Physiological And Molecular Mechanisms Of Pre-Storage Cold Acclimation Induces Chilling Tolerance In Harvested Cucumber

Low temperature storage is one of the most effective ways to maintain quality of harvested fruit and vegetables.Cucumber is a chilling sensitive vegetable,whose fruit are susceptible to chilling injury at temperatures below 7-10℃.Chilling injury seriously affects the quality of harvested fruit and vegetables,and is an important factor leading to postharvest losses.Studies have shown that pre-storage cold acclimation（PsCA）enhanced chilling tolerance of harvested fruit and vegetables,however,the underlying mechanism largely remains unclear.In this study,the relationship between PsCA and antioxidant defense system,the proteomic changes induced by PsCA,the functions of two PsCA-induced differentially expressed proteins（CsGR-RBP3 and CsDDI1）and the roles of three important signal molecules including H₂O₂,ABA and NO in cold acclimation were investigated using harvested cucumber as experimental materials,aiming to deepen the understanding of the mechanism of cold acclimation in harvested fruit and vegetables.The main results are as follows:（1）Following PsCA at 10℃for 6-72 h,the induced chilling tolerance increased with PsCA duration.PsCA（72 h）reduced MDA and reactive oxygen species（ROS）levels,enhanced the expression and activities of five enzymatic ROS scavengers,superoxide dismutase,peroxidase,ascorbate peroxidase,catalase,phenylalanine ammonia lyase and three non-enzymatic ROS scavengers,AsA,glutathione and total phenolic contents,suggesting that PsCA maintained cell membrane integrity and reduced ROS through comprehensively activating antioxidant system,and thereby reduced chilling injury in cucumber fruit.However,different ROS scavengers had variable transition points before they were consistently activated,suggesting that ROS scavengers were differentially activated in an order way.The activated ROS scavengers increased with PsCA duration,suggesting that the chilling tolerance was positively correlated with the number of activated ROS scavengers,and the induction of chilling tolerance required enough number of ROS scavengers to be effective.（2）The differentially expressed proteins induced by PsCA were identified through two dimensional electrophoresis coupled with MS/MS and the possible mechanism was also proposed.Compared with the non acclimation（NA）,PsCA（72 h）induced 21significant differentially-accumulated proteins（SDAPs）;Compared with the control,PsCA induced 23 SDAPs after prolonged cold storage.These proteins are mainly related to stress response and defense（SRD）,protein metabolism,signal transduction,primary metabolism,and transcription.The SRD proteins,which made up 37%of the 21 and 47%of the 23,respectively,represented the largest class of SDAPs,and all but one protein were up-regulated,suggesting accumulation of proteins involved in defense response is central feature of proteomic profile changes brought about by PsCA.In fruit just after PsCA treatment,the identified SDAPs were related to responses to various stresses,including chilling,fungi,bacteria,insects,dehydration,salt stress,and DNA damage.However,after prolonged cold storage,the targeted proteins in acclimated fruit were narrowed down in scope to those involved in defense against chilling and pathogens.These results suggested that PsCA initiated comprehensive defense responses in cucumber fruit at first,while the long term storage thereafter altered the responses more specifically to chilling.（3）The function of the differentially expressed protein CsGR-RBP3 induced by PsCA was verified.The expression patterns of six genes from CsGR-RBP genes family in response to low temperatures showed that chilling stress（control）only upregulated CsGR-RBP2 expression,whereas the other five genes including CsGR-RBP3 were highly upregulated by PsCA.Bioinformatics analysis showed that CsGR-RBP3 is a typical RNA binding protein and may function as an RNA chaperone.Transient expression in tobacco showed that CsGR-RBP3 was located in mitochondrion,implying a role for CsGR-RBP3 in maintaining mitochondria-related functions under low temperature.Overexpression of CsGR-RBP3 in Arabidopsis lines accelerated plants growth,enhanced chilling and freezing tolerance,upregulated eleven Arabidopsis genes involved in defense responses to various stresses,suggesting that CsGR-RBP3 plays a positive role in defense against chilling stress.（4）The function of the differentially expressed protein CsDDI1 induced by PsCA was verified.Analysis of CsDDI1 expression showed that both chilling stress（control）and PsCA could induce CsDDI1,whereas the expression level after PsCA was higher than that of in the control during chilling temperature storage.Amino acid sequence of CsDDI1contains UBA,UBQ and RVP conserved domains.Transient expression in tobacco showed that CsDDI1 was located in the nucleus and cytoplasm.Overexpression of CsDDI1 in Arabidopsis lines accelerated plants growth,enhanced chilling tolerance,increased catalase and superoxide dismutase activities,reduced ROS levels and upregulated the expression of eleven defense genes,suggesting that CsDDI1 plays a positive role in defense against chilling stress in cucumber.（5）To study the roles of H₂O₂,NO and ABA in PsCA-induced chilling tolerance,three sets of different experiments were conducted and effects of inhibition of endogenous H₂O₂,NO and ABA synthesis on chilling tolerance induced by PsCA were investigated,respectively.Fruit treated with TS（ABA biosynthesis inhibitor）or L-NAME（NO biosynthesis inhibitor）before cold acclimation showed increased chilling injury index,relative electrical conductivity and secondary disease index compared with PsCA alone,suggesting that PsCA-induced chilling tolerance is dependent on endogenous NO and ABA synthesis in cucumber.Inhibition of endogenous NO or ABA biosynthesis after cold acclimation for 12 h reduced chilling tolerance induced by PsCA,suggesting that PsCA-induced chilling tolerance was positively correlated with NO and ABA accumulation.Inhibition of endogenous NO after cold acclimation for 12 h followed by application of exogenous NO or ABA could restore PsCA-induced chilling tolerance,whereas inhibition of ABA followed by application of exogenous NO did not restore chilling tolerance,suggesting that NO functions upstream of ABA.Inhibition of endogenous H₂O₂ before cold acclimation reduced chilling tolerance,downregulated the expression of defense genes including AS-RP1,GSH-Px,Prd-2B,SOD（Cu-Zn）,L-APX6 and POD in cucumber,suggesting that H₂O₂ is the key signal molecule in initiating cold acclimation.Inhibition of endogenous H₂O₂ followed by application of exogenous NO or ABA could restore tolerance,suggesting that H₂O₂ functions upstream of NO and ABA.These results suggest that H₂O₂,NO and ABA are all involved in signaling pathway for cold acclimation,which goes from H₂O₂ to NO,and then to ABA.