| As one of the world’s most important economic crops, cotton is not only an important source of natural fiber, but also an excellent source of edible oil. Even cotton has a higher tolerance to salt compared with other major crops, the growth, yield and fiber quality of cotton are seriously affected by high concentration salt stress, especially when cotton at germination and young seedling stage. Therefore,it is helpful to study the molecular mechanism of cotton tolerance to salt stress and to improve the salt tolerance of cotton. To reveal the mechanism of salt stress in upland cotton(Gossypium hirsutum L.), relative and absolute quantitation(iTRAQ)-based proteomics technique was employed to identify the early salt stress response proteins in seedling roots. The full-length cDNA of GhCYPL and GhCaM which related to salt tolerance have been cloned and the function of GhCYPL has been preliminarily verified.In this present study, upland cotton “ZMS23”, a salt-tolerance variety, was used to study the early salt stress response proteins in seedling roots. 128 differentially expressed proteins(DEPs)in cotton seeding roots were identified by iTRAQ-based proteomic technique. Among them, 76(59.4%) were up-regulated, and 52(40.6%) were down-regulated under salt stress. According to the classification of the protein function, the main biological functions of the 128 DEPs were classified as: stress and defense(23.4%), carbohydrate and energy metabolism(13.3%), transcription related(4.7%), protein metabolism(15.6%), signal transduction(3.1%), cell wall and cytoskeleton metabolism(12.5%), membrane and transport(10.9%), some other metabolic processes(4.7%) and unknown function(11.7%). The diversity of these salt-stress response proteins indicate that the flexibility in cotton root metabolism improve the adaptability of cotton under salt-stress. It is worth emphasizing that some novel salt-responsive proteins were identified which are involved in cell cytoskeleton metabolism(actin-related protein2, ARP2, and fasciclin-like arabinogalactan proteins, FLAs), membrane transport(tonoplast intrinsic proteins, TIPs, and plasma membrane intrinsic proteins, PIPs), signal transduction(leucine-rich repeat receptor-like kinase encoding genes, LRR-RLKs) and stress responses(thaumatin-like protein, TLP, universal stress protein, USP, dirigent-like protein, DIR, desiccation-related protein PCC13-62), these proteins provide a good start for the further study in molecular mechanism of cotton tolerance to salt stress. The enzyme activity analysis of some altered proteins(superoxide dismutase, SOD, peroxidase, POD, glutathione S-transferase, GST, monodehydroascorbate reductase, MDAR, and malate dehydrogenase, MDH) showed that there were a high positive correlation between the enzyme activity and the protein abundance. The results demonstrated that the iTRAQ-based proteomic technique is reliable for identifying and quantifying a large number of cotton root proteins. qRT-PCR was used to study the gene expression levels of the five above-mentioned proteins: The RNA expression pattern of four gene were consistent with their protein expression. These results showed the complexity of the proteome of cotton seeding roots under salt-stress. The comparative proteomic analysis of roots under salinity improved the understanding of the molecular mechanisms involved in the tolerance of plants to salt stress.The CYP(Cyclophilin) gene of upland cotton, GhCYPL, was cloned. Its full length cDNA was 911 bp, encluding a 621 bp ORF which putatively code for a 207 amino acid residues. The polypeptide chain sequence of this gene contains a unique plant cyclophilin amino acid residues(KSGKPLH), 11 phosphorylation sites, 1 N-glycosylation sites and a peptidyl-prolyl cis-transisomerase activity site(PPIase FKGSAFHRIIPNFMIQGG). These structures suggested that the gene of GhCYPL may involved in a wide variety of processes including protein folding, protein transport, cell signaling, and stress response. qRT-PCR analysis showed that the expression of Gh CYPL was up-regulated under salt stress, it meant that the gene may play a role in enhancing resistance to salt stress in cotton.The cDNA sequence of GhCaM was cloned and showed that GhCaM cDNA was 806 bp, containing a 86 bp 5’-UTR, a 279 bp 3’-UTR and an ORF of 441 bp encoding a putative 147 amino acid residues polypeptide which has a predicted molecular mass of 16.61 KD and isoelectric point of 4.81. Its secondary structure contain four EF-hand domain and four Ca2+ ions binding sites, showed that it was highly homological with CaM protein in other plants. We predicted that GhCaM gene might be involved in cellular processes that are critical for cell growth and development, regulate Ca2+-dependent signal transduction, etc. qRT-PCR analysis showed that the gene expression of GhCaM was induced under salt stress, it meant that GhCaM genes may be involved in signal transduction processes under salt stress.To investigate the function of Gh CYPL gene, we constructed a loss of-function mutant of upland cotton ZMS23 by virus induced genes silencing(VIGS). qRT-PCR results demonstrated that compared with negative control plants, the relative expression level of endogenous GhCYPL in GhCYPL-silenced cotton plants was reduced more than 88.02%(Virus with pTRV2-00 was served as negative control), the results showed that the pTRV2-GhCYPL-silencing was successful in cotton ZMS23 and can be used as an efficient tool to study single gene function. Under salt-stress condition, the relative contents of H2O2 and MDA were lower in the true leaves of negative control plants than in GhCYPL-silencing plants, it suggested that the expression of GhCYPL gene was involved in the process of H2O2 accumulation and lipid peroxidation. The enzyme activities of the antioxidant enzymes superoxide dismutase(SOD), peroxidas(POD), catalase(CAT) and ascorbate peroxidase(APX) in GhCYPL-silencing plants and negative control plants had been detected, and the results showed that H2O2 concentration was controlled by GhCYPL upregulating the activities of SOD, CAT, and CAT at post-translation level rather than transcriptional level, and MDA was reduced as a result. Therefore, Gh CYPL might be a key regulator controlled ROS by modulating the activities of antioxidant enzymes. |
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