Quantitative Proteomic Analysis Provides Novel Insights into Cold Stress Responses in Petunia Seedlings

Abstract

Low temperature is a major adverse environmental factor that impairs petunia growth and development. To better understand the molecular mechanisms of cold stress adaptation of petunia plants, a quantitative proteomic analysis using iTRAQ technology was performed to detect the effects of cold stress on protein expression profiles in petunia seedlings which had been subjected to 2°C for 5 days. Of the 2430 proteins whose levels were quantitated, a total of 117 proteins were discovered to be differentially expressed under low temperature stress in comparison to unstressed controls. As an initial study, 44 proteins including well known and novel cold-responsive proteins were successfully annotated. By integrating the results of two independent Gene Ontology (GO) enrichment analyses, seven common GO terms were found of which "oxidation-reduction process" was the most notable for the cold-responsive proteins. By using the subcellular localization tool Plant-mPLoc predictor, as much as 40.2% of the cold-responsive protein group was found to be located within chloroplasts, suggesting that the chloroplast proteome is particularly affected by cold stress. Gene expression analyses of 11 cold-responsive proteins by real time PCR demonstrated that the mRNA levels were not strongly correlated with the respective protein levels. Further activity assay of anti-oxidative enzymes showed different alterations in cold treated petunia seedlings. Our investigation has highlighted the role of antioxidation mechanisms and also epigenetic factors in the regulation of cold stress responses. Our work has provided novel insights into the plant response to cold stress and should facilitate further studies regarding the molecular mechanisms which determine how plant cells cope with environmental perturbation. The data have been deposited to the ProteomeXchange with identifier PXD002189.

Keywords: Petunia hybrida; antioxidation mechanism; cold stress; cold-responsive protein; epigenetic factor; iTRAQ technology; proteomics.

Figure 1

High correlation among different replicates. (A) Relative quantification of CK. (B) Relative quantification of treatment. The “AveCK” represents the average quantification among control samples, and the “AveE” means the average quantification among experimental samples, and the “CK1~4” and “E1~4” are the corresponding different individuals.

Figure 2

Comparison of expression patterns at the mRNA and protein level of DEPs. (A) Up-regulated at both transcript and protein level. (B) Up-regulated at transcript level while down-regulated at protein level. (C) Down-regulated at transcript level while up-regulated at protein level. (D) No change at transcript level while up-regulated at protein level. (E) No change at transcript level while down-regulated at protein level. Relative transcript levels were calculated by real-time PCR with GAPDH as a standard.

Figure 3

Catalytic activities of anti-oxidative enzymes in control plants and cold-treated plants. Columns and bars represent the means and SE (n = 3), respectively.