Study On The Damage Mechanism Of Dendrobium Nobile Lindl. 'Hamana Lake Dream' Protocorm-Like Bodies During Vitrification-Cryopreservation

Cryopreservation is a safe and cost-effective method for long-term conservation of germplasm resources.However,there are still some germplasm resources that cannot survive cryopreservation,which is largely due to the unclear mechanism of cryopreservation.Therefore,revealing the damage mechanism of cryopreservation is important not only for providing theoretical basis of biological phenomena at ultra-low temperature,but also for optimizing cryopreservation technology.In this study,protocorm-like bodies(PLBs)of Dendrobium nobile Lindl.'Hamana Lake Dream',which exhibited extremely low regeneration rate(5.56%)after vitrification-cryopreservation,were used as materials.Proteome profiles of Dendrobium nobile PLBs during vitrification-cryopreservation were detected to find out the protein functions that related to PLB survival,and differentially expressed proteins that closely related to the loss of PLB survival were selected.On this basis,the role of key protein candidates of reactive oxygen species(ROS)-induced oxidative stress in PLBs during vitrification-cryopreservation was investigated.The main results were as follows1)The survival declines of Dendrobium nobile PLBs in each step of vitrification-cryopreservation were examined by TTC staining method.The results showed that PLB survival displayed a continuous decrease throughout the entire vitrification procedure.Preculture and cooling-rewarming steps resulted in the most obvious declines in PLB survival.Transmission electron microscope was adopted to observe the extent of cell structure changes in Dendrobium nobile PLBs during vitrification-cryopreservation,which showed that cell structures were changed and even damaged under cryopreservation treatments.However,after regeneration culture,most cell structures could be recovered and then presented the characteristics of living cells,suggesting that low regeneration of PLBs after cryopreservation might not result from cell structure changes.2)The isobaric tags for relative and absolute quantification(iTRAQ)-labeling method was used to evaluate proteome profiles of Dendrobium nobile PLBs under different steps of vitrification-cryopreservation.In total,1,747 proteins were detected.Of these,14,17,82,108 and 63 differentially expressed proteins were identified under the preculture,loading,PVS2-treated,cooling-rewarming and unloading steps,respectively.The differentially expressed proteins were mainly involved in functions of protein turnover,carbohydrate and energy metabolism,stress and defense,membrane and transport,which might be associated with PLB survival changes during cryopreservation.Among these,the differentially expressed proteins related to ROS metabolism were found to occupy a high proportion of identification coverage,which might be associated with the obvious loss of PLB survival.3)Changes of ROS,oxidative stress products and antioxidants in Dendrobium nobile PLBs were detected under different steps of vitrification-cryopreservation,the results showed that preculture and cooling-rewarming steps,which resulted in the most obvious declines in PLB survival,were the key steps to inspire oxidative stress in PLBs.Adding non-enzymatic ROS scavenger,ascorbic acid(AsA),to different steps of vitrification-cryopreservation all could improve survivals of cryopreserved PLBs.The addition of 0.1 mM AsA to the unloading step resulted in the most obvious improvement of PLB survival by 14.34%,and this improvement was associated with its role in reducing ROS,increasing antioxidant ability and relieving lipid peroxidation damage.The results indicated that ROS-induced oxidative stress was closely related to the reduced survival of Dendrobium nobile PLBs during vitrification-cryopreservation.4)Differentially expressed proteins involved in ROS homeostasis,such as exogenous superoxide dismutase(SOD),catalase(CAT)and pyruvate dehydrogenase(PDH)were added to different steps during vitrification-cryopreservation of Dendrobium nobile PLBs,the additions of SOD,CAT and PDH to the unloading step were found to significantly increase survivals of cryopreserved PLBs by 5.76%,33.49%and 2.49%,respectively,and these improvements were associated with their roles in reducing ROS and increasing antioxidant contents with various degrees;However,these exogenous proteins had different effects on endogenous SOD activity and malonaldehyde content,the effect of exogenous CAT on PLB survival was stronger than that of SOD and PDH,this discrepancy might be due to maintaining low endogenous SOD activity and increasing CAT activity by exogenous CAT addition,which was more conducive to reducing ROS and relieving lipid peroxidation damage.The results indicated that differentially expressed proteins involved in ROS homeostasis could mitigate oxidative stress in Dendrobium nobile PLBs during vitrification-cryopreservation.5)To explore the gene expression changes of ROS metabolism-related proteins in Dendrobium nobile PLBs during vitrification-cryopreservation,EF-1? and ACT genes were firstly select out from six candidate reference genes of Dendrobium nobile PLBs as the most optimal reference for the further gene expression analysis during vitrification-cryopreservation by the combined analysis of real-time quantitative PCR method with the softwares of geNorm,NormFinder,Bestkeeper and qBase plus.Using this reference gene as control,the gene expressions of 14 ROS metabolism-related proteins in PLBs were analyzed during vitrification-cryopreservation,the results showed that genes related to ROS-induced oxidative stress and cell apoptosis were significantly expressed under different steps,and the alleviation of damage in cryopreserved PLBs by adding non-enzymatic ROS scavenger,AsA,might be related to its role in controlling oxidative stress and cell apoptosis at transcriptional level.The expression changes of most ROS metabolism-induced proteins were inconsistent with their gene expressions,which might result from post-transcriptional,protein translational and post-translational regulations.The main conclusions of this study were that:the differentially expressed proteins involved in protein turnover,carbohydrate and energy metabolism,stress and defense as well as membrane and transport might be mainly associated with survival changes of Dendrobium nobile PLBs during vitrification-cryopreservation;Among these,ROS metabolism-related proteins might play the most critical roles in affecting PLB survival.ROS-induced oxidative stress was closely related to the reduced survival of Dendrobium nobile PLBs during vitrification-cryopreservation.Differentially expressed proteins involved in ROS homeostasis were verified to play protective roles in survival of Dendrobium nobile PLBs during vitrification-cryopreservation.