Pollen collection and cryopreservation
The P. lactiflora lactiflora 'Fen Yu Nu' pollen were all collected from the Luoyang International Peony Garden (Luoyang, Henan Province, China) in April 29, 2019. When the dry and humidity of air is suitable, the flower buds that are about to open are selected, the anthers are gently picked and brought back under the 4℃. Then, the anthers are naturally dried at room temperature (23±2℃) for 24h. The pollen is completely dispersed, and the pollen is collected with an 80 mm aperture sieve (Ren et al., 2019a).
Divide the collected pollen (0.1g/ package) into tin foil, place it in a 2 mL cryopreserved tube, and then directly put it into LN for storage. When needed, the pollen were frozen with running water (Ren et al., 2019a).
Determination of pollen moisture content and viability
Pollen moisture content was determined by 105℃ constant temperature drying method, the detailed procedure are shown in the existing studies (Ren et al., 2019a).
Pollen germination level was detected by suspension drop method, the culture medium contained 10% sucrose and 0.1% boric acid. Take 15 μL pollen culture medium on the cover glass, add trace amount of pollen and mix well. Turn the cover glass over immediately and place it in a glass groove (with 20 μL pollen culture medium). The culture was placed in the dark at 25℃ in a humid environment for 4h, the length of pollen tube was greater than twice the diameter of pollen grains as the criterion for pollen germination. Four replicates were set for each treatment, and three fields were randomly selected for each replicate. The germination number was counted with a 20X eyepiece (Leica DM-500), and the mean value of the results (Ren et al., 2019a).
Determination of pollen NO content
The content of NO was determined by DAF-FM DA fluorescence staining (Biyuntian Biotechnology Co., Ltd., S0019). Added 200 μL DAF-FM DA (100 μmol/L) into 0.01 g pollen, thoroughly mixed. Incubated at 37℃ for 30 min without light, centrifuged at 2000 rmp for 20s, discarded the dye solution. Washed twice with 500 μL PBS (pH=7.4), then diluted the precipitated pollen with 1 mL PBS (pH=7.4). Fluorescence value was measured at FITC channel by flow cytometer (FACSCalibur, Becton-Dickinson, Franklin Lakes, NJ, USA), the without adding fluorescent dye as control. The relative content of NO was taken as the actual fluorescence measurement value, and each treatment was repeated 3 times.
Determination of pollen mitochondrial membrane potential
Pollen mitochondrial membrane potential was detected by JC-1 fluorescence staining. 0.01 g pollen was added to 300 µL paraformaldehyde (4%), fixed at room temperature for 5 min, discarded the paraformaldehyde, 300 µL immunostaining permeable solution Triton X-100 (Biyuntian Biotechnology Co., Ltd., P0096) was added, permeable treatment at room temperature for 5 min. 500 µL PBS(pH=7.4) was used to wash the permeable pollen cells for twice.
0.5 mL of JC-1 staining solution (Biyuntian Biotechnology Co., Ltd., C2006) was added to the precipitated pollen, thoroughly mixed, incubated at 37℃ for 30 min without light. Fluorescent staining solution was removed and 500 µL JC-1 staining buffer (1X) was added to wash the stained pollen for twice, then 1 mL JC-1 staining buffer (1X) was added to resuspend the fluorescent loaded pollen. Fluorescence was detected by flow cytometry (BD-FACSAriaSORP, Becton-Dickinson, Franklin Lakes, NJ, USA). The negative control was the pollen without fluorescence loading, and the red-green fluorescence ratio was taken as the result. Each treatment repeated for 3 times.
Determination of pollen caspase-like protease activity
Caspase-3-like and caspase-9-like protease activities were detected using the kit provided by Biyuntian Biotechnology Co., Ltd (C1116, C1158). 0.03g pollen was homogenized with 300 μL lysate at 4℃, then cracked the homogenate in ice bath for 5min, and centrifuged at 12000 rmp at 4℃ for 20 min. Added 50 µL supernatant extract into 40 µL detection buffer solution, and 10 µL caspase-3-like protease reaction substrate Ac-DEVD-pNA (2mM) or caspase-9-like protease reaction substrate Ac-LEHD-pNA (2mM) was added to initiate the reaction, incubated in the dark at 37℃ for 2h. The absorbance was measured at the wavelength of 405 nm, the lysate as the control. Each treatment was repeated 3 times, and the average of the results was taken.
Determination of pollen cells apoptosis rate
Apoptosis rate was detected by Annexin V-FITC and PI double staining. 0.01 g pollen was fixed and transparent treated as mitochondrial membrane potential determination, then added 200 µL cell staining buffer and 10 µL Annexin V-FITC staining solution, incubated in darkness at 4℃ for 30min, after Annexin V-FITC staining, 15 µL PI was added in the dark for staining (15min). Removed the staining solution and 500 µL PBS（pH=7.4）was added to wash the stained pollen for twice, 600 µL PBS（pH=7.4）was added to resuspend the fluorescent loaded pollen. Pollen suspensions were used as control without any staining and separately stained by Annexin V-FITC or PI, fluorescence value was determined by flow cytometry (BD-FACSAriaSORP, Becton-Dickinson, Franklin Lakes, NJ,USA), and each treatment was repeated for 3 times.
qRT‑ PCR analysis of pollen PCD-related genes
Approximately 0.05 g pollen were ground into powder by LN. Total RNA was extracted using the Plant RNA Rapid Extraction Kit (RN38-EasySpin Plus) provided by Beijing Aidlab Biotechnologies Co., Ltd., and the procedure are detailed in the instructions. The cDNA was synthesized using the Rever Tra Ace® qPCR RT Master Mix with gDNA Remover kit (TOYOBO, Osaka, Japan), and the procedure according to the manufacturer’s instructions. The primers were designed using IDT online software (https://sg.idtdna.com/Primerquest/Home/Index) and synthesized by Beijing Ruiboxingke Biological Technology Co., Ltd (Tab. 1).
Real-time quantitative PCR according to the instructions of SYBR Premix EX Taq (Takara, Otsu, Japan) on the MiniOpticon™ Real-time PCR Detection System (Bio-Rad®, Hercules,CA). The amplification protocols for qRT-PCR included an initial denaturing step (95°C for 30 s), followed by 40 cycles of 95°C for 10 s, Tm(°C) for 15 s, and 72°C for 15 s, 65°C for 5 s and 95 °C for 5 s (Wan et al., 2019). The reaction system was 20 µL, including 1 µL cDNA, 2 µL forward primer (10 µM), 2 µL reverse primer (10 µM), 5 µL ddH2O, and 10 µL SYBR® Premix Ex Taq II (Tli RNaseH Plus) (2X). The expression level of the target gene was calculated by the 2-∆∆Cq method, and each treatment was repeated 3 times.
Addition of NO exogenous regulator
Different concentrations of NO carrier SNP (228710-5G, Sigma Chemical Co., St Louis, MO, USA) or NO scavenger c-PTIO (C221-10MG, Sigma Chemical Co., St Louis, MO, USA) solutions were added to the LN preserved pollen without thaw treatment at a ratio of 1:100 (w/v), mixed and incubated at 37 °C for 10 min in the dark. Centrifuged at 2000 rpm for 30 s to remove the regulator solution, then PBS（pH=7.4）was added to wash the pollen for 3 times. The precipitated pollen was used for the determination of various indexes, and 3 replicates for each sample.
Flow data was analyzed with FlowJo software. SPSS 17.0 (Version 17.0 SPSS Inc., Chicago, IL, USA) was used for one-way ANOVA analysis and correlation analysis. Microsoft Excel 2013 software (Microsoft Corp., Richmond, CA, USA) and Origin 2019b (Origin Lab, Northampton, Massachusetts, USA) for chart and table drawing.