| Vitrification-based cryopreservation is considered to be an important tool for long-term germplasm conservation. In vitrification procedures, materials are treated with plant vitrification solution(PVS) to reduce cellular water content and allow formation of a glass from the remaining water. This process avoids ice-caused damage during cryogenic storage. Stress plays a pivotal role in vitrification, and reducing the stress injury by exogenous compounds is an effective way to improve recovery after cryogenic treatment. Therefore, uncovering the dynamic processes of metabolism and stress response has important significance in cryopreservation. In this thesis, Arabidopsis thaliana seedlings germinated over two time periods were used to analyze metabolites and differentially expressed genes(DEGs) during cryopreservation. These processes revealed the regulatory mechanisms of exogenous compounds in the improved cryopreservation at the physiological and transcriptional levels. This study aims to further enrich cryobiological theory and provide a theoretical basis for the practical operations of plant cryopreservation.1) In Arabidopsis seedling cryopreservation, there was a significant negative correlation between seedling age and survival ratio. The logistic equation of this correlation curve was constructed, and the curve inflection was 58.74 h. After cryopreservation, FDA and Evan’s blue viability staining showed that 48-h seedlings had survival ratio of 96.8% with higher cell activity and intact plasma membranes. These seedlings recovered sooner from the cryo-injury than the 72-h seedlings. In contrast, 72-h had comparatively lower viability throughout the cryopreservation process, and the damage mainly occurred in the maturation and elongation zone of the roots.2) The physiological and biochemical indices indicated different responses for several endogenous substances in the two seedling age groups during cryopreservation. The levels of reactive oxygen species(ROS) increased in 72-h seedlings during osmoprotection and dehydration. In particular, the OH· generation activity increased by 1.62 times, and the content of malondialdehyde(MDA, the membrane lipid peroxidation end product) increased approximately 2-fold in 72-h seedlings. There was a significant negative correlation between MDA content and survival ratio. The membrane lipids of 72-h seedlings were severely injured after osmoprotection and dehydration, which might be responsible for the death of the 72-h seedlings after cryopreservation. The content of soluble saccharides rose in both the 48- and 72-h seedlings during osmoprotection and dehydration. Bioactive cis-ABA content was not significantly different during cryopreservation, but 48-h seedlings had more content than 72-h seedlings. IAA nearly doubled after osmoprotection and dehydration in 48-h seedlings, but there was no significant change in 72-h seedlings. From immunohistochemistry analysis, ABA signals were low, but IAA signals were significantly enhanced by osmoprotection and dehydration in 48-h seedlings, and mainly distributed in cotyledons, maturation and elongation zone of roots. During osmoprotection and dehydration, 72-h seedlings suffered severe membrane lipid peroxidation; in contrast, more endogenous substances(IAA, soluble saccharides) may provide 48-h seedlings protection against oxidative stress. They may be the reason for the different survival ratio between two age seedlings.3) To further understand the mechanism of the negative correlation between seedling age and survival ratio, cDNA-AFLP was performed to screen DEGs between 48-h and 72-h seedlings after exposure to PVS. Using 64 primer combinations, the transcriptional profile derived 145 TDFs showing significant changes in gene expression. All these ESTs were submitted to GenBank as the first plant cryopreservation related sequences. Transcriptional comparison of 48-h seedlings with and without treatments showed that 53% of the DEGs were involved in stress response, protein synthesis and metabolism, and energy metabolism. In contrast, the DEGs in 72-h seedlings were mainly involved in transport, signaling, cell organization and biogenesis. It indicated that transcriptional response had significant differences between two age seedlings cryopreservation. Quantitative analysis using qRT-PCR was used on 54 genes with significant differential expression among four samples. These genes were further analyzed using the hierarchical mean linkage clustering, and 5 major expression groups were distinguished. Among DEGs, oxidative stress response genes played an important role in cryoprotectant treatment, and oxidative stress was a key factor related to cell survival. Peroxidation injured cell membrane systems leading to photophosphorylation and oxidative phosphorylation damage. Strikingly, the DAD1 which encodes the protein involved in suppression of apoptosis was up regulated in 48-h seedlings and did not change expression level in 72-h seedlings after osmoprotection and dehydration. This indicated that cryopreservation induced apoptosis in 72-h seedlings, and that 48-h seedlings had inhibited apoptosis initiation. Both the 48-h and 72-h seedlings underwent oxidative stress, and 48-h seedlings effectively resisted oxidative stress, resulting in cell survival after cryopreservation.4) Using exogenous compounds to reduce injury from oxidative stress was an effective method to improve cryopreservation success. The curve inflection occurs with the 60-h seedlings and provided a model to improve the cryopreservation procedure. Nine exogenous compounds were added to PVS2. VC was the best compound added to PVS2, and improved survival ratio from 23.1% to 58.2%. This improved cryopreservation procedure was used to study the antioxidant system response in 60-h seedlings. The main ROS leading to oxidative damage in cryopreservation was H2O2, and it was mainly distributed in cotyledons, shoot tips, and the maturation and elongation zones of roots as shown by staining. Through a comparative analysis of antioxidant systems between control and improved cryopreservation, it was shown that some oxidative stress related indices continuously rose and reached a peak after rapid warming in the control procedure. The high level of ROS induced the antioxidant system response during cryopreservation. Due to the serious oxidative stress, the elevated endogenous VC content and CAT activity failed to reduce membrane lipid peroxidation effectively, and MDA content still reached the maximum after rewarming. In the control cryopreservation procedure, 60-h seedlings had serious membrane damage leading to cell death. But adding exogenous VC increased the total seedling VC content, and improved the POD activity in the dehydration step, which reduced oxidative stress damage. Rapid cooling, warming, dilution and recovery of the improved cryopreservation also remained at lower levels of ROS and lipid peroxidation than the control cryopreservation. This change resulted in differential survival ratio between the two cryopreservation protocols.5) The comparative transcriptomic analysis of 60-h seedling cryopreservation with and without exogenous VC was used to study the molecular regulatory mechanism of the exogenous antioxidant. Venn diagrams showed that the number of DEGs in the improved dehydration was almost 10 times that of the control dehydration. However, the number of DEGs was significantly decreased during rapid cooling, warming and dilution of the improved cryopreservation compared with the control process. Mapman and KEGG pathway analysis was used to study the metabolism and regulation processes during cryopreservation. Cell wall and lipid related genes were up regulated by osmoprotection. In the control procedure, phenylpropanoid and terpenoid biosynthesis related genes were up regulated, and amino acid biosynthesis and metabolism related genes were down regulated before rapid cooling. Biosynthesis of secondary metabolites, amino acids, lipids and energy metabolism related genes were differentially expressed during rapid cooling and warming. After dilution, photosynthesis and metabolism of xenobiotics by CYPs related genes were down regulated. However, abiotic stress response related genes had no differential expression during dilution. In the initial stage of re-growth, most DEGs were up regulated, but photosynthesis related genes were still down regulated slightly. Carbohydrate, glutathione, proteasome, cofactor and vitamin metabolism were involved in recovery. In the improved cryopreservation protocol, cell wall, lipid, photosynthesis and secondary metabolism related genes were down regulated during dehydration. The exogenous antioxidant, VC, influenced the early stage of regrowth, and more carbohydrates, energy and amino acid metabolism-related genes were down regulated. These changes are conducive to repairing cryo-injury, and adjusting to normal metabolic processes as soon as possible.6) In Arabidopsis seedling cryopreservation, abiotic stresses are perceived by diverse members of membrane-localized protein receptors, which convey physicochemical signals intracellularly via their cytosolic kinase domains, such as the histidine kinases(HKs). HKs were up regulated during osmoprotection, dehydration and cooling-warming in the control cryopreservation, whereas they were only up regulated in the osmoprotection and dehydration steps of the improved cryopreservation. The mitogen-activated protein kinase(MAPK) signaling pathways are involved in a variety of abiotic stresses. MAPK11 was involved in osmoprotection stress signal transduction; furthermore, MAPK20 participated in the response to the cooling-warming process. MAPKKK13 was specifically up regulated in the improved dehydration step. In the initial stage of regrowth, the two procedures both had many MAPKs participate and most of them were up regulated. In plant cells, the incoming stress signal triggers adaptive responses by reprogramming gene expression governed by transcription factors. DNA-binding proteins associated with abiotic stresses include the dehydration-responsive binding proteins/C-repeat binding factors(DREBs/CBFs) which regulate the expression of cold- and drought-responsive genes. During cryopreservation, CBF2 reached a peak after dehydration in both procedures, and other DREBs were specifically down regulated in control dehydration, but these DREBs were continuously up regulated in the improved procedure. Therefore, the abiotic stress response in the improved cryopreservation procedure was more active than the control procedure, especially the dehydration step. This allowed seedlings to adapt to the stress environment, and allowed them to cope with rapid cooling and other treatments.Exogenous VC also played an important role in ROS signal transduction during cryopreservation. Detection of ROS by unknown receptors resulted in the generation of Ca2+ signals. Ca2+ signal-related genes were mainly down regulated in the control cryopreservation; contrarily, calcium binding protein was highly up regulated in the improved cryopreservation. The protein kinase OXI1 played a central role in ROS signal transduction, and was down regulated in the control dilution step, but up regulated through the improved cryopreservation. Some important transcription factors related to ROS signal transduction were up regulated in the improved cryopreservation protocol. The activation of transcription factors regulated the ROS-producing and ROS-scavenging pathways. The improved cryopreservation protocol activated the amplification loop and ROS-scavenging network, and kept the ROS signal balanced and stable during cryopreservation. The result was major differences in oxidative damage and survival ratio between the control and improved cryopreservation protocols. |
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