The Programmed Cell Death Signaling Molecules And Their Relationships In Paeonia Lactiflora Pollen Cryopreservation

Cryopreservation,one of the important techniques for safe,effective and long-term preservation of plant germplasm resources.However,the low survival rate and regeneration rate of some biomaterials after liquid nitrogen（LN）freezing is the bottleneck problem that restricts the large-scale application of this technology.At present,programmed cell death（PCD）has been found in the cryopreservation of animal and human tissues and cells,which is one of the reasons for the decreased survival rate of some materials after cryopreservation.But,there are relatively few studies on PCD in plant cryopreservation.Exploring the damage mechanism of plant cryopreservation from the perspective of PCD signal molecules and their roles will not only help to develop new methods to reduce the mortality after plant preservation in LN,but also enrich the theory of cryobiology.Therefore,this study studied the role of signaling molecules of reactive oxygen species（ROS）,calcium ions(Ca²⁺)and nitric oxide（NO）in the occurrence of PCD and their interaction in cryopreservation of Paeonia lactiflora‘Feng Yu Nu’pollen.The main research results are as follows:（1）The occurrence of PCD directly affected pollen viability and which was regulated by genes during cryopreservation.When PCD was inhibited,pollen germination rate was up to 30.06%（16.42%in the control）.After cryopreservation,the pollen germination rate of Paeonia lactiflora‘Feng Yu Nu’decreased,the mitochondrial membrane potential decreased,caspase-3-like and caspase-9-like protease activation,and the early apoptosis rate decreased significantly,while the late apoptosis rate and the total apoptosis level significantly increased.Correlation analysis showed that these indexes were significantly related to pollen viability.The addition of exogenous PCD regulators significantly affected pollen viability.The germination rate of pollen with the mitochondrial membrane potential permeability conversion hole inhibitor cyclosporin A treatment was 83.06%higher than that of the control.Compared with the control,the specific inhibitor of caspase-3-like protease and the broad-spectrum inhibitor of caspase-like protease increased the germination rate by 60.69%and 76.72%,respectively.Mitochondrial membrane potential loss agent and caspase-like protease activator significantly reduced pollen germination rate by 30.75%and 38.56%compared with the control,respectively.The expression levels of pro-PCD genes SBT3,RTNLB2,AMC9,BAG6,PDCD4,PDCD2,ATG8CL and ACD11 were up-regulated,while the expression levels of anti-PCD genes DADI,BI-1 and LSD1 were down-regulated.（2）The content of ROS in pollen after preserved in LN was significantly increased by 47.38%compared with the control,and was significantly correlated with PCD,particularly,H2O2 was highly correlated with PCD and viability.The content of endogenous ROS decreased with appropriate concentration of ROS scavengers（CAT,ASA,GSH）,which were 29.97%,33.97%,32.15%of the control,and the germination rate of pollen increased by 30.80%,50.06%,33.52%.The caspase-3-like protease activity decreased by 5.47%,26.61%and 11.87%,and the caspase-9-like protease activity decreased by10.44%,25.54%and 14.32%,respectively;the late apoptosis rate was reduced by 22.08%,26.82%and19.00%,and the total apoptosis rate was reduced by 16.08%,20.96%and 18.29%,respectively.At the same time,the expression of pro-PCD genes PDCD4,BAG6 and RTNLB2 were down-regulated.These results indicate that signal molecule ROS is involved in PCD in pollen cryopreservation.（3）After cryopreservation,the intracellular Ca²⁺content of pollen was significantly increased,which was 84.94%higher than that of fresh pollen.Correlation analysis showed that Ca²⁺content was significantly correlated with PCD and pollen germination rate.After the addition of Ca²⁺carrier A23187in LN storaged pollen,with the increase of endogenous Ca²⁺content,the activities of caspase-3-like and caspase-9-like proteases were respectively increased by 7.78%and 9.05%,mitochondrial membrane potential level was decreased by 63.25%,late cell apoptosis rate and total cell apoptosis rate were respectively increased by 56.52%and 52.94%,the expression levels of pro-PCD genes PDCD2,PDCD4and SBT3 were significantly up-regulated,and the expression levels of anti-PCD gene BI-1 were significantly down-regulated.While,the addition of Ca²⁺chelating agent EGTA had the opposite effect,which decreased the endogenous Ca²⁺content by 6.43%,the activity of caspase-3-like and caspase-9-like proteases respectively decreased by 22.77%and 27.41%,the late apoptosis rate and total apoptosis rate respectively decreased by 25.00%and 23.53%,the expression of three pro-PCD genes was significantly down-regulated,and the expression of anti-PCD gene BI-1 was significantly up-regulated.The results showed that the signaling molecule Ca²⁺was involved in the occurrence of PCD during pollen cryopreservation.（4）The content of NO in pollen increased significantly after cryopreservation,and the correlation analysis showed that it was highly correlated with PCD.Adding suitable concentration of NO carrier SNP,pollen germination rate decreased by 16.81%,the activities of caspase-3-like protease and caspase-9-like protease were respectively increased by 12.20%and 4.9%,mitochondrial membrane potential decreased by 53.88%,late apoptosis rate and total apoptosis rate were respectively increased by 17.65%and 15.79%,the expression levels of pro-PCD genes PDCD2 and ATG8CL were significantly up-regulated,while the expression levels of anti-PCD genes DAD1,BI-1 and LSD1 were significantly down-regulated,and the viability of pollen was reduced by 16.81%after LN storage.However,adding NO scavenging agent c-PTIO had the opposite effect.Pollen germination rate was increased by 55.36%and endogenous NO content was decreased by 32.92%after LN storage;the activity of caspase-3-like protease and caspase-9-like protease respectively decreased by 12.92%and 20.99%,late apoptosis rate and total apoptosis rate respectively decreased by 25.53%and 21.05%;the expression levels of three pro-PCD genes were significantly up-regulated,and the expression levels of three anti-PCD genes were significantly down-regulated.The results showed that NO was involved in the occurrence of PCD during pollen cryopreservation.（5）There is a bidirectional relationship among ROS,Ca²⁺and NO in pollen cryopreservation.After cryopreservation,the contents of Ca²⁺,NO and the related regulatory indexes of production were significantly changed under the action of ROS regulator;with the addition of exogenous Ca²⁺regulator,the contents of ROS and NO and their related indexes changed significantly;while,ROS and Ca²⁺contents and their related indexes changed significantly under the action of NO regulator.Among them,Ca²⁺channels regulated by IP3 and Ca²⁺-ATPases may be one of the pathways through which ROS and NO regulated endogenous Ca²⁺concentration.While,the regulation of Ca²⁺and NO on ROS may be through the activated the NADPH oxidase,and inhibited enzymes and non-enzymatic antioxidant substances,especially the ASA-GSH antioxidant cycle system.NOS-like enzymes involved in NO biosynthesis may be one of the pathways by which ROS and Ca²⁺regulated intracellular NO content.（6）The addition of Ca²⁺inhibitors in cryopreservation of pollen can significantly inhibit the caspase-9-like protease involved in the initiation of PCD,while ROS inhibitors had significant inhibitory effects on caspase-3-like proteases,late apoptosis rate and pro-PCD genes involved in PCD execution.In the two-step method,added Ca²⁺inhibitors first followed by added ROS or NO inhibitors had higher inhibitory effects on the two caspase-like proteases than added inhibitors alone or sequential treatments.These results indicated that Ca²⁺acted upstream of ROS and NO during the occurrence of PCD in pollen cryopreservation,which was closely related to PCD initiation;while ROS plays an important role in the execution of PCD by affecting the expression of caspase-3-like protease and PCD-related genes;and NO plays an auxiliary role in PCD by affected ROS and Ca²⁺content.The existence of PCD signal regulation network is worthy of further study.Based on cell morphological characteristics,biochemical level and molecular level,this study determined the occurrence of PCD in pollen cryopreservation.Signal molecules ROS,Ca²⁺and NO were closely related to the occurrence of PCD in Paeonia lactiflora‘Feng Yu Nu’pollen cryopreservation.In addition,Ca²⁺plays a more important role in PCD initiation,ROS plays a more important role in PCD execution,and NO plays a major auxiliary role.At the same time,there is a two-way interaction between the three signal molecules.