A B C Supplementary Figure S1. GO enrichment analysis of statistically significant DEGs in osmads13 mutant. (A) GO test for molecular function of annotated DEGs showing enrichment of transcription factor activity and transcription regulator activity. (B) Over-representation of the category transcription factor activity (red) in genes upregulated in osmads13 mutant. (C) Over-representation of the category protein binding in genes downregulated in osmads13 mutant. Singular Enrichment Analysis (SEA) for molecular function was carried out using Rice (Oryza sativa Japonica) MSU7.0 nonTE transcript ID as Reference in AgriGO. A B C DC OP OP SP FM FM Supplementary Figure S2. Basipetal gradient of flower development along the rice inflorescence. (A) Immature inflorescence dissected from wild-type plants 3 weeks after floral induction. Young flowers with Floret Meristem (FM) at the base, differentiating flower with Ovule Primordium (OP) in the middle, closed flowers with mature ovule at the top. Bar represents 1 mm. (B) and (C) FM enclosed by Stamen Primordia (SP) and OP protected by the Developing Carpel (DC) before and after Laser assisted Micro-Dissection (LMD). Stages In6 and In7 of inflorescence development correspond to stages Sp7 and Sp8 of spikelet development (carpel primordium the former, ovule and pollen the latter), and stage Ov1 of ovule development. A B C D Supplementary Figure S3. Expression analyses of known regulators of rice reproductive development. (A) and (B), relative expression levels of OsFD1 and OsSPL14 in immature inflorescences, FM and OP of wild-type (white) and osmads13 mutant (green). Expression data are represented as the mean values of three biological replicates ±SD. Differences were analyzed using Student’s t-test and asterisks indicate statistical significance (*P < 0.033). (C) in silico expression profiles of OsFD1 in developing inflorescences and reproductive organs inferred using Gramene database. (D) in silico expression profiles of OsSPL14 throughout plant development (left), and specifically in reproductive organs (right), inferred using RiceXPro database. A B Supplementary Figure S4. In silico analysis of OsbZIP52. (A) Phylogram representing the relationship between OsbZIP52 (Rice Seed B Zipper 5, in red) and homologous genes from sequenced plant species. Related rice genes of the bZIP family are reported in blue. The phylogenomics tree was obtained using the Compara Tree tool (Gramene). (B) in silico expression of OsbZIP52 in roots and reproductive organs (left), specifically during early seed development (right), inferred using the RiceXPro database. A B C Supplementary Figure S5. Phylogenomic and expression analysis of OsbHLH6. (A) Phylogram representing relationship between OsbHLH6 (Os04g0301500, in red) and homologous genes from sequenced plant species, obtained using the Compara Tree tool of Gramene. Paralogous rice genes are reported in blue (OsbHLH007, Os04g0300600; UDT1, Os07g0549600; TDR, Os02g0120500). (B) In situ analysis of OsbHLH6 expression in developing inflorescences at different stages of flower formation. Top, wild-type flower (bar, 20 μm); bottom, osmads13 inflorescence (bar, 100 μm). (C) Predicted expression patterns of OsbHLH6 in reproductive organs, inferred using the RiceXPro database. A B C Supplementary Figure S6. In silico analysis of OsbHLH10. (A) Phylogram representing relationship between OsbHLH10 (OsMYC2-like1, in red) and homologous genes from sequenced plant species. Related rice genes are reported in blue (OsMYC2, Os10g0575000; OsMYC2-like1, Os01g0235700). The Arabidopsis closest orthologous genes are AtbHLH3/JAM3 (AT4G16430), AtbHLH17/AtAIB/JAM1 (AT2G46510) and AtbHLH13/JAM2 (AT1G01260). The phylogenomics tree was obtained using the Compara Tree tool. (B) and (C) predicted expression profile of OsbHLH10 in all plant tissues but developing embryos and endosperms, and specifically in floral organs, inferred using the RiceXPro database. A B C Supplementary Figure S7. In silico analysis of OsbHLH39. (A) Phylogram representing relationship between OsbHLH39 (in red) and homologous genes from sequenced plant species. Related rice genes are reported in blue (OsbHLH38, Os08g0432800, OsbHLH40, Os03g0260600). The Arabidopsis closest orthologous genes are AtbHLH32/TMO5, (AT3G25710) and AtbHLH30/ABS5/TMO5-LIKE1 (AT1G68810). The phylogenomics tree was obtained using the Compara Tree tool. (B) and (C) Predicted expression profile of OsbHLH39 in all tissues but in anther, inferred using the RiceXPro database. A B ChIP-seq DEGs OsMADS1 osmads13 Supplementary Figure S8. In silico identification of genes acting downstream of OsMADS1 and OsMADS13. (A) Over-representation of the category transcription factor activity in GO analysis for molecular function of genes upregulated in osmads13 and downregulated in osmads1. (B) modified Venn diagram showing common genes bound by OsMADS1 (ChIP-sequencing, Khanday et al 2016) and mis-regulated in osmads13 inflorescences. A LOC_Os01g50940 PFG_1B-24315 ATG TGA B wild-type osbhlh10 C LOC_Os01g49830 PFG_2A-10680 PFG_3A-03612 ATG TAG Supplementary Figure S9. T-DNA insertions in OsbHLH10 and OsRAV11. (A) Schematic representation of OsbHLH10, with T-DNA insertion (yellow triangle) in the 5’UTR, 250 bp upstream of the ATG. (B) Morphological analysis by optical microscopy of carpels dissected from wild-type and osbhlh10 mutant flower. Bar, 1 mm. (C) Schematic representation of OsRAV11 gene, with T-DNA insertion (yellow triangle) in the 5’UTR, 100 bp upstream of the ATG. In black, coding sequence; in white, 5’ and 3’ UTRs; in grey, promoter; in pink, CArG boxes; bar, 1 Kb. A B C Locus ID Gene name ZF domain LATE KNU ATZFP1 C2H2 ATZFP7 ATZFP4 ATZFP2 ATZFP6 ATZFP3 C2H2 C2H2 GIS C2H2 NUB GIS3 JAG ATZFP8 GIS2 C2H2 URO D FM FM E LOC_Os03g57240 (ZOS3-19/DST) s s s C2H2 s C C 500 bp wt mut Supplementary Figure S10. Analysis of ZOS3-19/DST. (A) Phylogram representing paralogous genes in O. sativa subspecies japonica, obtained using Gramene website. (B) Graphic results of the Multiple Sequence Alignment of ZOS3-19/DST and Arabidopsis Zinc-Finger Proteins (ZFP) with highest similarities. In green, factors involved in trichome formation; in violet, factors involved in reproductive organs formation. (C) In silico expression profiles of ZOS3-19/DST in different vegetative and reproductive tissues, inferred using the Rice XPro tool. (D) In situ analysis of DST expression in developing inflorescences. Top, hybridization with sense (left) and antisense (right) probes at early stages of development in wild-type flowers (bar, 20 μm). Bottom, detection of DST transcripts in wild-type (left) and osmads13 mutant (right) mature flowers (bar, 50 μm). FM, floral meristem; s, stamen; C, carpel. (E) Analysis of DST expression (by standard RT-PCR) in pistils dissected from wild type and zos3-19/dst mutant. The 530 bp band was amplified only in wild-type plants. In black, coding sequence; in white, 3’ UTRs; in grey, promoter. Grey arrow, forward primer used for genotyping; black arrows, primers specific for 5’ of the gene. A B Gene ZF domain (N-term) ATZHD5 ATZHD2 ATZHD4 OsZHD3 ATZHD1 OsZHD6 ATZHD2 OsZHD5 ATZHD3 OsZHD7 ATZHD4 ATZHD6 ATZHD12 OsZHD11 ATZHD9 ATZHD10 ATZHD7 MIF1 MIF3 MIF2 OsZHD8 ATZHD14 ATZHD13 Gene HD domain (C-term) ATZHD5 ATZHD2 ATZHD4 ATZHD1 ATZHD2 ATZHD3 ATZHD4 ATZHD6 ATZHD12 OsZHD1 ATZHD9 OsZHD2 ATZHD10 ATZHD7 MIF1 MIF3 MIF2 ATZHD14 ATZHD13 OsZHD4 Supplementary Figure S11. In silico analysis of OsZHD8. (A) Phylogram representing paralogous genes of OsZHD8 in O.sativa subspecies japonica (in blue), and orthologous genes in sequenced land plant species. All the information was retrieved by Gramene website. (B) Graphic results of the Multiple Sequence Alignment of OsZHD8 and Arabidopsis Zinc-Finger Homeo-Domain (ZHD) proteins with highest similarities. Proteins belonging to this family are characterized by a Zinc-Finger (ZF) domain at the N-term, and Homeo-Domain at the C-term. A small cluster of Mini ZF proteins (MIF) play a regulatory role as they contain protein-protein interaction ZF domain but lack the HD DNA binding domain. A B s s s s FM FM C C C C C D s LOC_Os02g27060 E H PFG_3A-00989 LOC_Os04g35500 ZF HD 500 bp ATG TAG F G wild-type oszhd8 wild-type oszhd8 Supplementary Figure S12. Molecular and phenotypic analysis of OsZHD8. (A) and (B) In situ analysis of OsZHD8 expression in developing inflorescences. (A) Hybridization with sense (left) and antisense (right) probes at early stages of development in wild-type flowers (bar, 20 μm). (B) Detection of OsZHD8 transcripts in wild-type (left) and osmads13 mutant (right) mature flowers (bar, 50 μm). FM, floral meristem; s, stamen; C, carpel. (C) Upregulation of OsZHD8 in immature inflorescences and OP of osmads13 (green) compared to wild type (white). (D) and (E) Schematic representations of T-DNA insertions (in yellow) in the coding sequence of LOC_Os02g27060/OsARID1 (red asterisk represents stop codon) and in the 3’UTR of LOC_Os04g35500/OsZHD8 (cds in black, UTRs in white, and promoter in grey). (F) Downregulation of OsZHD8 in immature inflorescences of the T-DNA insertion mutant (yellow) compared to wild type (white). (G) Morphological alterations of carpels from dissected oszhd8 mutant flowers compared to wild type. Bar,1 mm. (H) Morphological alterations of oszhd8 mutant inflorescence compared to wild type. Expression data are presented as the mean values of three biological replicates ±SD. Differences were analyzed using Student’s t-test and asterisks indicate statistical significance (*P < 0.033). A B endogenous OsMADS13 locus UTR TOS -17 ProOsMADS13:OsMADS13(gene):GFP GFP wild-type OsMADS13 (cds) GFP C OsMADS13 (cds)-GFP OsMADS13 (gene)-GFP wild-type D wild-type homo #1 Supplementary FigureS13. Generation and analysis of OsMADS13-GFP lines. (A) Clearing analysis of mature pistils dissected from wild-type and ProOsMADS13-OsMADS13 (cds)-GFP in osmads13 background. Arrow indicates tracheid-like structure. (B) Schematic representations of endogenous gene (top) with the Tos-17 inserted in the upstream regulatory region, and transgenic cassette containing ProOsMADS13-OsMADS13 (gene)-GFP, including introns (bottom). (C) Fluorescent microscopy analysis of GFP expression in ovaries of transgenic lines transformed with ProOsMADS13-OsMADS13 (cds)-GFP and ProOsMADS13-OsMADS13 (gene)-GFP. Wild- type pistils were used as negative control (right). For each genotype, bright field on the left and FITC on the right. (D) Molecular and morphological analyses of plants transformed with ProOsMADS13-OsMADS13 (gene)-GFP. Left, determination of transgene copy number, reported as transgene/osmads13 gene ratio, in different transgenic lines by qPCR; the ratio osmads13 mutant allele/osmads13 wt allele is 0 for the wt, 0.5 for heterozygotes and 1 for homozygotes. Right, optical microscopy analysis of gynoecia dissected from wild type and a selected transgenic line showing defects in apical tissues of the gynoecium. . A B Supplementary Figure S14. In silico analysis of regulatory sequences of putative downstream target. Sequence information of OsZHD8 (A), ZOS3-19/DST (B) and DL (C), obtained by using SnapGene. In pink (CArG box), sequences containing consensus element bound by MADS domain proteins. C