supplemental Figures and Legends

3 Figure S1. Development of ring-like outgrowths following auxin treatments on NPA pins. Related to Figure 1 in the main text (A) Time-course imaging of ring-like primordia development upon treatments with IAA in 35S::GFP-LTI6b. The images show 3D reconstructions of the outer surface of NPA pins imaged from the side at the indicated times. Coloured dots mark the same cells over time. (B) Time-course of MONOPTEROS/AUXIN RESPONSE FACTOR 5 (MP/ARF5) expression during the development of ring-like primordia upon treatments with IAA in pmp::3xgfp plants. The images show 3D reconstructions of the inner surface of NPA pins imaged from the side at the indicated times. Inset at t=96h shows the same meristem imaged from the top. Notice ARF5/MP expression throughout the cells of the ring, marking organ primordium identity. (C) Time-course imaging of untreated 35S::GFP-MBD NPA pins. CMT alignment and pin shape are stably maintained over 96h. (D) Time-course imaging of IAA-treated 35S::GFP-MBD NPA pins. Before auxin treatment (t=0) CMT are circumferentially aligned around the periphery of the SAM. Twenty-four h after the initial auxin application (t=24h), CMT become disorganized in each cells and the circumferential supracellular alignment is lost (see also Fig.1A-E). At this stage no outgrowth is visible at the SAM periphery. Seventy-two h after the initial auxin application (t=72h), a circular outgrowth starts bulging out from the SAM periphery. At this stage CMT of the cells belonging to the outgrowth are still disorganized, whereas circumferential CMT alignment begins to be restored around the summit of the SAM. This leads to the formation of a crease between the ring-like outgrowth and the SAM center at t=96h. The upper images display CMT alignment over the meristem surface. The lower images display the XZ orthogonal projection passing through the center of the meristem.

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5 Figure S2. Genetic manipulation of CMT organization alters auxin responsiveness and organ formation at the SAM. Related to Figure 2 in the main text (A) CMT disorganization precedes outgrowth formation in NPA-grown bot1-7 mutants. Upper panel: time-course analysis of spontaneous outgrowth formation (indicated by the arrowhead) in the SAM of a bot1-7 35S::GFP-MBD plant constantly grown on NPA. Images were taken at the indicated times. Lower panel: higher magnification details of the regions highlighted by white boxes at t=0, t=24h and t=48h in the upper panel, displaying that CMT disorganization on the cell surface precedes the bulging of the outgrowth. (B) Full images of the representative meristems of WT, bot1-7 and rop6-1 before (t=0) and 24h after the beginning of the auxin treatment, displayed in Figures 2F, 2H and 2J of the main text. (C) Expression of ABP1, KTN1, ROP6 and RIC1 in inflorescences of Col-0 wild-type plants. The graph shows the expression levels of the indicated genes relative to the expression of the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) obtained by qpcr.

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7 Figure S3. Mutations in ABP1 alter CMT organization, auxin responsiveness and organ formation at the SAM. Related to Figure 3 in the main text (A) Full images of the representative meristems of WT, abp1-5 and abp1-1s/abp1-5 mutants, before (t=0) and 24h after the beginning of the auxin treatment, displayed in Figures 3C, 3E and 3G of the main text. (B and C) abp1-5 mutation delays auxin-induced organ initiation. (F) WT SAM showing major outgrowth formation 96h after the beginning of the auxin treatment. Left panel, surface reconstruction of the SAM viewed from the top; right panel, orthogonal projection of the same meristem drawn along the white line in the left panel. (G) abp1-5 SAM showing reduced outgrowth formation 98h after the beginning of the auxin treatment. Left panel, surface reconstruction of the SAM viewed from the top; right panel, orthogonal projection of the same meristem drawn along the white line in the left panel. The red asterisks mark the center of the meristem; the arrowheads mark the auxin-induced outgrowths at the flank of the meristem. (D) Spontaneous outgrowth formation in absence of auxin transport in abp1-1/abp1-5 meristems. Time-course analysis of spontaneous outgrowth formation (indicated by red arrowheads) in the SAM of a abp1-1s/abp1-5 35S::GFP-MBD plant constantly grown on NPA. Images were taken at the indicated times. (E) Surface reconstructions of confocal images of NPA-grown abp1-5, abp1-5 bot1-7 and bot 1-7 shoot apices stained with FM4-64. Seeds of the segregating F2 population of the abp1-5xbot1-7 cross were germinated on NPA-containing medium and grown until bolting. When the SAM became visible plants were stained with FM4-64, imaged and further processed for DNA extraction and subsequent genotyping. Notice that the abp1-5 bot1-7 double mutant displays organ outgrowths on NPA as the parental bot1-7 suggesting that KTN1 acts downstream of ABP1. Asterisks mark the center of the SAMs; arrowheads mark the spontaneous outgrowths at the flank of the meristems.

8 Figure S4. Variable changes in outer cell wall rigidity measured by AFM. Related to Figure 4 in the main text The graph shows the values of Young s Modulus obtained by AFM analysis of 8 independent meristems probed 72h after the beginning of the auxin treatments (see Fig. 4G-H). Notice the variability of the response. The highest reduction in cell wall rigidity was recorded for SAM 1 and 3, where the rigidity of the peripheral cells was reduced of 26% and 30%, respectively, compared to the apex. Error bars represent standard deviation.

9 Supplemental Experimental Procedures Plant material and growth condition Arabidopsis thaliana Col-0 and Ws-2 were the wild types in this study. Growth conditions were previously described [S1]. All the transgenic lines and mutant used in this studies were previously described: 35S::GFP-MBD [S1]; 35S::GFP-LTI6b [S2]; bot1-7 35S::GFP-MBD [S3]; pmp::3x-gfp [S4]; abp1-5 [S5], rop6-1 and ric1-1 [S6]; DR5::VENUS-N7 [S7]; pin1-1 (introgressed in Col-0) [S8], pin1-6 [S9], abp1-1s ( [S10]. The DR5::VENUS-N7 35S::GFP-MBD, rop6-1 35S::GFP-MBD, abp1-1s/abp1-5, abp1-5 35S::GFP-MBD, bot1-7 pin1-6 and abp1-5 bot1-7 lines were generated by crossing and further selected by genotyping or antibiotic selection. Chemical treatments NPA treatments were carried out as previously described [S11]. Plants were further kept on NPA-containing medium for all the duration of the experiments. To induce organ formation plants were treated in petri dishes with 1mM IAA liquid solution for 3h after imaging the t=0 time point and again the next day after imaging the t=24h time point. No other IAA was added after the 24h time point until the completion of the experiment. Lanolin paste for local SAM treatments was prepared as follows: 1 volume of chemical stock solution (3x concentrated) was added to 2 volumes of melted (55 C) lanolin and thoroughly mixed until the formation of a homogenous emulsion. Stocks concentration were 3mM for IAA; 666 µg/ml for oryzalin. Local applications were made with a pipette tip under a binocular. After lanolin application, plants were kept in humid conditions in a transparent plastic box for 4 days before further analyses. Equal amounts of DMSO were used for untreated controls Microscopy Confocal microscopy was carried out with a Zeiss LSM700 upright microscope equipped with 40x water immersion lens. Live imaging of NPA pins was carried out directly in petri dishes, after submerging plantlets with water. SAMs of plant grown on soil were imaged as previously described [S12] or in whole mount preparations. Briefly, for whole mount preparations plants were grown on soil in flat vessels until bolting. Plants were imaged after the removal of older flower buds to expose the SAM surface with a droplet of water between the objective and the SAM surface. Plants were kept in humid transparent boxes for all the duration of the experiments to prevent SAM desiccation.

10 Scanning electron microscopy was carried out with a Hirox SH-3000 table-top SEM on fresh plant material at -20 C, with an accelerating voltage of 5kV. Images of SAMs treated with lanolin paste were taken with a Leica MZ12 stereomicroscope equipped with a Leica DFC320 camera. Image analyses Image processing for GFP-LTI6b, pmp::3x-gfp and FM4-64 stained SAMs was carried out with the ZEN software (Zeiss) using the 3D transparent projection. For MT visualization, confocal stacks were further processed with MerryProj [S13] to obtain the projection of MTs on the L1 layer. Measurements of MT organization were carried out with the MT plug-in for ImageJ [S3]. Briefly, for each meristem cell contours were inferred from the original confocal stack (loaded onto the FIJI release of ImageJ; and further saved in a single Region Of Interest (ROI) file. The ROI file was then overlaid on the corresponding MerryProj-derived image and used as template to measure MT organization with the MT plug-in as previously described [S3]. Supracellular organization of MT in the SAM was assessed with FIJI by calculating the angle between the radius of the SAM and the average MT orientation from each cell as obtained by MT plug-in. At least three meristems were analysed for each condition. Graphs and statistics were obtained with Microsoft Excel software. Gene expression analysis Total RNA was extracted from inflorescences of Arabidopsis Col-0 ecotype using the Spectrum Plant Total RNA kit (Sigma-Aldrich). Total RNA was processed with processed with DNAse (kit Turbo DNA free, Ambion), and further reverse transcribed with M-MulV reverse transcriptase (Fermentas). Quantitative PCR was performed with the FastStart Universal SYBR Green Master (Roche) using a StepOne Plus (Applied Biosystem) according to manufactures instructions. Each reaction was performed in triplicates. Data were normalized according to Pfaffl [S14]. Details on primer sequences are given below. Atomic Force Microscopy Sample preparation for AFM analysis was as follow: in vitro grown plantlets (untreated or treated with auxin as described above) were individually transferred onto 35x10mm round tissue culture dish (BD Falcon) containing solid (2% agar) culture medium and subsequently fixed by using 1.5% of lukewarm low-melting agarose (Sigma-Aldrich), to prevent vibrations

11 during AFM scanning. Before AFM analysis each meristem was imaged by confocal microscopy to correctly determine position of the sample. Atomic force microscopy was carried out with a JPK NanoWizard3 system loaded with a SCANASYST-AIR tip (Bruker; tip radius: 2 nm; K: 0.4 N/m), by using a constant force of 70nN. This resulted in an average indentation of nm on the SAM surface for each contact point. Data analysis was carried out with the JPK Data Processing Software (v. 4.2). The Young s Modulus was estimated using the contact model for a four-sided pyramidal indenter [S15]. Numerical Simulations Mechanical simulations were designed with a previously developed modelling framework based on the SOFA software platform [S16] for FEM simulation and specific data structure for cellular representation. This framework aimed at computing mechanically-induced growth of plant tissues and is described in details elsewhere (Boudon et al., submitted manuscript). A synthetic, schematic, pin-like meristem was designed by concatenating a cylinder and a hemisphere. Each wall of the epidermis of the structure is transformed into triangular finite elements. Mechanically these finite elements displayed a linear anisotropic elastic behavior characterized by two Young s Moduli defined in two perpendicular directions. The synthetic meristem was divided in four different zones, featuring specific values of the Young s moduli: the central zone, the periphery, the primordium and the frontier zone, on the top border of primordium (see Figure 4A in the main text). The elastic properties of the periphery were set anisotropic: a Young s modulus of 1000 MPa circumferentially and 400 MPa vertically; the central zone displayed an isotropic Young s modulus of 700 MPa. The frontier zone featured the same values of Young s moduli that the periphery but with a different orientation: the stronger direction defined tangential to the contour of the primordium region and the softer one toward the primordium. Concerning the primordium, we compared 4 scenarios. In the first case the primordium cells have the same material properties than the ones of the periphery to model a pin shape. In another case, the overall rigidity of the primordium is decreased by a 70% (cells within the primordium are assigned Young s moduli of 300 MPa circumferentially and 120 MPa vertically). In the last two cases, the overall rigidity of the primordium is decreased by 50% but in two different ways. In one way the original anisotropy of rigidity is preserved (the circumferential Young s modulus is set to 500 MPa and the vertical one to 200 MPa). In the other way, the cells within the primordium were assigned the same average value (350 MPa) of Young s modulus in both directions. A turgor pressure of 0.4 MPa was set inside all cells and gave rise to stresses within the cell walls.

12 Bottom of the structure is attached to a plane. Given the turgor pressure and an initial shape, the simulation framework, first, computes the deformation of the structure satisfying mechanical equilibrium and then, if strain reaches a given threshold, initiates the growth phase. For that specific study, the strain threshold (0.03) was chosen so that it inhibits growth in the circumferential direction but allows it in the vertical direction, in the periphery. Mechanical equilibrium and growth equations will be given elsewhere (Boudon et al., submitted manuscript). List of primers used in this study Primer Sequence use KTN F 5 -AGGGAGCTGCCTCAAAATCT-3 qpcr KTN R 5 -CATAGCAGCCAGGTCCTCAT-3 qpcr ABP1 F 5 -TCTTGGCCAACAGTACAATTCA-3 qpcr ABP1R 5 -CGGCCGAGATATGATAACCA-3 qpcr ROP6 F 5 -ACGGTGCTGTTGGAAAGACT-3 qpcr ROP6 R 5 -TCCCACAATCCCAAGTTGAT-3 qpcr RIC1 F 5 -AGGATTTCCGACCGATGTAA-3 qpcr RIC1 R 5 -CTTGCGGGTTGTATTTGTTG-3 qpcr bot1-7 F 5 -TCTCCGAGATCGAAGCTCTTGC-3 Genotyping bot1-7 R 5 -TGCCAAAAGGGGTGACAAAA-3 Genotyping abp1-5 F 5 -TGACCTTCCTCAGGATAACTATGG-3 Genotyping* abp1-5 R 5 -CCAACACCTGCAGGTCCTCATGAC-3 Genotyping* abp1-1s F 5 -ATTTGCAATAGCGCATTGAAC-3 Genotyping abp1-1s R 5 -GATGGGACAAGGAGCTCCTAG-3 Genotyping *the genotyping of abp1-5 mutation is obtained by further restriction of PCR fragments with RsaI as described in [S5] Supplemental References S1. Hamant, O., Heisler, M. G., Jönsson, H., Krupinski, P., Uyttewaal, M., Bokov, P., Corson, F., Sahlin, P., Boudaoud, A., Meyerowitz, E. M., et al. (2008). Developmental patterning by mechanical signals in Arabidopsis. Science 322, S2. Cutler, S. R., Ehrhardt, D. W., Griffitts, J. S., and Somerville, C. R. (2000). Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc. Natl. Acad. Sci. U. S. A. 97, S3. Uyttewaal, M., Burian, A., Alim, K., Landrein, B., Borowska-Wykręt, D., Dedieu, A., Peaucelle, A., Ludynia, M., Traas, J., Boudaoud, A., et al. (2012). Mechanical stress

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