Pollen collection and cryopreservation
Pollen of P. lactiflora 'Fen Yu Nu' was collected at the International Peony Garden in Luoyang, Henan province, on April 29, 2019. When the dry and humidity of air was appropriate, selected the bud that is about to open, gently picked the anther and brought it back under the 4℃. Then placed the anthers at room temperature (23±2℃) for 24 h to completely dispersed the pollen. The pollen was collected with an 80 mm aperture sieve (Ren et al., 2019b).
Divide the collected pollen (0.1 g/ package) into tin foil, placed it in a 2 mL cryopreserved tube, and then directly put it into LN for storage. When needed, the pollen was thawed by running water (Thaw) or thawing at room temperature (LN).
Pollen moisture content and viability determination
Pollen moisture content was determined by 105℃ constant temperature drying method (Ren et al., 2019b).
Pollen germination rate was detected by hanging drop culture method. The medium was 10% sucrose and 0.1% boric acid. The culture condition was under 25℃ constant temperature for 4 h. Pollen tube length greater than twice of the pollen grains diameter was the criterion for pollen germination. Four replicates every 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 was taken (Ren et al., 2019b).
Pollen NO content determination
NO content was determined by DAF-FM DA fluorescence staining. The pollen (0.01 g) was added with 200 μL 100 μmol/L DAF-FM DA fluorescent probe (Biyuntian Biotechnology Co., Ltd., S0019) in the dark state and thoroughly mixed. Then incubated at 37℃ for 30 min without light, centrifuged at 2000 rmp for 20 s, then discarded the dye. Washed the fluorescent loading pollen twice with 500 μL PBS (pH= 7.4), 1 mL PBS (pH= 7.4) resuspended the precipitation pollen. Used flow cytometer (FACSCalibur, Becton-Dickinson, Franklin Lakes, NJ, USA) to measure the fluorescence values at FITC channels, the actual fluorescence measurement value as the relative content of NO, and each treatment repeated 3 times.
Pollen ROS content determination
ROS content determined by fluorescence staining with DCFH-DA (Xu, 2014). A weight of 0.01 g pollen added with 200 μL 100 μmol/L DCFH-DA fluorescent probe (Sigma Chemical Co., St Louis, MO, USA, D6883-50MG) without light and thoroughly mixed. The pollen was incubated at 37℃ for 30 min in the dark, centrifuged at 2000 rmp for 20 s, and discarded the dye solution. Washed the pollen twice with 500 μL PBS (pH=7.4), then 1mL PBS (pH=7.4) resuspended the fluorescent loading pollen. Fluorescence was measured at FITC channel by flow cytometry (FACSCalibur, Becton-Dickinson, Franklin Lakes, NJ, USA), taken the actual fluorescence measurement value as the NO content, and every treatment repeated 3 times.
Pollen NOS-like activity determination
NOS-like activity was determined by a kit (S0025) provided by Biyuntian Biotechnology Co., Ltd. Pollen (0.01 g) was added to 100 µL NOS detection buffer, and then added 79.6 µL Milli-Q water, 10 µL arginine solution, 10 µL NADPH solution (0.1 mM) and 0.4 µL DAF-FM DA solution (5 mM), then incubated at 37℃ for 30 min without light. Used the flow cytometry (FACSCalibur, Becton-Dickinson, Franklin Lakes, NJ, USA) detected the fluorescence value at the FITC channel, taken the actual fluorescence measurement value as the NOS-like activity, and each treatment repeated 3 times.
Pollen NDAPH oxidase activity determination
NDAPH enzyme activity was determined according to the NADP+/NADPH detection kit (S0179) provided by Biyuntian Biotechnology Co., Ltd. Ground the pollen (0.01 g) to homogenate in ice bath with 400 µL NADP+/NADPH extract, and centrifuged at 12,000 rpm at 4°C for 10 min. Added with 200 µL G6PDH working solution into 100 µL extraction solution, incubate at 37°C for 10 min without light. Then add 10 µL chromogen solution, mixed well and incubated for 60 min under the constant temperature of 37°C without light. Measured the absorbance at 450 nm with an ultraviolet spectrophotometer, each treatment repeated three times, and the average of the results was taken.
Pollen antioxidants determination
SOD determined by nitrogen blue tetrazole (NBT) reduction method (Li et al., 2000). Homogenated 0.05 g pollen with 1 mL of 0.05 mol/L phosphoric acid buffer in ice bath, and 4℃ centrifuged at 10,000 rpm for 15 min. Added 1.5 mL phosphoric acid buffer (0.05 mol/L), 1.5 mL methionine solution (130 mmol/L), 300 µL NBT solution (750 mol/L), 300 µL EDTA-Na2 (100 mol/L) and 300 µL riboflavin solution (20 µmol/L) into 300 µL extract solution without light. Mixed and subjected to 4000 Lx ray reaction for 15 min, and the reaction was terminated in darkness. Determined the absorbance value by spectrophotometer at the wavelength of 560 nm, every treatment repeated three times, and the average of the results was taken.
CAT was measured according to the Prochakova’s method and with slight modifications (Prochazkova et al., 2001). Pollen (0.05 g) was homogenated with 1 mL 0.05mol/L phosphate buffer in ice bath, and centrifuged at 10,000 rpm at 4℃ for 15 min. Added 1.5 mL phosphoric acid buffer (pH= 7.0) and 1.0 mL distilled water into 200 µL extract solution. Mixed and added 300 µL hydrogen peroxide solution (0.1 mol/L) to initiate the reaction. The absorbance value was measured at the wavelength of 240nm by the spectrophotometer. Each treatment repeated three times, and the mean value was taken.
GR activity was determined according to the glutathione reductase assay kit (S0055) provided by Biyuntian Biotechnology Co., Ltd. and with slight modifications. A liquid of 1 mL phosphate buffer solution (0.01 mol/L) was taken to ground 0.03 g pollen into homogenate in ice bath, and then centrifuged at 4℃ at 10,000 rpm for 15min. The supernatant was collected for the determination of GR activity, every treatment repeated three times.
APX activity was determined using the method of Nakano and Asada with slight modification (Nakano and Asada, 1981). A liquid of 1 mL APX enzyme extraction reagent (50 mmol/L PBS+2 mmol/L AsA+5 mmol/L EDTA) was taken to ground 0.03 g pollen into homogenate in ice bath, and centrifuged at 10,000 rpm at 4℃ for 20 min. In 50 µL supernatant extract, added 2 mL PBS (50 mmol/L, pH=7.0), 500 µL AsA solution (5 mmol/L) and 500 µL EDTA-Na2 (1 mmol/L), then added 50 µL H2O2 (30%) initiated the reaction. Measured the absorbance at 290 nm with a spectrophotometer, each treatment repeated three times, and the results were averaged.
AsA content was determined by Kampfenkel’s method and with some modifications (Kampfenkel et al., 1995). Ground 0.03 g pollen with 1.5 mL trichloroacetic acid (10%) into homogenate in ice bath, and centrifuged at 10,000 rpm at 4℃ for 10 min. Added 200 µL sodium dihydrogen phosphate solution (150 mmol/L) and 200 µL ddH2O into 200 µL supernatant extract, mixed at least 30 s. Then added 10% trichloroacetic acid solution, 44% phosphoric acid solution and 4% 2, 2-dipyridine solution in turn, 400 µL each. Mixed and initiated reaction by added 200 µL FeCl3 (3%), then incubated at 37℃ for 1 h. Measured the absorbance value at 525 nm by spectrophotometer, each treatment repeated 3 times, and the mean value of the results was taken.
GSH content was determined by 2-nitrobenzoic acid (DTNB) reduction method (Griffith, 1980). A liquid of 1.5 mL trichloroacetic acid (10%) was taken to ground 0.03 g pollen into homogenate in ice bath, and centrifuged at 10,000 rpm at 4℃ for 10 min. Added 0.5 mL ddH2O, 1 mL phosphate buffer (1 mmol/L, pH= 7.7) and 0.5 mL DTNB (4 mmol/L) into 0.5 mL supernatant extract, mixed well and reacted for 10 min at 25℃. Measured the absorbance at 412 nm by spectrophotometer, each treatment repeated three times, and the average of the results was taken
qRT‑ PCR analysis of pollen ROS and NO related genes
Pollen total RNA extracted by the Plant RNA Rapid Extraction Kit (RN38-EasySpin Plus) provided by Beijing Aidlab Biotechnologies Co., Ltd.; cDNA was synthesized by the Rever Tra Ace® qPCR RT Master Mix with gDNA Remover kit (TOYOBO, Osaka, Japan); primers were designed by the IDT online software (https://sg.idtdna.com/Primerquest/Home/Index) and synthesized by Beijing Ruiboxingke Biological Technology Co., Ltd (Tab. S1 and Tab. S2).
According to the instructions of SYBR Premix EX Taq (Takara, Otsu, Japan) on the MiniOpticon™ real-time PCR detection system (Bio-Rad®, Hercules, CA) performed the real-time quantitative PCR. 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 µmol/L), 2 µL reverse primer (10 µmol/L), 5 µL ddH2O, and 10 µL SYBR® Premix Ex Taq II (Tli RNase 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 ROS and NO exogenous regulator
The pollen without running water treatment after preserved in LN was respectively added with 0.8 mmol/L NO carrier SNP (228710-5G, Sigma Chemical Co., St Louis, MO, USA), 0.8 mmol/L NO scavenger c-PTIO (C221-10MG, Sigma Chemical Co., St Louis, MO, USA), 200 mmol/L ROS scavenger AsA (A7506-25G, Sigma Chemical Co., St Louis, MO, USA) or 0.16 mmol/L ROS scavenger GSH (G6013-5G, Sigma Chemical Co., St Louis, MO, USA) solution at a ratio of 1:100 (w/v), and was thoroughly mixed. Incubated for 10 min in the dark at 37°C, then washed the pollen incubated with the regulator three times with phosphate buffer of 0.01 mol/L (PBS, pH= 7.4). After centrifugated 2000 rpm for 30 s, the precipitated pollen was used for the determination of various indexes, three repeats were set for each treatment.
Statistical analysis
Flow data was processed and 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) was used to draw charts and tables.