Effects of Programmed Cell Death Induced by NO on Pollen Viability After Cryopreservation

Low activity level of biomaterials after cryopreservation is a bottleneck problem that limited the application of this technology. At present, the mechanism of viability decline after cryopreservation is not fully understood. In this study, the effects of nitric oxide (NO) on programmed cell death (PCD) and its relationship with viability were investigated, using Paeonia lactiora 'Fen Yu Nu' pollen with signicantly decreased viability after cryopreservation. The results showed that: the activity of caspase-3-like and caspase-9-like protease and the apoptosis rate of pollen cells were signicantly increased, the expression level of promoting PCD (pro-PCD) gene was up-regulated, while the expression level of inhibiting PCD (anti-PCD) gene was down-regulated after preserved in liquid nitrogen (LN); the NO content in pollen cells increased signicantly after LN preserved. The results of correlation analysis showed that NO was signicantly correlated with pollen viability and all indicators of PCD. The addition of NO carrier SNP after LN storage reduced pollen viability, increased endogenous NO content, decreased mitochondrial membrane potential level, activated caspase-3-like and caspase-9-like protease in pollen cells, and increased cell apoptosis rate, and the expression levels of pro-PCD genes PDCD2 and ATG8CL were signicantly up-regulated, while the expression levels of anti-PCD genes DAD1, BI-1 and LSD1 were signicantly down-regulated; and the addition of NO scavenger c-PTIO produced the opposite effect of SNP. It was suggested that NO induced the PCD during the cryopreservation of pollen, which was one of the reasons for the signicant decrease of pollen viability after cryopreservation.


Introduction
Cryopreservation is one of the important biotechniques for long-term, effective and safe preservation of plant germplasm resources, which can safely store biomaterials in LN (-196℃), and certain methods can be used to make them return to room temperature for normal growth and development when needed (Baust, 2002;Engelmann, 2004). Pollen, as an important carrier of plant germplasm resources, is one of the main forms of plant germplasm resources conservation. However, in the process of practical application, it has been found that pollen viability after cryopreservation have variety changes, but in most cases, the viability is at a declining level (Ren et al., 2019a), and the changes is probably due to the various stresses involved in the preservation process, such as osmotic and low temperature.
Nitric oxide (NO) is a kind of short-life free radicals of gaseous, which is produced in various stress response, and plays a role in a variety of physiological and biochemical processes, which mainly as a messenger and effector molecule (Delledonne, 2005 (He et al., 2017); Ye found the intracellular NO accumulation in Arabidopsis thaliana under cadmium ion stress (Ye et al., 2013). Similarly, it was also found that the NO content changed signi cantly after cryopreservation of plants. In the cryopreservation of Dendrobium nobile protocorms by vitri cation, the accumulation of NO content in cells was accelerated by LN preservation (Jiang et al., 2019b).
Programmed cell death (PCD) is an active and orderly cell death controlled by genes (Kerr et al., 1972).
Current studies have shown that, NO as a direct inducer of PCD response, is a major factor that determines whether the PCD occurrence or not (Mira et al., 2016), and it has been found that NO has a dual effect on the occurrence of stress-induced PCD in plants (Wang et al., 2010). Cadmium induced PCD in suspension cells of Nicotiana tobacillus (Ma et al., 2010) and aluminum induced PCD in root tip of Arachis hypogaea (He et al., 2017), NO is negatively involved in regulating the occurrence of PCD. On the contrary, NO plays a positive regulatory role in GA induced PCD in aleurone layer of barley (Beligni et al., 2002) and PCD of plant hypersensitivity induced by proenzyme (Zaninotto et al., 2006). Similarly, the double role of NO in PCD was also found in plant cryopreservation. Jiang found that in the vitri cation cryopreservation of Dendrobium nobile protocorm, NO played a dual role in the occurrence of PCD, the content of NO increased signi cantly in the loading process, and it was negatively involved in the regulation of PCD; but in the pre-culture stage, although the content of NO increased signi cantly, it was positively involved in regulating the occurrence of PCD (Jiang,2019).
As an important method to preserve plant germplasm resources, existing studies have shown that there is a certain relationship between the changes of pollen viability and the occurrence of PCD after LN storage.
In six P. suffruticosa cultivars, PCD was found to be involved in the changes of pollen viability after cryopreservation (Ren et al., 2020a). In the study of cryopreservation of P. lacti ora pollen, it was found that the exogenous additives catalase, pyruvate dehydrogenase and heat shock protein-70 had certain inhibitory effects on the activity of caspase-3-like protease and the apoptosis rate in PCD (Jiang et al., 2019a;Ren et al., 2019b). Further studies on cryopreservation of P. lacti ora 'Zi Feng Chao Yang' pollen showed that, ROS-induced PCD was one of the reasons for the signi cant decrease in pollen viability after LN preserved (Ren et al., 2020b). But, the role of NO in the occurrence of PCD in pollen cryopreservation has not been studied so far. Therefore, in this study, the P. lacti ora lacti ora 'Fen Yu Nu' pollen with signi cantly decreased viability after cryopreservation as material. Investigated the changes of NO content, the occurrence of PCD, and the effects of exogenous NO regulator on pollen viability and PCD after cryopreservation, to explore the relationship between NO and PCD in pollen cryopreservation and the role of that in the decline of viability after cryopreservation.

Materials And Methods
Pollen collection and cryopreservation The P. lacti ora lacti ora '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 ower 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 elds 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 uorescence 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 ow cytometer (FACSCalibur, Becton-Dickinson, Franklin Lakes, NJ, USA), the without adding uorescent dye as control. The relative content of NO was taken as the actual uorescence measurement value, and each treatment was repeated 3 times.
Determination of pollen mitochondrial membrane potential Pollen mitochondrial membrane potential was detected by JC-1 uorescence staining. 0.01 g pollen was added to 300 µL paraformaldehyde (4%), xed 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 uorescent loaded pollen. Fluorescence was detected by ow cytometry (BD-FACSAriaSORP, Becton-Dickinson, Franklin Lakes, NJ, USA). The negative control was the pollen without uorescence loading, and the red-green uorescence 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 xed 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 uorescent loaded pollen. Pollen suspensions were used as control without any staining and separately stained by Annexin V-FITC or PI, uorescence value was determined by ow 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 ampli cation protocols for qRT-PCR included an initial denaturing step (95°C for 30 s), followed by 40 cycles of 95°C for 10 s,

Moisture content and viability changes of pollen after cryopreservation
The natural moisture content of fresh pollen was 8.10%, and the viability of it was 22.73%. The cryopreservation process signi cantly reduced the pollen viability (P< 0.05), especially the viability of pollen without water thawing treatment after LN storage, which was only 16.42, signi cantly lower than that of the fresh pollen (Tab. 2).
The occurrence of PCD in pollen during cryopreservation As a uorescent indicator of mitochondrial membrane potential, JC-1 is often used to indicate the change level of membrane potential by its presence form in mitochondrial matrix. The number of pollen JC-1 transformed from polymer to monomer after LN preservation was signi cantly increased ( Fig. 1-A2, A3), thus the ratio of monomer to polymer was signi cantly decreased (P< 0.05), respectively decreased by 55.76% and 54.85% compared with the control (Fig. 1-B). The results showed that cryopreservation signi cantly reduced the mitochondrial membrane potential level of P. lacti ora 'Fen Yu Nu' pollen.
Cryopreservation had a signi cant effect on the activity of pollen caspase-like protease. The activity of caspase-3-like protease in pollen without and with thawing treatment after LN storaged was signi cantly higher than that of control (P< 0.05), which was increased by 12.35% and 5.67%, respectively ( Fig. 2-A), moreover, the activity of caspase-9-like protease also increased signi cantly after LN storaged (P< 0.05), which respectively increased by 26.89% and 12.30% compared with the fresh pollen ( Fig. 2-B).
Cryopreservation signi cantly affected the apoptosis rate of pollen cells (Fig. 3). The apoptotic rate of pollen cells increased signi cantly after LN preservation ( Fig. 3-B, C, D), especially without thawing treatment after preserved in LN (LN), the apoptosis rate of pollen cells was relatively high (Fig. 3-B, D).
The expression levels of PCD related genes were signi cantly changed before and after cryopreservation. The expression of pro-PCD genes PDCD4, PDCD2, ATG8CL and ACD11 were signi cantly up-regulated in the pollen without thawing treatment after preserved in LN, while the expression of pro-PCD genes SBT3, RTNLB2, AMC9, BAG6 was signi cantly up-regulated in pollen without and with thawing treatments after LN preservation (Fig. 4), the expression of anti-PCD genes DADI, BI-1 and LSD1 was signi cantly downregulated in the pollen after preserved in LN (Fig. 4).
The changes of NO content in pollen during cryopreservation Cryopreservation has a signi cant effect on NO content in pollen (Fig. 5). The NO content of pollen without and with thawing treatment after LN storage were all signi cantly higher than that of the control level (P < 0.05), increased by 94.14% and 92.71%, respectively( Fig. 5-D).

Correlation analysis of pollen viability, PCD indexes and NO content in cryopreservation
There was a certain correlation among NO content, PCD indexes and viability of pollen before and after cryopreservation (Fig. 6). Pollen viability was negatively correlated with caspase-3-like protease activity, caspase-9-like protease activity and apoptosis rate (P< 0.01), and positively correlated with the mitochondrial membrane potential (P < 0.05).
The content of NO was positively correlated with the apoptosis rate, the activity of caspase-3-like and caspase-9-like protease (P< 0.01), and negatively correlated with the mitochondrial membrane potential (P < 0.01), while, there was a signi cantly negative correlation between NO and viability in pollen cryopreservation (P < 0.05).

Effects of regulating NO on pollen viability after cryopreservation
The addition of different concentrations of exogenous NO regulators after LN storage had outstanding effects on pollen viability (Fig. 7). Added 0.6 mM and 0.8 mM NO carrier SNP signi cantly inhibited pollen viability after LN storaged, especially 0.6 mM SNP had a remarkable inhibitory effect on pollen viability ( Fig. 7-A), while, No scavenger c-PTIO at 0.4 mM, 0.6 mM and 0.8 mM signi cantly promoted pollen viability after LN storaged, and the optimization effect of 0.8 mM c-PTIO on pollen viability was the most obvious ( Fig. 7-B).
Effects of regulating NO on pollen endogenous NO content after cryopreservation The addition of exogenous NO regulators had a certain effect on the endogenous NO content of pollen after LN storage (Fig. 8). After LN storaged, added the SNP of NO carrier signi cantly increased the endogenous NO content, especially the SNP at 0.8mM, which increased by 31.05% compared with the control (Fig. 8-A). While, the addition of different concentrations of NO scavenger c-PTIO signi cantly reduced the endogenous NO content, and the 0.6mM c-PTIO decreased by 32.92%, which showed the most obvious inhibition effect (Fig. 8-B).
Effects of regulating NO on the occurrence of PCD of pollen after cryopreservation The regulation of NO content had an effect on mitochondrial membrane potential of pollen after cryopreservation (Fig. 9). The addition of NO carrier SNP of appropriate concentration (0.8mM) after LN storaged signi cantly reduced the ratio of JC-1 polymers to monomers, that is, accelerated the decrease of mitochondrial membrane potential level ( Fig. 9-A3, B), but the addition of c-PTIO of appropriate concentration (0.8mM) after cryopreserved did't have signi cant effect on the ratio of pollen JC-1 polymers to monomers ( Fig. 9-A2, B).
The addition of NO regulators signi cantly affected the activities of caspase-3-like and caspase-9-like proteases (Fig. 10). After LN preservation, the appropriate concentration (0.8mM) of SNP not only improved the activity of caspase-3-like protease, but also signi cantly enhance the activity of caspase-9like protease. However, the addition of appropriate concentration (0.8mM) of c-PTIO signi cantly inhibited the activity of caspase-3-like and caspase-9-like proteases ( Fig. 10 -A, B).
The apoptosis rate of pollen cells was signi cantly affected by the regulation of NO content (Fig. 11). After preservation of LN, the addition of NO carrier SNP at an appropriate concentration (0.8mM) increased the apoptosis rate of pollen cells ( Fig. 11-C, D), while the NO scavenging agent c-PTIO at 0.8mM signi cantly decreased the apoptosis rate of pollen cells (Fig. 11-B, D).
Exogenous NO regulator also signi cantly affected the expression of some PCD-related genes (Fig. 12). The addition of NO carrier SNP signi cantly up-regulated the expression levels of pro-PCD genes PDCD2, ATG8CL, ACD11 and AMC9, down-regulated the expression levels of anti-PCD genes DADI, BI-1, HXK1, LSD1 and pro-PCD genes PDCD4, BAG6, and RTNLB2 after preserved in LN. The addition of NO scavenger C-PTIO signi cantly down-regulated the expression levels of PDCD4, PDCD2, SBT3, ATG8CL, ACD11, BAG6 and RTNLB2 of pro-PCD genes, and signi cantly up-regulated the expression of DADI, BI-1 and LSD1, which inhibited the occurrence of PCD.

Discussion
In this study, the viability of P. lacti ora 'Fen Yu Nu' pollen decreased signi cantly after cryopreservation whether they were treated with or without water thawing. This is consistent with the results of a large number of studies. Xu found that the viability of 26 species/cultivars of ornamental plant pollen PCD is a gene-controlled, active and orderly process of cell death, as a normal physiological response of an organism in the environment and its own metabolism, which can be induced under a variety of stresses (Kerr et al., 1972). Studies have shown that low temperature and osmotic stress involved in cryopreservation induced PCD in plant materials. Jiang in the cryopreservation of Dendrobium nobile protocorms and Ren in the Paeonia suffruticosa pollen cryopreservation found that the activity of caspase-3-like protease and cell apoptosis rate were increased to a certain extent, and some PCD regulatory genes were signi cantly changed, indicated that cryopreservation induced PCD (Jiang et al., 2019b;Ren et al., 2020a). In this study, after cryoppreservation, pollen mitochondrial membrane potential signi cantly decreased, the activities of caspase-3-like and caspase-9-like protease and the apoptosis rate of pollen cells signi cantly increased, the expression levels of pro-PCD genes RTNLB2, AMC9 and BAG6 were signi cantly up-regulated, and the expression of anti-PCD genes DADI, BI-1 and LSD1 were signi cantly down-regulated. The results showed that PCD also occurred in the cryopreservation of pollen of P. lacti ora 'Fen Yu Nu'.
Studies have shown that the occurrence of PCD affected the change of viability of biomaterials after cryopreservation. Zhang found that the changes of PCD-related genes in vitri cation cryopreservation were related to the changes of cell viability in the cryopreservation of embryogenic callus of Agapanthus praecox (Zhang et al., 2015); and in the cryopreservation of Dendrobium nobile protocorms, Jiang found that the activity of caspase-3-like protease increased, which indicated PCD occurred, and that some pro-PCD genes were signi cantly up-regulated during the viability signi cantly decreased periods (Jiang et al., 2019b); Ren found that the occurrence of PCD was related to the changes of pollen viability in the cryopreservation of 6 cultivars of Paeonia suffruticosa (Ren et al., 2020a). This study is consistent with these ndings. During the cryopreservation, pollen viability was negatively correlated with caspase-3-like protease activity, caspase-9-like protease activity and apoptosis rate, and positively correlated with the mitochondrial membrane potential, all these indicators are involved in the occurrence of PCD. The results indicated that PCD contributed to the decrease of pollen viability after cryopreservation in P. lacti ora 'Fen Yu Nu'.
Nitric oxide (NO) is a short-lived gaseous free radicals. In this study, the NO content of P. lacti ora 'Fen Yu Nu' pollen increased signi cantly after LN storage. These results are consistent with the results of cryopreservation of Dendrobium nobile protocorms by vitri cation (Jiang et al., 2019b). This suggested that the increase of endogenous NO content after cryopreservation may be a common phenomenon in preserved of plant materials.
The binding of NO with cysteine mercaptan of protein to form s-nitrosolation is an important posttranslational modi cation, which is considered to be an important pathway for NO to regulated cellular redox signaling. In recent years, a large number of proteins in plants have been identi ed as the targets of s-nitrosoylation, especially in the process of PCD, which directly affected the occurrence of PCD mainly by regulating the activity of cell death related enzymes or by reconstructing the conformation of several functional proteins, either positively or negatively involved in the PCD (Huang et  Correlation analysis results of this study showed that the NO content of pollen before and after LN storaged was positively correlated with the activities of caspase-3-like protease, caspase-9-like protease and cell apoptosis rate, while negatively correlated with mitochondrial membrane potential; and signi cantly negative correlation with pollen viability. In addition, in this study, added appropriate concentration of NO-speci c scavenger c-PTIO decreased the content of endogenous NO, inhibited the PCD indexes of pollen, including the activities of caspase-3-like protease, caspase-9-like protease and the apoptosis rate, and improved the viability of pollen; while the addition of NO carrier SNP signi cantly reduced the mitochondrial membrane, promoted the activation of caspase-3-like and caspase-9-like protease as well as the generation of apoptosis rate, and signi cantly reduced the pollen viability. This is consistent with the results of Jiang in the vitri cation cryopreservation of Dendrobium nobile protocorms, the addition of NO-speci c scavenger (c-PTIO) in the pre-culture process can signi cantly reduced the activity of caspase-3-like protease; while the addition of NO carrier SNP in the pre-culture process  (Makishima et al., 2000). In this study, the appropriate concentration of NO speci c scavenger c-PTIO added after preserved in LN signi cantly down-regulated the expression of pro-PCD gene PDCD2, and signi cantly up-regulated the expression of anti-PCD gene DAD1 and BI-1; while the addition of NO carrier SNP signi cantly up-regulated the expression of pro-PCD gene PDCD2, and decreased the expression of anti-PCD gene DAD1 and BI-1. Among them, BI-1 is the related regulation gene of endoplasmic reticulum pathway PCD, and PDCD2 is the key regulation gene of mitochondrial pathway PCD. These results indicated that NO was not only involved in the regulation of endoplasmic reticulum pathway PCD, but also involved in the mitochondrial pathway PCD. However, the speci c mechanism of its effect is still not fully answered in this study and further study is needed.
In this study, the NO scavenger c-PTIO signi cantly up-regulated the expression of LSD1 gene, while the NO carrier SNP signi cantly down-regulated its expression. LSD1 protein encodes a negative regulatory gene for plant PCD, which contains three zinc ngers, antagonizes the accumulation of copper and zinc superoxide dismutase, and control the cell death by maintaining the homeostasis of reactive oxygen species (ROS) intermediates (Dietrich et al., 1997;Epple at al., 2003). These results suggest that NO may participated the regulation of PCD by acting on ROS during pollen cryopreservation. As in the process of root tip PCD induced by aluminum stress in Arachis hypogaea, NO regulates PCD by acting on H 2 O 2 (He et al., 2019); In the vitri cation cryopreservation of Dendrobium nobile protocorms, NO was also found to be involved in PCD by positively regulating ROS (Jiang et al., 2019b).
In addition, in this study, the NO-speci c scavenger c-PTIO signi cantly down-regulated the expression of pro-PCD gene ATG8CL; the SNP of NO carrier has the opposite effect. As a gene family involved in autophagy, ATG8CL participated the occurrence of autophagy by acting on the microtubule skeleton to mediate the formation of autophagosomes, or acting on mitochondrial membrane proteins (Broda et al., 2018). However, existing studies have shown that it also plays an important role in the process of PCD (Shibuya et al., 2011). In the cryopreservation of Dendrobium nobile protocorms by vitri cation, Jiang also found that NO regulator had a signi cant effect on the expression of ATG8CL gene (Jiang,2019). It may be that NO plays a certain role in the occurrence of PCD by participated the regulation of autophagosome formation during cryopreservation of pollen, but the speci c mechanism of effect needs to be further explored.

Conclusion
This study rst investigated the role of NO in the occurrence of PCD during cryopreservation of pollen and the relationship with pollen viability. It was found that NO-induced PCD played an important role in the decline of pollen viability after cryopreservation. The pollen viability of P. lacti ora 'Fen Yu Nu' decreased after LN preservation, and PCD occurred, accompanied by signi cant increase of intracellular NO content; the production of NO was signi cantly correlated with the occurrence of PCD and the decline of pollen viability; and regulation of NO content regulated PCD-related cellular, physiological and gene expression,   Figure 1 Changes of mitochondrial membrane potential in P. lacti ora pollen before and after cryopreservation. Notes: Values represent the mean (± SE) over 3 detections. Different letters indicate signi cant differences among different treatments (P< 0.05). A1: control (fresh pollen/CK); A2 the LN preserved pollen without thaw treatment (LN); A3 the LN preserved pollen with thaw treatment (Thaw); B: the changes of JC-1 monomer and polymer ratio of pollen.

Figure 2
Changes of caspase-like protease activity in P. lacti ora pollen before and after cryopreservation. Notes: Values represent the mean (± SE) over 3 detections. Different letters indicate signi cant differences among different treatments (P< 0.05). CK: control (fresh pollen); LN the LN preserved pollen without thaw treatment; Thaw the LN preserved pollen with thaw treatment. A: caspase-3-like protease activity; B: caspase-9-like protease activity.   Changes of NO content in P. lacti ora pollen before and after cryopreservation. Notes: Values represent the mean (± SE) over 3 detections. Different letters indicate signi cant differences among different treatments (P< 0.05). A: fresh pollen (CK); B: LN preserved pollen without thaw treatment (LN); C: the LN preserved pollen with thaw treatment (Thaw); D: the NO content of pollen; 1: the ow cytometry maps; 2: the ow spectrogram of DAF-FM DA uorescence (NO uorescent probe); 3: the comparison spectrograms.

Figure 6
Correlation analysis among NO, PCD indexes and viability of P. lacti ora pollen cryopreservation. Note: Data is the correlation coe cient among different indicators. * indicates signi cant correlation at P < 0.05, and ** indicates extremely signi cant correlation at P < 0. 01. NO: nitric oxide; MMP: mitochondrial membrane potential.    Changes of caspase-like protease in P. lacti ora pollen with and without NO regulter treatment after cryopreservation. Notes: Values represent the mean (± SE) over 3 detections. Different letters indicate