Transcriptome Analysis Of Camellia Sinensis Under Heat And Drought Stress And Functional Characterization Of Histone H1 Gene

Tea plant [Camellia sinensis (L.) O. Kuntze] is an important perennial, evergreen,woody crop,and the leaves are processed to prepare a popular non-alcoholic beverage known as 'tea'. C. sinensis experienced various abiotic stresses during their lifecycle, such as drought, cold, heat, salinity, heavy-metal stress and soil nutrient deficiency. Among these stresses, heat and drought are important factors and significantly constrain the yield and quality of tea products. Moreover, the damage caused by heat and drought has become frequently and unpredictably due to global climate changes and serious water scarcity.Therefore, improving the heat and drought tolerance of C. sinensis and breeding resistant varieties deserves more attetions. So, understanding the molecular.mechanisms of C.sinensis responding to heat and drought stress and obtaining resistant genes are highly valuable for the rapid breeding of fine varieties of C. sinensis.In response to the above scientific questions, the cultivar of C. sinensis'Longjingchangye' was used to reveal the response mechanism of transcriptional level under heat and drought stress, the molecular mechanisms of drought stress affected the accumulation of major bioactive ingredients in leaves, and the functional mechanisms of CsHis-H1 in the process responding to abiotic stress by high-throughput sequencing,bioinformatics and molecular biology technology. The main results and conclusions are summarized as follows:1. The transcriptome of C. sinensis under heat and drought stress was acquired through RNA-Seq technology based on Illumina HiSeqTM 2000 platform. The results showed that a large number of differentially expressed genes (DEGs) related to Ca2+ signaling pathway were doMwn-regulated expressed under heat stress, while most of corresponding DEGs were up-regulated expressed under drought stress,especially CIPKs and CMLs genes,indicating Ca2+ signaling pathway plays an important role in responses to heat and drought stress in C.sinensis, although their molecular regulation mechanisms exists a certain differences. In addition, six major transcription factors (HSF, WRKY, AP2/EREBP, bZIP, NAC and MYB)were also confirmed in the process of C. sinensis responses to heat and drought stress by different regulatory response patterns. For examples, most of the HSF family genes in DEGs are up-regulated expressed under heat stress,but down-regulated expressed under heat stress. Similar differences were also found in the WRKY,bZIP,MYB and NAC family genes. Furthermore, heat and drought stress induced the expression of HSPs and AQPs genes, respectively, and both inhibit the expression of some LEAs genes.2. The effects of drought stress on the phenotype,physiological characteristics and major bioactive ingredients accumulation in leaves of C. sinensis were examined, and the results showed that drought stress resulted in dehydration and wilt of the leaves, significant decreased in the total polyphenols and free amino acids and increased in the total flavonoids. In addition, HPLC analysis showed that the catechins, caffeine, theanine and some free amino acids were significantly reduced in response to drought stress,implying that drought stress severely affected the accumulation of major bioactive ingredients in leaves of C. sinensis. Furthermore, DEGs related to amino acid metabolism and secondary metabolism were identified and quantified under drought 'stress based on RNA-Seq data,especially the key regulatory genes involved in the catechins, caffeine and theanine biosynthesis pathways. The expression levels of key regulatory genes were consistent with the results from the HPLC analysis, which reveals a potential molecular mechanism of drought stress affected the accumulation of major bioactive ingredients in leaves of C.sinensis.3. The expression levels of CsHis-H1 in leaves of C. sinensis under various inverse environments were investigated by qRT-PCR, and the results indicated that CsHis-H1 was strongly induced by various abiotic stresses, such as low temperature, high salinity, ABA, drought and oxidative stress. Overexpression of CsHis-Hl in tobacco (Nicotiana tabacum) promoted chromatin condensation, while there were almost no changes in the growth and development of transgenic tobacco plants. Phylogenetic analysis showed that CsHis-H1 protein belongs to the H1C and HID variants of Histone H1, which are stress-induced variants and not the key variants required for growth and development in plants. Stress tolerance analysis indicated that the transgenic tobacco plants exhibited higher tolerance than the WT plants upon exposure to various abiotic stresses; the transgenic plants displayed reduced wilting and senescence and exhibited greater net photosynthetic rate (Pn), stomatal conductance (Gs) and maximal photochemical efficiency (Fv/Fm) values,implying that CsHis-H1 can improve the tolerance to various abiotic stresses in the transgenic tobacco plants, possibly through the maintenance of photosynthetic efficiency.