Plant material and growing conditions
Rice (Oryza sativa L., subspecies Japonica, cultivar Nipponbare) was used in this study. We obtained Nipponbare seeds from the National Institute of Genetics of Japan. The Nipponbare genome was sequenced by the International Rice Genome Sequencing Project and is available in the Rice Annotation Project Database (RAP-DB; https://rapdb.dna.affrc.go.jp /).
Plants were grown in growth chambers at 70% humidity with a daily cycle of 14 h light at 29.5–30°C and 10 h dark at 25°C for 40 days, then transferred to short-day conditions, with 10 h of light and 14 h of darkness, to favor the induction of the reproductive phase and to adjust the sampling steps.
Generation of MEL1 and ZEP1 antibodies and H3K9dM antibodies
Two oligopeptides, MEL1-1 (CVYGAPMPAAHHQGAYQ) and MEL1-2 (GQAVAREGPVEVRQLPKC), were used to produce antibodies in rabbits (Cosmo Bio). Rabbit antisera were purified by affinity chromatography (Cosmo Bio).
An N-terminal region of ZEP1 cDNA was amplified by PCR using the primer pair 5′-gacaagcttgcggccATGCAGAAGCTGGGTTTATC-3′ and 5′-tgctcgagtgcggcctgTTCAGCAGATCTAGAATCCTCC-3′, where lowercase letters indicate vector sequences for cloning of the ZEP1 amplicon in pET24(+) (Novagen, 69772) using an infusion system. Transformation of BL21(DE3)pLys, protein purification and immunization of rats were performed commercially (Scrum). To detect the localization of histone 3 K9 dimethylation, an anti-dimethyl histone H3 (Lys9) mouse monoclonal antibody (FUJIFILM, 308-32361) was used for 3D immunoimaging, Fig. 2 extra.
Wes is an automated Western analysis using capillary blotting rather than gel blotting. Results are displayed in a digital image similar to a western blot and in graphs using raw data. Proteins were extracted from anthers 0.6-0.7 mm long. Anthers were ground and mixed with extraction buffer (150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 0.1% Tween 20, 10% glycerol, 5 mM DTT, 1 mM Pefabloc SC ( Roche), 1 × cOmplete Protease Cocktail Inhibitor). After two rounds of centrifugation (5800g for 10 min at 4°C and 20,400g for 10 min at 4°C) and removal of debris, total proteins were extracted. MEL1 and ZEP1 antibodies (1/20 dilution) were used for initial immune reactions.
The anthers were fixed as previously described12. PFA fixative was prepared fresh immediately before use and 4% PFA (Alfa Aesar) was added in 1× PMEG buffer (50 mM PIPES (Dojindo Molecular Technologies), 10 mM EGTA, 5 mM MgSO47 hours20.4% glycerol, 0.2% DMSO; pH 6.8). Inflorescences in PFA fixative were degassed at 0.09–0.1 MPa for 20 min on ice (Supplementary Fig. S4), and this step was repeated three more times. Samples were incubated for 100 min at 25°C and washed in 1X PMEG buffer for 20 min at 25°C; this washing step was repeated five more times. Fixed inflorescences can be stored at 4°C for 1 year.
Immunostaining using pollen mother cells for antibody estimation
To release the meiocytes, the anthers of the fixed inflorescences were incubated in an enzyme cocktail containing 2% Onozuka-RS cellulase (Yakult Honsha), 0.3% pectolyase Y-23 (Kikkoman) and 0.5% Macerozyme- R10 (Yakult Honsha) in PME buffer (50 mM PIPES, 5 mM EGTA and 5 mM MgSO4; pH 6.9)17 on a microscope slide coated with MAS, for 1 min at 25°C. Anthers were washed with PME, crushed in distilled water using a needle to release meiocytes and incubated at 25° C for 30 mins. Meiocytes were then blocked with 3% BSA in PME for 60 min and incubated at 4°C overnight with either rabbit anti-MEL1 or rat anti-ZEP1 antibody, diluted 1:500 with 3% BSA in SMEs. After three washes with PME for 5 min, the slide was incubated in a dark room for 3 h at 25°C with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A11034 ) and Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Invitrogen, A21247), diluted 1:200 with 3% BSA/PME, followed by three washes with PME for 5 min each. The MEL1 sample was then mounted in Vectashield mounting medium with DAPI (Vector Laboratories, H-1200), and images were captured using an LSM 780 microscope (Carl Zeiss). ZEP1 samples were incubated for 15 min at 25°C in DAPI (Sigma, MBD0015), then washed three times with PME buffer. Samples were mounted in ProLong Gold antifade reagent (Invitrogen, P10144) and images were captured using an LSM 880 microscope (Carl Zeiss).
Immunostaining using whole mounts of anthers
Anthers were recovered from inflorescences fixed in 4% PFA in PME buffer under a stereomicroscope. Then, the anthers were transferred into distilled water in a circle drawn with a PAP pen (Daido Sangyo) on a microscope slide coated with MAS (Matsunami Glass) and divided into two parts using a scalpel. (see Fig. 2B–D); samples were incubated for 30 min at 25°C. After blocking with 3% BSA in PME for 60 min at 25°C, the cut anther samples were immersed in a primary antibody solution (rabbit anti-MEL1, rat anti-ZEP1 or mouse anti-dimethyl H3 diluted 1:500 with 3% BSA in PME), placed in a vacuum desiccator (Nalgene) and degassed at 0.05 MPa for 2 min at 25°C, which was repeated four more times, and incubated overnight at 4°C. After three washes with PME for 5 min, the slide was placed in a solution of secondary antibodies, anti-Rabbit IgG, Alexa Fluor 568 (Invitrogen, A11036), anti-Rat IgG, Alexa Fluor 647 (Invitrogen, A21247) or anti-Mouse IgG, Alexa Fluor 488 (Invitrogen, A11001) diluted 1:200 with 3% BSA in PME and degassed at 0.05 MPa for 2 min at 25°C, repeated four more times . The slide was incubated in a dark, humid chamber for 2 h at 25°C and then incubated overnight at 4°C. After three washes with PME buffer for 5 min, the samples were incubated for 15 min at 25°C in DAPI (Sigma, MBD0015), followed by three washes with PME buffer. Specimens were mounted in ProLong Gold Antifade Reagent (Invitrogen, P10144) with 0.13–0.17 mm thick coverslips (MATSUNAMI Micro Cover Glass 13 mm #1).
Visualization of 3D immunostaining of whole anthers
Images were captured using an LSM 880 microscope (Carl Zeiss) under the following conditions: 40× Plan Apochromat objective (1.3 oil) (Fig. 3 and Movie 1) and 63× Plan Apochromat objective ( 1,4 oil) (Fig. 4) for detection; 405, 561 and 633 nm laser lines for DAPI, Alexa Fluor 568 and Alexa Fluor 647 excitation; and 410–483 nm (DAPI), 571–633 nm (Alexa Fluor 568), and 638–755 nm (Alexa Fluor 647). Images and animation were created using ZEN (Carl Zeiss) or Imaris 9 (Bitplane AG) software (Figs. 3, 4; Movie 1).
3D histochemical staining of whole anthers and imaging
Anthers fixed in 4% PFA were divided into two parts as described above. Anthers were stained with PI for 15 min at 25°C, then transferred to 20% iTOMEI solution (20% caprylyl sulfobetaine in 100 mM sodium phosphate buffer) and incubated for 10 min at 25°C. Samples were then transferred to 50% iTOMEI solution (50% caprylyl sulfobetaine in 100 mM sodium phosphate buffer) and incubated for 10 min at 25°C. Finally, samples were transferred and incubated in 70.4% iTOMEI (70.4% iohexol in PBS) for 1 h at 25°C14, and mounted with 70.4% iTOMEI. Images were captured with a confocal microscope (LSM 880; Carl Zeiss) under the following conditions: 40× Plan Apochromat lens (1.3 oil) for detection, 561 nm laser lines for PI excitation and filter emission from 571 to 633 nm. Images were created using ZEN (Carl Zeiss) (Supplementary Fig. S3).
Ethical approval and consent to participate
The experimental research reported here complies with relevant institutional, national and international guidelines and legislation.