Studies On The Molecular Mechanism Of Winter Bud Dormancy In Tea Plant(Camellia Sinensis(L.) O. Kuntze)

Tea beverage is consumed by the largest population in the world. An increasing number of healthy benefits of tea drinking are recognized and accepted currently. Tea plant is evergrowing woody plant, which develops dormant buds in the fall to survival cold temperature in winter except for the tea plants grown in tropical region. Therefore winter dormancy is an important survival strategy for tea plant, which also extremely affects tea production and economic value. However, so far little study has been done on the mechanism of tea plant winter dormancy. In this study, on the basis of comprehensive transcriptome analysis of tea plant axillary buds under different dormant statuses, we focus on the roles of auxin and dormancy related genes in winter bud dormancy to illuminate the dormancy mechanism of tea plant. The main results are listed as below:1. Identification and evaluation of reliable references genes for expression analysis under different conditions, especially for dormancy induction and auxin treatment. In this study, we examined the expression stability of eleven candidate reference genes based on six experimental series with 94 experimental samples. The expression stabilities of these candidate genes were ranked using four different computation programs. Results showed that the three commonly used housekeeping genes of Cs TUBUIN1, Cs ACTIN1 and Cs18 S r RNA1 together with Cs UBQ1 were the most unstable genes in all the ranking orders. However, Cs PTB1, Cs EF1, Cs SAND1, Cs CLATHRIN1 and Cs UBC1 were the top five appropriate reference genes for q RT-PCR analysis in complex experiential conditions.2. Transcriptome analysis of tea plant axillary buds under different dormant statuses by RNA-seq. 16,125 differentially expressed genes were discovered from transcriptome data among the comparisons between different dormant and sprouting stages. Gene ontology analysis results indicated that the differentially expressed genes were mainly grouped into cellular process, single-organism process, metabolic process, biological regulation, response to stimulus in biological process; cell, organelle, membrane and macromolecular complex in cellular component; binding, catalytic activity nucleic acid binding transcription factor activity, transporter activity and structural molecule activity in molecular function. Extremely differentially expressed gene analysis, gene set enrichment analysis(GSEA) and gene association network analysis results showed that plant hormones and dormancy related genes(MADS-BOXs and PEBP family) may pay important roles in winter bud dormancy formation and release in tea plant.3. The roles of Cs ARF1 in tea plant winter bud dormancy. The full-length c DNA of Cs ARF1 gene encoding 820 amino acids was firstly cloned from tea plants by RACE. Bioinformatics analysis results indicated that Cs ARF1 is a soluble protein which functions in the cytoplasm and consisted of an amino-terminal DNA-binding domain(B3), a carboxy-terminal dimerization domain(IAA_ARF), and a Gln, Ser and Leu-rich middle region, which was proposed to function as an activation domain. The expression analysis showed that Cs ARF1 had a marked rise in the expression level at deep dormant stage and sprouting stage, which demonstrated Cs ARF1 was relevant in the regulation of tea plant bud dormancy and bud break.4. The correlations between the expression changes of auxin related genes and the formation and release of winter bud dormancy in tea plant were discovered. To fully understand the roles of auxin in tea plant winter dormancy, 12 genes were selected from auxin signal pathway based on transcriptome analysis and previous studies. The expression pattern of chosen auxin related genes were detected under different conditions or treatments, including the axillary buds under different dormant and sprouting stages, dormancy induction treatment and auxinole treatment. Then the IAA concentration was also detected in the apical buds, axillary buds and the second leaves from top of tea branches. Results showed that most detected auxin related genes had significant expression differences under different conditions and treatments. Moreover, the IAA concentration in apical buds and axillary buds went down when tea plant went into endodormancy or ecodormancy from paradormancy, while the IAA concentration quickly increased during bud spouting stage in the spring. These indicated that auxin signal plays important roles in tea plant winter dormancy transition.5. Cs FLOWERING LOCUS T(Cs FT) gene played key roles in controlling flowering and growth cessation in tea plant. Cs FT is an important member of PEBP family. Two kinds of Cs FT transcripts named Cs FTa and Cs FTb were cloned successfully. Their expression was closely related to tea plant dormancy formation and release. Ectopic expression of Cs FTa in poplar showed extremely early flowering phenotype, and ectopic expression of Cs FTb in poplar showed functions in the inhibition of growth cessation and bud set induced by short day condition. Further study on FTs’ amino acid sequence indicated that the 100 th amino acid in Cs FT may play crucial role in binding to 14-3-3 to control flowering.6. Cs MADS-BOX genes were engaged into tea plant dormancy regulation. We cloned two Cs MADS-BOX genes from tea plant and performed further expression analysis and functional identification. Results show that the expression of Cs MADS-BOX1 was significantly down-regulated during dormancy formation and was up-regulated during dormancy release. Ectopic expression of Cs MADS-BOX1 in poplar inhibited the growth cessation of meristem during short day induction and showed sensitive feature to low temperature. Therefore Cs MADS-BOX1 may have important roles in dormancy controlling in tea plant in response to low temperature in the fall.