The Mechanism Of Nitric Oxide Participated In Regulating Cell Death During Ageing Of Elm(Ulmus Pumila L.)Seeds

Seed aging is a serious problem in germplasm conservation.Exploring the molecular mechanism of seed aging and optimizing seed storage methods are major challenges facing scientific research.Studies have shown that Nitric Oxide(NO),an important signaling molecule,plays important roles in plants alleviating stress and delaying senescence,but whether it is involved in regulation of seed aging has not been reported.In this study,Ulmus pumila L.seeds were used as experimental materials to study the mechanism of NO regulating seed aging.The main research contents and results are as follows:The results of seed vigor test of elm seeds pretreated with SNP and c-PTIO showed that NO could delay the loss of seed vigor during the aging of elm seeds.TTC staining results showed that NO could delay cell death during seed aging.The results of DNA laddering and Caspase-3 enzyme activity showed that NO could delay the PCD process in the aging of elm seeds.The results of NO specific fluorescence probe staining showed that NO increased first and then decreased during seed aging.Nitrite reductase(NR)and nitric oxide synthase(NOS)related reagents treatments showed that both NR and NOS pathways were involved in the regulation of seed aging.Further staining with NO specific fluorescent probe showed that NR and NOS could delay seed aging by regulating NO content.Overexpression of UpNOA1 gene in Arabidopsis thaliana showed that overexpression of UpNOA1 could delay seed aging.These results suggest that NO produced by NR and NOS pathway can delay cell death during aging of elm seeds.In order to study the mechanism of NO delaying cell death of ageing seeds,we examined the metabolome of seeds before and after aging and treated with NO donors or inhibitors.By comparing the metabolites of six treatments,we found 550 potential marker metabolites were significantly regulated by aging,of which 244 metabolites increased significantly and 306 metabolites decreased significantly after aging.371 metabolites were significantly affected by NO,of which 155 were up-regulated and 216 were down-regulated.163 metabolites regulated by aging can be regulated by NO,indicating that NO may be involved in the regulation of seed aging.KEGG functional analysis of 163 metabolites showed that glutathione metabolism,carbonhydrate metabolism and methionine metabolism could be affected by NO during seed aging.The results of qRT-PCR showed that NO could increase the expression of glutathione metabolism and methionine metabolism-related genes,but had no significant effect on the expression of carbonhydrate metabolism pathway-related genes.It is speculated that NO may regulate carbonhydrate metabolism through post-translational modification.Protein S-nitrosylation modification is an important post-translational modification mediated by NO.In order to study whether S-nitrosylation modification play roles in aging of elm seeds,we used Saville-Griess method to detect the content of SNO groups,and Biotin-Switch method to detect the S-nitrosylated proteins in aging seeds.The results showed that the change of SNO groups and S-nitrosylated proteins were consistent with the change of NO content in seeds during aging.These results suggest that the S-nitrosylation modification induced by NO may be involved in the regulation of seed aging.Eighty-two S-nitrosylated proteins were detected by S-nitrosylated proteome analysis before and after aging.Further analysis revealed that 11 carbonhydrate metabolism related proteins were modified by S-nitrosylation.It was found that S-nitrosylation modification could regulat carbonhydrate metabolism through activating the activities of pyrophosphate fructose-6-phosphate-1-phosphate transferase and phosphoglucose mutase in glycolysis pathway,inhibiting the activity of 6-gluconate dehydrogenase in pentose phosphate pathway.At the same time,we also found that glyceraldehyde 3-phosphate dehydrogenase(GAPDH),which plays an important role in animal cell death,also undergoes S-nitrosylation modification.In order to study whether GAPDH is involved in the regulation of cell death during the aging of elm seeds,we cloned UpGAPDH gene and mutated three Cys of UpGAPDH by Overlap method.Four UpGAPDH proteins were expressed and purified in prokaryotic.Biotin-Switch method was used to detect the S-nitrosylation modification degree of four proteins.It was found that UpGAPDH had only one S-nitrosylation modification site,C154.The activity of GAPDH was determined by spectrophotometry.It was found that S-nitrosylation modification could inhibit the activity of GAPDH.Overexpression of UpGAPDH gene on Arabidopsis thaliana showed that S-nitrosylation modification of UpGAPDH could accelerate seed aging.Subcellular localization of UpGAPDH showed that under normal conditions,UpGAPDH was distributed in the cytoplasm.But after S-nitrosylation treatment,UpGAPDH was aggregated.Some studies have shown that GAPDH aggregation may be involved in cell death regulation.It is speculated that S-nitrosylation modification induced UpGAPDH protein aggregation may be involved in the regulation of cell death during aging of elm seeds.In conclusion,a model of NO delaying cell death in elm seed aging through transcriptional regulation and post-translational modification was established.This study will lay a foundation for further exploring the molecular mechanism of NO regulating plant cell death.