Differentially Expressed Proteome Under Cold Stress In Arabidopsis Thaliana

Low temperature stress is one of the major abiotic factors affecting plant growth and the geographical distribution of many economically important crops. Proteomic techniques are effective approaches that have in recent years been adopted to study the mechanism of plants responce to cold stress and, to discover cold responsive proteins. To investigate low-temperature resistance mechanism for Arabidopsis thaliana, we used several techniques including prefractionation of protein samples, two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).2-DE is one of the key techniques used in proteomic studies and, production of a high quality 2-DE image is crucial for analysis of the whole-cell proteins. In this experiment, an optimized protocol was developed paying special attention to the whole-cell protein analyses of Arabidopsis based on 2-DE. The optimization included sample preparation, electrophoresis conditions, gel staining, protein spots resolution and reproducibility. The results indicated that, in combination with high-voltage and long-running time in isoelectric focus (IEF), precipitation of proteins using TCA/acetone method gave rise to satisfactory 2-DE images. The maximal detectable protein spots number were estimated to be more than 1000 per gel under silver staining. This protocol may be applicable to other plant materials for 2-DE studies.Poor detection of low-abundant proteins is a common problem in 2-DE in proteome analysis. This is at least attributed partially to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants, which engage the majority of total plant proteins limiting the detectability of low-abundant proteins. In our work, we developed a straightforward protocol for preparation of the whole-cell plant proteins, through differential polyethylene glycol (PEG) precipitation, aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of proteins prepared using a conventional TCA/acetone method, a relatively higher reproducibility was achieved using the PEG fractionation protocol in protein yield and population. As expected, the large subunit of Rubisco was precipitated exclusively in the 16% PEG fraction. This allowed proteins in the Rubisco containing fraction to be analyzed separately from other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which ca. 80% are proteins undetectable using the TCA/acetone method without prefractionation, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants.Using the PEG prefractionation protocol developed, 4 week-old Arabidopsis plants were treated at 4℃for 24 h. The proteome was separated by 2-DE and analysed by MS. Under cold stress, 67 differentially expressed protein spots with higher than 2-fold spot density were detected in gel image comparison using sivil staining. 57 protein spots were detectable by CBB staining and sampled for further MS analysis. The results showed that 42 proteins were successfully identified by MALDI-TOF-MS, among which 9 proteins were cold-inducible proteins previously reported, indicating our results are consistent with others work. The others identified by MS were novel proteins appeared under cold stress previously unreported. These included acetyl-CoA carboxylase, coproporphyrinogen oxidase, Agmatinase, uracil phosphoribosyltransferase, hydroxymethylbilane synthase, protein phosphatase type 2C etc. According to MIPS function category, TAIR database and related references, these proteins may approximately be classified into 9 functional categories such as, proteins involved in regulation of metabolism, energy, protein binding, cell fate, etc. The analysis also elucidated that 43.3% of proteins detected under cold stresss are located in chloroplast, suggesting that the chloroplast is likely play important role in the cold-response in plants.To investigate the gene expression of those identifyied proteins induced un cold stresss, so as to examine the vadility of proteomic analyses performed in this study, we carried out real-time fluorescent quantitative PCR (qPCR) analysis for selected set of 32 genes that are significantly change in their expression under cold stress. The results indicated that the mRNA of only 14 genes (which accunt for ca. 43.8 % genes) coincided with the corresponding protein translates for 24 h cold treatment. Our results suggested that transcription and translation of genes under cold stres may not necessarily correlated each other. This phenomenon is also observed by other laboratories who have done similar work that, gene regulatory mechanisms at both transcriptional and translational levels are likely involved in cold adaptation in plants.