Abstract
Liquid nitrogen (LN) exposure, a crucial step in cryopreservation, is mainly responsible for the reduced viability of protocorm-like bodies (PLBs) in Dendrobium nobile Lindl. To characterize the stress and damage mechanisms during pre- and post-LN exposure, the isobaric tags for relative and absolute quantification (iTRAQ)-labeling method was used to evaluate proteome profiles of PLBs in D. nobile ‘Hamana Lake Dream’ undergoing the cooling and rewarming process. In total, 1747 proteins were detected; a cluster of orthologous group (COG) analysis of the identified proteins revealed that posttranslational modification, protein turnover, and chaperone-related proteins were the predominant protein classes. Of these, 108 proteins were differentially changed and grouped into different functional categories, which included protein turnover, stress and defense, carbohydrate and energy metabolism, signaling transduction, metabolism, membrane, and transport. Furthermore, a quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that some proteins, such as annexin, l-ascorbate peroxidase 1, copper/zinc superoxide dismutase, glutathione S-transferase dehydroascorbate reductase 2-like, and an adenosine triphosphate (ATP) synthase, were regulated at their transcriptional levels. Based on functional analysis of these proteins, it was found that protein synthesis, processing, and degradation might be the main strategies used to reestablish cell balance after the cooling and rewarming process. Moreover, the production of reactive oxygen species (ROS) and the decline in energy production, signaling transduction, and membrane transport during pre- and post-LN exposure might be responsible for the viability loss of the PLBs. This work provides potential protein candidates for exploring the stress and cryo-injury mechanism in cryopreservation.
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This work was supported by the National Natural Science Foundation of China (No. 31370693).
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WD prepared the PLB cultures of D. nobile Lindl. ‘Hamana Lake Dream’ for the iTRAQ-based proteomic analysis. W.D. and Y.L. performed the general statistical analysis of the proteomics data. W.D., X.R.J., M.X.J., and Y.L. were involved in the proteomics analysis and wrote the manuscript. Y.L., J.X., and B.L.L. conceptualized the design and approach used in the entire study.
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Table S1
Overview of identified proteins from Dendrobium nobile protocorm-like bodies pre- and post-liquid nitrogen exposure. (a) The accession number of the NCBI database of translated sequences from nucleic acids of Dendrobium nobile transcriptome. (b) Significant difference. (c) Geometric mean ratio corresponding to the protein reporter ion intensity originating from post-liquid nitrogen chilled–rewarmed (C-RM) group protein samples relative to pre-liquid nitrogen plant vitrification solution 2 (PVS2) group protein samples. (d) The accession number of Oryza sativa subsp. japonica protein database (XLSX 662 kb)
Table S2
Quantification of abundances of proteins from Dendrobium nobile protocorm-like bodies pre- and post-liquid nitrogen exposure. (a) The accession number of the NCBI database of translated sequences from nucleic acids of Dendrobium nobile transcriptome. (b) Significant difference. (c) Geometric mean ratio corresponding to the protein reporter ion intensity originating from post-liquid nitrogen chilled–rewarmed (C-RM) group protein samples relative to pre-liquid nitrogen plant vitrification solution 2 (PVS2) group protein samples. (d) The accession of Oryza sativa subsp. japonica protein database (XLSX 244 kb)
Table S3
Detailed information regarding the functional annotations of the differentially expressed proteins from Dendrobium nobile protocorm-like bodies during the pre- and post-LN exposure. (a) The accession of the NCBI database of translated sequences from nucleic acids of Dendrobium nobile transcriptome. (b) Geometric mean ratio corresponds to the protein reporter ion intensity originating from post-liquid nitrogen chilled–rewarmed (C-RM) group protein samples relative to pre-liquid nitrogen plant vitrification solution 2 (PVS2) group protein samples. (XLSX 28 kb)
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Di, W., Jiang, X., Xu, J. et al. Stress and damage mechanisms in Dendrobium nobile Lindl. protocorm-like bodies during pre- and post-liquid nitrogen exposure in cryopreservation revealed by iTRAQ proteomic analysis. In Vitro Cell.Dev.Biol.-Plant 54, 253–272 (2018). https://doi.org/10.1007/s11627-018-9898-x
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DOI: https://doi.org/10.1007/s11627-018-9898-x