Functional Analysis Of Green Leaf Volatile And Histone H3K4 Methylation In Response To Drought Stress In Tea Plant

Tea plant is an important economical crop in south China.During the growth process of tea plant,it is often affected by abiotic stresses such as drought,which leads to the decline of tea quality and yield,and even the large-scale death of tea plant.At present,researches on drought tolerance of tea plant mainly focused on the physiological and biochemical changes such as morphological changes,photosynthesis,osmotic pressure regulators and related molecular mechanisms,while the regulation of volatile compound and histone methylation was largely unknown.In this study,we screened volatile pathways response to drought stress via transcriptome and volatile component analysis,quantitative real-time PCR（qRT-PCR）was used to verify the changes in volatile metabolic pathways,volatile component pretreatment was performed to verify its function in drought tolerance,exogenous ABA and volatile component spraying were used to explore the molecular regulatory pathways of green leaf volatiles.Meanwhile,the functions of histone methyltransferase genes（CsATX4s）under drought stress were investigated by CDS full-length sequence cloning,Arabidopsis thaliana mutant functional complements and Arabidopsis thaliana wild-type overexpression.The results are as follows:（1）It was found that 7-day drought treatment significantly decreased total volatile content in tea leaves.Nevertheless,（Z）-3-hexen-1-ol,（E）-2-hexenal and methyl salicylate were accumulated.Transcriptome analysis suggested the activation of linolenic acid degradation and methyl salicylate processes.The increased expression of enzyme and transcription factor genes,including 13-lipoxygenase（13-LOX）,hydroperoxide lyase（HPL）,alcohol dehydrogenase（ADH）,hexenal isomerase（HI）,NPR1 and TGA,was further confirmed by qRT-PCR analysis after drought treatment.Surprisingly,exogenous（Z）-3-hexen-1-ol pretreatment dramatically enhanced drought tolerance of tea plants,whereas（E）-2-hexenal and methyl salicylate did not exhibit such a function.qRT-PCR analysis revealed that exogenous ABA did not induce the expression of enzyme gene related to（Z）-3-hexen-1-ol production.Drought and exogenous（Z）-3-hexen-1-ol could up-regulate DREB2A-2 expression,which might be a key ABA-independent transcription factor in drought response of tea plants.Moreover,exogenous（Z）-3-hexen-1-ol tremendously induced the expression of specific LOX and ADH genes within 24h.（2）With genome and transcriptome data,we identified ATX4 homologous gene in tea plant,analyzed CsATX4-1 and CsATX4-2 protein structure domain.Phylogenetic tree was used to analyze the evolutionary relationship of ATX4 protein in various species,and the CDS full-length sequences of CsATX4-1 and CsATX4-2genes were cloned.The overexpression vectors of CsATX4-1 and CsATX4-2 genes were constructed and transferred into Arabidopsis thaliana wild type and atx4 mutant,and 5 T₃-generation homozygous lines were screened.Overexpressed CsATX4-1 and CsATX4-2 transgenic Arabidopsis thaliana plants showed negatively drought-tolerant phenotype.