Cloning Of Wheat Histone Variants And Comparison Of Wheat Chloroplast Proteomes

Salt, drought and other abiotic stress adversely affect crop yield. Therefore, it is urgent to isolate stress-tolerant gene and elucidate the mechanism of stress-tolerance, with the aim to breed stress-tolerance crops. The response to abiotic stress is achieved by the comprehensively physiological alteration of chloroplast and other organelles, which is genetically and epigenetically orchestrated within the signaling network. In this study, we (1) isolated histone variant genes that is associated with epigenetic modulation from wheat introgression cultivar SR3and its parent common wheat JN177, and analyzed their relationship with the response to abiotic stress;(2) compared the difference in chloroplast proteomes between SR3and JN177to understand the linking between the SR3’s salt and drought tolerance ability and chloroplast.1. Isolation of histone variant genes and analysis of their role in the response to abitic stressHistones are a class of evolutionarily conserved proteins, and are the main components of nucleosomes. Histone variants are the special chromosome histone types required for chromatin assembly at specific chromosome region, and it can replace the conventional histone via chromatin remodeling to exercise its function to regulate gene transcription under epigenetic level. In the previous proteomic work of our lab, a set of probes annotated as histone variants in the wheat cDNA microarray showed salt responsive expression profiles, some of which had differentially signals between SR3and JN177, suggesting their crucial role in the response to salt stress.In this paper, based on the transcriptomic data, we isolated eight histone variant genes, including H2A variants TaH2A-1～4and TaH2A.Z-1, H3variants TaH3-1～2, and H4variant TaH4-1. The cDNA and genomic sequences of each of these genes was absolutely identical between SR3and JN177, among which TaH2A-4and TaH3-2had one intron, respectively. Their deduced protein sequences possessed typical α-helix and nuclear localization signal sequences, and subcellular localization assay indicated that these proteins functioned in the nucleus. RT-PCR analysis indicated that, these genes showed salt-responsive transcription profiles, and some were specifically transcribed in roots.To know their roles in the response to abitic stress, we transformed five genes intro Arabidopsis and three of them were found to ectopically express. No difference was found between the overexpression lines (OE) and the wild-type (WT) during the whole period of growth. Under the treatment of NaCl, H2O2, mannitol and several phytohormones, the TaH2A-2/TaH2A.Z-1OE lines were comparable to WT with respective to germination rate, leaf size and root length. Similarly, no phenotypic difference was found between TaH2A-3OE lines and WT after exposure to NaCl, NAA, ACC and GA3. However, in comparison with WT, TaH2A-3OE lines had higher drought tolerance, lower tolerance to H2O2, delayed germination under ABA treatment, and fewer lateral roots under mannitol treatment. These results suggest that histone variants perform different roles in the response to abiotic stress, and the difference may be resulted from the specificity of the exerting rules during chromatin remodeling, e.g. TaH2A-2and TaH2A.Z-1need nucleosome assembly co-factors to be recruited onto chromosome, while TaH2A-3dose not; TaH2A-2and TaH2A.Z-1perform similar function to their homologues of Arabidopsis, while TaH2A-2play its special role.2. The comparison of chloroplast proteomes between SR3and JN177Chloroplast is the place of photosynthesis of plant cells and is the key for plant autotrophic survival. In the previous proteomic analysis of our lab, a close relationship was found between the vigorous growth and abiotic stress tolerance of SR3and its assimilation ability in chloroplast. To further understand the relationship, we conducted the chloroplast proteomic analysis. We improved the published wheat chloroplast extraction method and obtained high purity chloroplast that is suitable for proteomic analysis. Totally,28differentially expressed protein spots were found by two-dimensional electrophoresis, including eighteen with differential abundance between SR3and JN177under the control and two under NaCl treatment, and eight between the control and NaCl treatment. Of which,21spots were identified by MALDI-TOF MS, which are involved in photosynthesis, photosynthesis stability, and protein synthesis and transporting. Of them, two key enzymes of Carlvin cycle showed differential patterns between SR3and JN177; photosynthesis stability associated proteins had higher abundance in SR3than in JN177. This difference may lead to the higher photosynthesis efficiency of SR3under the control and stressful conditions, which provides evidence for further understanding the mechanism of SR3’s tolerance to abioti stress.