Differential Proteomic Study On Chilling Stress Response In Cotton And Tobacco

During the long-term evolution, plants have developed sophisticated biochemical and physiological mechanisms to adapt and resistance to various environmental stresses. Among different stress, cold is a major environmental limitation to crop productivity and to the distribution of wild species. Plants acquire resistance to stress environment by reprogramming metabolism and gene expression, gaining a new equilibrium between growth, development and survival. But the mechanism of low temperature stress on growth and the accompanying metabolic changes remain to be elucidated in detail. In this study, we carried out comparative proteomic analyses to investigate the proteomic response of cotton (Gossypium hirsutum L.) and Nicotiana tabacum cell suspension culture under chilling stress and the proteomic changes of total proteins in Nicotiana tabacum cell suspension culture when the cotton CBF1A were expressed in them. The aim was to provide a better understanding about the underlying molecular mechanisms for plant response to low temperature.To gain a better understanding of chilling stress responses in cotton (Gossypium hirsutum L.), we carried out a comparative proteomic analysis. Cotyledon proteins of 1-week-old cotton seedlings treated with or without chilling treatment at 4℃were extracted, separated by a two-dimensional gel electrophoresis and compared. Among 1500 protein spots reproducibly detected on each gel, 25 protein spots were down-regulated and 29 were up-regulated. Seven cotton proteins were identified by mass spectrometry analysis as beta-globulin A precursor fragments. One was identified as a Rubisco large subunit fragment, providing evidence of both seed storage protein and photosynthetic protein destruction by chilling stress. The related physiological and biochemical analysis showed that there was a significant increase in endopeptidase activity and activated oxygen generation rate, and an obvious decrease in carboxylation efficiency and maximum net photosynthetic rate of cotton seedlings under chilling stress. Based on the above results, the degradation mechanisms of beta-globulin and Rubisco under chilling stress were discussed. In conclusion, our study provides new insights into chilling-stress responses in cotton and better understanding of the degradation mechanisms of seed storage proteins and photosynthetic proteins under chilling stress.In this study, differential proteomic analysis was conducted to characterize the proteins in Nicotiana tabacum cell suspension culture that were differently expressed in responsive to chilling stress with 2-DE and MS/MS. The gels were analyzed by ImageMasterTM 2D Platinum software. About five hundred reproducible protein spots were detected, among which one protein spot was up-regulated in the chilling stressed samples. MALDI-TOF-TOF MS analysis followed by database searching helped to identify the spot as late embryogenesis abundant (LEA)–like protein. To investigate the change of gene expression at the mRNA level, we performed qPCR analysis. The results showed that the mRNA level is correlated well with protein level. A full-length cDNA encoding the protein (designated as NtLEA7-3, GenBank accession No. EF532409) was cloned from Nicotiana tabacum cell suspension culture by rapid amplification of cDNA ends. The cDNA was 1 267 bp containing a 969 bp open reading frame which was deduced to encode a peptide of 322 amino acids whose predicted molecular mass was 35.7 kDa and isoelectric point was 4.86. It was predicted that the structure of NtLEA7-3 was rich in coils, and was poor in helix and strands. The coding region of the NtLEA7-3 was inserted into an expression vector, pET30a (+), and transformed into Escherichia coli BL2l. The fusion protein was successfully expressed with IPTG induction. The plant expression vector pBI121-LEA with this fragment under the control of 35S promoter was constructed and transformed into Nicotiana tabacum plants and cell suspension culture. Northern blot and Western blot analysis indicated that the NtLEA7-3 was expressed successfully in them. The related physiological and biochemical analysis showed that the NtLEA7-3 was a very efficient gene for improving chilling tolerance of Nicotiana tabacum. The coding region of the NtLEA7-3 was inserted into an expression vector pBI121 containin GFP and transformed into Arabidopsis thaliana plants and Nicotiana tabacum cell suspension culture. Then the transgenic Arabidopsis thaliana plant roots and cell suspension culture were observed through fluorescence microscopy and the results showed the NtLEA7-3 were expressed in the nucleus. This research not only provides a better understanding for the molecular mechanisms of plant response to low temperature, but also a useful candidate gene for resistance breeding. Furthermore, this study may be helpful to study the structure, function and regulation of the NtLEA7-3.The cotton CBF1A gene under the control of 35S promoter was transformed into Nicotiana tabacum cell suspension culture successfully. A comparative proteomic analysis was carried out to gain a better understanding of the proteins differently expressed in responsive to CBF1A expression in Nicotiana tabacum cell suspension culture. Among the protein spots detected, 7 protein spots displayed differential expression. There were 4 up-regulated and 3 down-regulated protein spots in the transgenic cell suspension culture. MS analysis followed by database searching helped to identify 6 spots representing 5 different proteins. The identified proteins include putative chloroplast cysteine synthase 1 precursor, malate dehydrogenase like-protein, short chain dehydrogenase and two unknown protein. This research provides a better understanding for the molecular regulation mechanisms of CBF1A.