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1 PRC2 represses dedifferentiation of mature somatic cells in Arabidopsis Momoko Ikeuchi 1 *, Akira Iwase 1 *, Bart Rymen 1, Hirofumi Harashima 1, Michitaro Shibata 1, Mariko Ohnuma 1, Christian Breuer 1, Ana Karina Morao 2, Miguel de Lucas 3, Lieven De Veylder 4,5, Justin Goodrich 6, Siobhan M. Brady 3, François Roudier 2 and Keiko Sugimoto 1 1 RIKEN Center for Sustainable Resource Science, Suehiro-cho, Tsurumi, Yokohama, Kanagawa , Japan. 2 Institut de Biologie de l Ecole Normale Supérieure, CNRS UMR8197, INSERM U1024, 46 rue d Ulm, Paris Cedex 05, France. 3 Department of Plant Biology and Genome Center, University of California, Davis, 1002 Life Sciences, One Shields Avenue, Davis, CA Department of Plant Systems Biology, VIB, B-9052, Gent, Belgium, 5 Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052, Gent, Belgium, 6 Institute of Molecular Plant Sciences, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK. *These authors contributed equally to this work. Correspondence should be addressed to KS (keiko.sugimoto@riken.jp). NATURE PLANTS 1

2 Supplementary Figure 1 Confocal microscopy images of LTI6-GFP and H2B-YFP marker lines. a, 4-day-old WT-like root (left) and the same root visualised at day 5 (right). Dotted lines mark division zone where most cells have 2C or 4C nuclei and differentiation zone where most cells, including root hairs, have 4C to 16C nuclei. b, 12-day-old emf2-3 vrn2-1 root (left) and the same root visualised at day 15 (right). Figure panels in Figure 1d are reproduced from this dataset. Bar = 0.25 mm. 2 NATURE PLANTS

3 SUPPLEMENTARY INFORMATION Supplementary Figure 2 Root-hair specific activity of the EXPANSIN7 promoter. a, Confocal microscopy image of pexp7:nls-gfp in WT-like plants. Note that the GFP expression is only detectable in developing and mature root hairs. Bar = 0.1 mm. b, Flow cytometry analysis of DAPI-stained nuclei from pexp7:gtl1-gfp expressing roots using DAPI (left) or GFP signal (right). Note that the GFP signal is detected predominantly in 8C and 16C nuclei. NATURE PLANTS 3

4 Supplementary Figure 3 Ploidy analysis of clf-28 swn-7, fie and emf2-3 vrn2-1 roots. a, Flow cytometry analysis of DAPI-stained nuclei from 10-day-old WT, clf-28 swn-7, fie and emf2-3 vrn2-1 roots. b, Nuclear size as measured by the average projected area of DAPI-stained nuclei in unicellular root hairs of 7-day-old WT, clf-28 swn-7, fie and emf2-3 vrn2-1 roots. Average projected area of DAPI-stained nuclei in root cap cells is shown as a 2C control. c, Average projected area of chromocentres in SYBR GREEN-stained nuclei from unicellular root hairs of 7-day-old WT, clf-28 swn-7, fie and emf2-3 vrn2-1 roots. Average projected area of SYBR GREEN-stained chromocentres in root cap cells is shown as a 2C control. d, Average projected area of 4 NATURE PLANTS

5 SUPPLEMENTARY INFORMATION H2B-YFP-labelled nuclei in unicellular root hairs of 7-day-old WT-like and emf2-3 vrn2-1 roots. e, Average projected area of H2B-YFP-labelled nuclei in unicellular root hairs of 15-day-old WT-like roots and multicellular root hairs of emf2-3 vrn2-1 roots. Error bars represent S.E. of the means (n = 400 for b, 250 for c, 50 for d, 30 for e for each genotype). NATURE PLANTS 5

6 Supplementary Figure 4 Live imaging of CYCB1;2-YFP-expressing root cells in clf-28 swn-7. a, Mitotic division pattern of CYCB1;2-YFP-labelled nuclei within the root meristem. b, CYCB1;2-YFP fusion protein being ectopically produced in mature cortex cells. Nuclear size indicates endoreduplication preceding the nuclear division. Note that the CYCB1-YFP signal persists after nuclear division in both meristematic cells and mature cortex cells, and this is likely due to the nature of the marker. Bar = 0.1 mm. 6 NATURE PLANTS

7 SUPPLEMENTARY INFORMATION NATURE PLANTS 7

8 Supplementary Figure 5 Expression of PRC2 subunits in roots. a, Confocal microscopy images of SWN-GFP expression in 7- and 14 day-old roots. b, Confocal microscopy images of EMF2-GFP expression in 7- and 14 day-old roots. c, Bright-field micrograph of VRN2 expression in 7- and 14 day-old pvrn2:vrn2-gus plants. Bars = 0.1 mm (a, b, c). 8 NATURE PLANTS

9 SUPPLEMENTARY INFORMATION Supplementary Figure 6 Ectopic WIND1 expression in emf2-3 vrn2-1 roots. Confocal microscopy analysis of WIND1-GFP expression, driven by its own promoter, in multicellular root hairs (a) and (b) root hair-derived callus of 14-day-old emf2-3 vrn2-1 transgenic plants. Note that the WIND1-GFP signal is not detectable in unicellular root hairs (c) of the same plant. Bars = 0.1 mm. NATURE PLANTS 9

10 Supplementary Figure 7 H3K4me3 marking at WIND loci. ChIP-chip measurements of H3K4me3 enrichment in WT seedlings, roots, root tips, epidermal root hair and non-hair cells. Green bars highlight probes reporting significant enrichment in H3K4me3 (log2 IP/INPUT). Red arrows show the position and orientation of WIND genes according to the TAIR NATURE PLANTS

11 SUPPLEMENTARY INFORMATION Supplementary Figure 8 Enhanced multicellularisation of root hairs by co-expression of WIND1 and LEC2 The multicellular phenotype was scored using 500 root hairs from XVE:WIND1 35S:LEC2-GR double transgenic plants treated with 10 µm 17β-estradiol and/or 10 µm dexamethasone (DEX). WT plants treated with 10 µm 17β-estradiol and 10 µm DEX as well as XVE:WIND1 35S:LEC2-GR plants treated with ethanol (EtOH) and dimethyl sulfoxide (DMSO) are shown as a control. NATURE PLANTS 11

12 Supplementary Video Legends Supplementary Video 1 Time-lapse imaging of nuclear and cellular division in emf2-3 vrn2-1 root hairs. Confocal microscopy of H2B-YFP-labelled nuclei in emf2-3 vrn2-1 root hairs. Root hair nuclei normally endoreduplicate up to 16C and elongate up to 50 m along the long axis of the cell. In emf2-3 vrn2-1 root hairs these fully endoreduplicated nuclei reenter into the mitotic cell cycle and undergo nuclear and cellular division. Newly formed cell plates are visualised by LTI6-GFP localised to the plasma membrane. Figure panels in Figure 1e are reproduced from this dataset. Supplementary Video 2 Time-lapse imaging of nuclear division in clf-28 swn-7 root hairs. Confocal microscopy of CYCB1;2-YFP-labelled, endoreduplicated nuclei in clf-28 swn-7 root hairs. The mitotic B-type cyclin, CYCB1;2, is ectopically expressed in fully differentiated clf-28 swn-7 root hairs and these CYCB1;2-YFP-labelled nuclei, which are substantially larger than 2-4C nuclei undergoing the mitotic cycle in the meristem, divide to produce two sister nuclei. Figure panels in Figure 1g are reproduced from this dataset. Supplementary Video 3 Time-lapse imaging of nuclear division in mature cortex cells of clf-28 swn-7 roots. Confocal microscopy of CYCB1;2-YFP-labelled, endoreduplicated nuclei in clf-28 swn-7 roots. Note that CYCB1;2-YFP-positive nuclei in root cortex cells divide to produce two sister nuclei. Figure panels in Supplemental Figure 4b are reproduced from this dataset. 12 NATURE PLANTS