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Additional file 9: Table S2. OsRopGEF isogene-specific primers for qPCR and cloning.

Additional file 2: Figure S2. Meta-expression analysis of entire AtRopGEF genes. The heatmap was prepared using the Genevestigator. We chose five representative tissues, including pollen. It revealed that five Arabidopsis RopGEFs were highly expressed in pollen. The dark red color of the heatmap indicated the highest expression; white color, lowest expression.

Additional file 3: Figure S3. Protein sequence alignment domain analysis and conserved phosphorylated amino acid residues of C-termini of RopGEF genes. Every OsRopGEF and AtRopGEF protein sequence was collected and aligned, followed by the PRONE domain (C1, C2, C3) and the WW-motif. At the end part of the sequence, we found some conserved regions. According to previous studies, S510 in the C-terminus of AtRopGEF12 is involved in the C-terminal inhibition of GEF activity. As in AtRopGEF12, the serine residue is conserved in each of the OsRopGEF2, OsRopGEF3, and OsRopGEF8 genes but not in OsRopGEF6. However, K (Lysine) can also be phosphorylated.

Additional file 6: Figure S6. Single mutant assay using CRISPR-Cas9 system. (a-b, d-e) In-vitro pollen germination test of wild-type (a, d), ropgef2 (b, c), and ropgef3 (e-f) single mutants on solid pollen media, Bar = 50 μm; (c,f) enlarged pollen pictures, Bar = 20 μm. Each control and mutant pollen in-vitro assays were performed in the same environment on the same day. (g) The ratio of pollen germination. Black bar indicates wild type; grey bar indicates ropgef2; white bar indicates ropgef3 mutant pollen. Error bars are standard deviation of three technical repeats. (h) Fertility ratio comparison between wild-type and ropgef2 single mutant plants. Error bars are standard deviation of more than three panicles in the plants.

Additional file 1: Figure S1. Meta-expression analysis and genome-wide identification of RopGEF in rice. The expressions from various rice tissues were examined using Microarray database. Yellow color in the heatmap indicates high level of expression; dark blue, low level of expression. Numeric values indicate the average of the normalized log2 intensity of microarray data.

Additional file 7: Figure S7. Meta-expression analysis and genome-wide identification of the seven OsRac and ten AtRop genes. (a) Heatmap expression analysis of OsRac gene. (b) Heatmap of AtRop using Genevestigator. (c) Phylogenetic tree constructs including every OsRac and AtRop.

Additional file 5: Figure S5. Promoter analysis of each OsRopGEF gene. Specific cis-acting element (CRE) known to affect expression in pollen were identified using PLACE database, namely three pollen-CREs and the TATA box. The number above the yellow bar shows the upstream position of the promoter base pair when taken at + 1 of ATG. We analyzed up to the upstream 2000-base pair. The p-values indicate how significantly the four genes exhibiting high expression in pollen differed from the other seven genes.

Table S4. Primers used in this work. (DOCX 17 kb)

Table S2. Detailed wax constituents in WT and ostkpr1â 2 anthers. (DOCX 18 kb)

Table S3. Detailed cutin composition of the WT and ostkpr1â 2 anthers. (DOCX 17 kb)

Table S1. Allelism test of ostkpr1â 2 with ostkpr1. (DOCX 22 kb)