An Auxin Minimum Triggers the Developmental Switch from Cell Division to Cell Differentiation in the Arabidopsis Root

AN AUXIN MINIMUM TRIGGERS THE DEVELOPMENTAL SWITCH FROM CELL DIVISION TO CELL DIFFERENTIATION IN THE ARABIDOPSIS ROOT Riccardo Di Mambro1,*,#, Micol De Ruvo1,6,7,*, Elena Pacifici1, Elena Salvi1, Ross Sozzani2, Philip N. Benfey3, Wolfgang Busch4, Ondrej Novak5, Karin Ljung5, Luisa Di Paola6, Athanasius F. M. Marée7, Paolo Costantino1, Verônica A. Grieneisen7,†, Sabrina Sabatini1,8,†. 1: Dipartimento di Biologia e Biotecnologie, Laboratory of Functional Genomics and Proteomics of Model Systems, Università di Roma, Sapienza - via dei Sardi, 70 - 00185 Rome, Italy. 2: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, United States. 3: Department of Biology and Howard Hughes Medical Institute, Duke University, Durham, North Carolina 27708, USA. 4: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria. 5: Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden. 6: Unit of Chemical-physics Fundamentals in Chemical Engineering, Department of Engineering, Università Campus Bio-Medico di Roma, via Álvaro del Portillo 21, 00128 Rome, Italy. 7: Computational and Systems Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. 8: Istituto Pasteur-Fondazione Cenci Bolognetti. * These authors contributed equally to this work # Present address: Dipartimento di Biologia, Università di Pisa - via Ghini, 13 - 56126 Pisa, Italy. † Authors for correspondence Tel: 39-06-49917916 Fax: 39-06-49917594 e-mail: [email protected] e-mail: [email protected] 1 Supporting Information Appendix Materials and methods Root Length, Meristem Size and Cell Size Analysis For root length measurements, plates were photographed and the resulting images were analysed using the image analysis software ImageJ 1.47v available online (http://rsbweb.nih.gov/ij/). Root meristem size for each plant was measured based on the number of cortex cells in a file extending from the quiescent centre to the first elongated cortex cell excluded, as described previously (1, 2). The cortex is the most suitable tissue to count meristematic cells, as its single cell type composition shows a conserved number of cells among different roots. The boundary between dividing and differentiating cells for each tissue is called transition boundary (TB), while the region including the different transition boundaries is called transition zone (1) (TZ) (Fig. 1). Cell-o-Tape (3), a Fiji (http://fiji.sc/Fiji) macro, was used to count and measure individual neighbouring cells along a defined file as well as to estimate the position of the last cortical cell at the TB (SI Appendix, Table S1). Images were obtained using a confocal laser scanning microscope (Zeiss LSM 780). For each experiment a minimum of 30 plants for two biological replicates were analysed. Chosen settings in Cell-o-Tape: threshold = 10; stringency = 2.5. Student’s t test, P < 0.05, n=30 (http://graphpad.com/quickcalcs/ttest1/). Differential Interference Contrast (DIC) with Nomarski technology microscopy (Zeiss Axio Imager A2) was used to count meristem cell number. Plants were mounted in a chloral hydrate solution (8:3:1 mixture of chloral hydrate:water:glycerol). 2 pGH3.17:GH3.17-GFP plants were analysed by confocal laser scanning. The cell wall was stained with 10 μM propidium iodide. A minimum of 20 roots for 12 independent transgenic lines were analysed. GUS Histochemical Assay To visualize pARR1::ARR1:GUS lines, GUS histochemical assay was performed using the β-glucuronidase substrate X-gluc (5-bromo-4-chloro-3-indolyl glucuronide, Duchefa) dissolved in N-N-dimethyl-formamide. X-gluc solution, composed of 100 mM Na2HPO4, 100 mM NaH2PO4, 0.5 mM K3 Fe(CN)6, 0.5 mM K4Fe(CN)6, 0.1% Triton X-100, and 1 mg/ml X-gluc, was prepared as previously described (2). Five-day old seedlings were incubated for 20 hours at 37°C in the dark and imaged using the Axio Imager.A2 (Zeiss) microscope. Generation and Characterization of Transgenic Plants Standard molecular biology techniques and the Gateway system (Invitrogen) were used for the cloning procedures. Genomic DNA from Arabidopsis ecotype Columbia (Col-0) was used as the template for amplification. For the translational fusion with the GFP, the promoter sequence of GH3.17 (AT1G28130) (2,128 base pairs (bp)) was amplified using the pGH3.17 primers (SI Appendix, Table S6) and the PCR product was then cloned into pENTR5’-TOPO TA vector. The genomic sequence of GH3.17 (2450 bp) was amplified using the gGH3.17 primers (SI Appendix, Table S6) and cloned in a pDONOR221. A LR reaction was then conducted by using the promoter and genomic sequence of GH3.17, and a c-term pDONORP2P3-GFP. For pUBQ10::GH3.17 transgenic plant, pDONORP4P1-pUBQ10 vector and genomic sequence of GH3.17 were used. The LR products were then sub cloned in the 3 Gateway pBm43GW destination vector. Plasmids were transformed into Col-0 plants by floral dipping (4). pGH3.17:GH3.17-GFP fusion was tested to be functional by rescuing the gh3.17-1 mutant phenotype. RNA Isolation and qRT-PCR Total RNA was isolated from root tissues of 5-day-old seedlings using RNeasy® Micro Kit (Quiagen) and the first strand cDNA was synthesized using the Superscript® III First Strand Synthesis System (Invitrogen). Transcript levels were monitored by qRT-PCR using gene-specific oligonucleotide primers (SI Appendix, Table S6). qRT-PCR reactions were performed with Sensi Fast SYBR (Bioline) using a 7500 Fast Real-Time PCR system (Applied Biosystems), according to the manufacturer’s instructions. Data were analysed using the ∆∆Ct (cycle threshold) method and normalized with the expression of the reference gene ACTIN2. For each analysis, three technical replicates of qRT-PCR were performed on two independent RNA batches. Results were comparable in all experiments. Student’s t-test was used for data significance (http://graphpad.com/quickcalcs/ttest2.cfm). Chromatin Immunoprecipitation, ChIP-chip and ChIP-qRT-PCR For the genome-wide identification of ARR1 direct targets, we first conducted Chromatin Immunoprecipitation and then hybridized the amplified precipitate to a custom Agilent microarray (ChIP-chip) that contains probes representing a large proportion of all Arabidopsis promoter regions (5). Two independent ChIP experiments were performed on the roots of either 5-day-old seedlings, expressing 4 ARR1 genomic sequence under the control of its own promoter fused to GFP (pARR1::ARR1-GFP plants) (4), or Col-0 plants. ChIP was performed as described in Sozzani et al. 2010 (6), except that DNA-free protein A agarose beads (Invitrogen) and a rabbit polyclonal antibody against GFP (ab290, Abcam) were used. DNA from these experiments was amplified using a random-primer-based genome amplification method described in http://cat.ucsf.edu/pdfs/22_Round_A_B_C_protocol.pdf, with minor modifications. After labelling with Cy3 and Cy5, respectively, following an amino-allyl-dye coupling protocol (http://camd.bio.indiana.edu/files/amino-allyl- protocol.pdf), DNA was cleaned up using the PCR purification kit (Qiagen) and 3 μg from the ChIP and from the mock samples were taken and mixed for hybridization to a custom long oligonucleotide (60 bases) Arabidopsis promoter microarray that has been described in Sozzani et al., 2010 (6). Two independent labeling reactions and subsequent hybridizations were performed from each biological replicate to perform dye-swaps. Hybridization was performed according to the Agilent ChIP-chip protocol, and images were obtained using an Agilent microarray scanner (model G2565BA) at a resolution of 5 μm. Signal extraction and initial data processing were done using the Agilent feature extraction software. For ChIP-chip analysis, to assess genome-wide binding for the processed signal ratio (SR = signal of ARR1-GFP / signal Col-0) of each probe on the array, a Z-Score (ZS) was calculated as follows: . These empirical ZS-values for each probe of the independent ChIP-chip experiments were added. Enrichment was scored by: (1) the length of DNA regions that were covered by probes above a defined ZS-value threshold; (2) a local ZS maximum (seed) and (3) the number of nucleotides allowed as gaps within called regions. For each combination of parameters, the detected regions were registered. A gene was assigned to an enriched region if that region was 5 present within 4,000 bp upstream or 300 bp downstream of the transcription start site in an intron, or 300 bp downstream of the gene model. Each parameter combination produced a list of called regions and thus of assigned genes. The proportion of genes that were classified as regulated by ARR1 was recorded for each list. The optimal enrichment detection criteria were defined by those parameters that yielded the highest proportion of ARR1-regulated genes and a major fraction of the already described ARR1 direct genes. These settings were: probe ZS seed > 13; probe ZS > 6; minimum length of hybridization above probe ZS threshold, 175 nucleotides; maximum gap, 125 nucleotides. Gene ontology enrichment categories were found using ChipEnrich software (6) (http://www.arexdb.org/software.jsp) (SI Appendix, SI Appendix, Fig. S3B). The enrichment of the GH3.17 target promoter-regions DNA was confirmed using RT-qPCR (SI Appendix, SI Appendix, Fig. S3A). A qPCR efficiency of 2-fold amplifications per cycle was assumed, and sequences from UBIQUITIN 10 were used to normalize the results between samples. Tiling along the GH3.17 (AT1G28130) was done using sets of adjacent specific amplified regions (SI Appendix, SI Appendix, Fig. S3A and SI Appendix, Table S6) along 2,1 Kb region of the GH3.17 promoter. Accession codes Gene Expression Omnibus: GSE70595. pGH3.17:GH3.17-GFP and DR5::GFP Fluorescence Quantification The fluorescence intensity of plants carrying pGH3.17:GH3.17-GFP (SI Appendix, SI Appendix, Fig. S3F – I) or DR5::GFP (SI Appendix, SI Appendix, Fig.

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