| Salt stress is a major abiotic stress in agriculture, more than 20% of the land and almost half of all irrigation land are affected by salt. So it is very important to look for salt stress related proteins or genes to understand the mechanisms of plant salt resistance and to improve plant salt resistance.Sugar beet is a kind of glycophyte and has strong salt resistant ability. Sugar beet M14 line is a special species and it is obtained from the intercross between Beta vulgaris L and Beta corolliflora Zoss. It exhibits tolerance to drought, cold and salt stresses. It has become rare materials to dig quality genes and proteins of sugar beet.Protein phosphorylation plays an important role in the regulation of many plant vital activities and produces many post-translational modifications which are generally involved in plant response to various abiotic and biotic stresses. Protein phosophorylation alters the conformation of proteins and regulates the activity of proteins by introducing phosphate groups to the protein. Here, quantitative proteomics approach was used to research the differentially expressed phosphorylated proteins of M14 in response to salt stress, which lead to improved understanding of the defense mechanisms of M14 in response to salt stress.For a more complete and accurate results, the differentially expressed phosphorylated proteins were obtained by employing two methods at the same time, namely two-dimensional electrophoresis and label free. On the one hand, Pro-Q Diamond fluorescence staining method combined with two-dimensional gel electrophoresis was used to separate phosphorylated proteins in M14 leaves. Compared with the control, 24 differentially expressed phosphorylated proteins were obtained in 200 mM NaCl treatment groups, 16 phosphorylated proteins were identified successfully, including 8 up regulated phosphorylated proteins. Compared with the control, 36 differentially expressed phosphorylated proteins were obtained in 400 mM NaCl treatment groups, 28 phosphorylated proteins were identified successfully, including 17 up regulated phosphorylated proteins. On the other hand, the proteins in control and NaCl treatment groups were digested in solution, desalted and enriched. In total 2182 phosphorylated proteins had been identidied by Q Exactive MS/MS, 189 phosphorylated proteins of them were differentially expressed, including 94 up regulated phosphorylated proteins and 95 down regulated phosphorylated proteins. In order to further study salt response mechanisms, according to the BLAST annotations, Uniprot database as well as the related publications, the differentially expressed phosphorylated protein in salt stress were divided into 10 groups based on their biological functions, which include transport(15%), metabolism(16%), photosynthesis(2%), signal transduction and kinase(14%), protein folding and degradation(3%), stress and defense(8%), protein synthesis(7%), transcription(13%) and cell structure(12%). On the basis of the identified functions of phosphorylated proteins, schematic presentation of processes regulated by phosphorylated proteins of M14 in response to salt stress was drawn. The results showed that the signal transduction and kinase phosphoproteins, metabolism related phosphoproteins and transcription related phosphoproteins accounted for a majority proportion.In order to study the effects of signal transduction pathways to salt-tolerant mechanism in sugar beet M14, the 15 phosphorylated proteins obtained above which involved in signal transduction pathways were verified at the transcriptional level by using RT-PCR. These results suggest that phosphoproteins can perceive salt stress signal and transmit signal to make various organelles phosphoproteins work together against salt stress. These processes work cooperatively to achieve homeostasis under salt stress conditions. This research has set the stage for further understanding of the salt tolerance mechanisms in sugar beet M14 line.
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