Additional file 8: of Ostkpr1 functions in anther cuticle development and pollen wall formation in rice

Figure S4. Weight/Surface area ratio of WT and ostkpr1–2 anthers. The weight/surface area ratio of the anthers in the WT (blue squares) and ostkpr1–2 (red squares). (JPG 49 kb

Additional file 1: of Ostkpr1 functions in anther cuticle development and pollen wall formation in rice

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

Additional file 5: of Ostkpr1 functions in anther cuticle development and pollen wall formation in rice

Table S2. Detailed wax constituents in WT and ostkpr1–2 anthers. (DOCX 18 kb

Additional file 7: of Ostkpr1 functions in anther cuticle development and pollen wall formation in rice

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

Additional file 6: of Ostkpr1 functions in anther cuticle development and pollen wall formation in rice

Table S3. Detailed cutin composition of the WT and ostkpr1–2 anthers. (DOCX 17 kb

Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination

Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ;6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice

Additional file 2: of Ostkpr1 functions in anther cuticle development and pollen wall formation in rice

Figure S1. Phenotypic comparison between WT and ostkpr1 T-DNA insertion mutant. a Plants after bolting. b Spikeltes after removal of the lemma and palea. c WT pollen grains stained with 2% I2-KI solution. d Stage 13 pollen grains of ostkpr1 stained with 2% I2-KI solution showing no pollen grains. e-j SEM observation for the WT (e, g, i) and ostkpr1 (f, h, j) anthers and pollens. e, f The epidermal surface of WT (e) and ostkpr1 (f) anthers. g, h SEM observation for the WT (g) and ostkpr1 (h) pollen grains. i, j The enlarged view of the surface of WT (i) and ostkpr1 pollen grains. Bars = 1 mm in b, 200 μm in c, d, 10 μm in e, f, 20 μm in g, h and 5 μm in i, j. (JPG 993 kb

DataSheet_1_OsPRD2 is essential for double-strand break formation, but not spindle assembly during rice meiosis.pdf

Meiotic recombination starts with the programmed formation of double-strand breaks (DSB) in DNA, which are catalyzed by SPO11, a type II topoisomerase that is evolutionarily conserved, and several other accessary proteins. Homologs of MEIOSIS INHIBITOR 4 (MEI4/REC24/PRD2) are proteins that are also essential for the generation of meiotic DSBs in budding yeast, mice and Arabidopsis thaliana. In Arabidopsis, the protein ARABIDOPSIS THALIANA PUTATIVE RECOMBINATION INITIATION DEFECTS 2/MULTIPOLAR SPINDLE 1 (AtPRD2/MPS1) has been shown to have additional roles in spindle assembly, indicating a functional diversification. Here we characterize the role of the rice MEI4/PRD2 homolog in meiosis. The osprd2 mutant was completely male and female sterile. In male meiocytes of osprd2, no γH2AX foci were detected and twenty-four univalents were produced at diakinesis, suggesting that OsPRD2 is essential for DSB generation. OsPRD2 showed a dynamic localization during meiosis. For instance, OsPRD2 foci first appeared as discrete signals across chromosome at leptotene, and then became confined to the centromeres during zygotene, suggesting that they might be involved in assembly of the spindle. However we did not observe any obvious aberrant morphologies in neither the organization of the bipolar spindle nor in the orientation of the kinetochore in the mutant. These findings suggest that in rice PRD2 might not be required for spindle assembly and organization, as it does in Arabidopsis. Taken together our results indicate that plant MEI4/PRD2 homologs do play a conserved role in the formation of meiotic DSBs in DNA, but that their involvement in bipolar spindle assembly is rather species-specific.</p

Additional file 4: of Ostkpr1 functions in anther cuticle development and pollen wall formation in rice

Figure S3. Amino acid sequences alignment of OsTKPR1 and AtTKPR1. Sequences were aligned using Clustal W. (DOCX 45 kb

Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination

Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ;6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice