Drought Response Mechanism Of Zanthoxylum Bungeanum And Functional Analysis Of The Key Genes

Zanthoxylum bungeanum Maxim.is a member of the family Rutaceae.Z.bungeanum pericarp is fragrant and numbed,which is an important condiment in cooking.Z.bungeanum pericarp is also a kind of traditional Chinese medicine with anti-inflammatory and analgesic effects and also health care value of anti-aging and anti-cancer.In recent years,Z.bungeanum has been widely planted in an area of 25 million mu nationwide,especially in drought areas in the north of China.Z.bungeanum plants have drought resistance,and there are obvious drought-resistant differences among cultivars.The molecular mechanism of drought response of Z.bungeanum is essential for breeding drought-resistant Z.bungeanum germplasm.In this study,the representative of southern germplasm resources"Fengjiao"and the representative of southern germplasm resources"Hanjiao"were applied.The effects of drought stress on physiological indicators of"Fengjiao"and"Hanjiao"were determined,and the metabolome and transcriptome were also conducted.The integrative analysis of transcriptome and metabolome revealed the drought response mechanism and sheltered drought-resistant genes.Additionally,the functions of two key genes（ZbCPD and ZbROT3）in Z.bungeanum were analyzed.The main research results are as follows:1.Drought treatment decreased stomatal aperture and intercellular CO₂ concentration,inhibiting the net photosynthetic efficiency;increased the contents of hydrogen peroxide（H₂O₂）and malondialdehyde（MDA）,and enhanced the activities of catalase（CAT）,superoxide dismutase（SOD）and peroxidase（POD）;decreased the relative water content and elevated the content of soluble sugar,soluble protein and proline.Compared with Fengjiao,the relative water content,chlorophyll content,osmotic substance content and intercellular carbon dioxide concentration in Hanjiao leaves were higher.Additionally,the antioxidant enzyme activity and net photosynthesis rate were stronger,while the H₂O₂ and MDA contents were lower in Hanjiao than in Fengjiao.The results indicated that Hanjiao were with higher drought resistance than Fengjiao.2.Transcriptome data showed that the differential genes（DEGs）under drought stress were mainly enriched in plant hormone signal transduction（ko04075）,starch and sucrose metabolism（ko00500）,photosynthetic signaling protein（ko00196）,photosynthesis（ko00195）,eukaryotic ribosome biogenesis（ko03008）,flavonoid biosynthesis（KO00195）.ko00941)and so on.Widely targeted metabolome identified 911 metabolic components in Z.bungeanum leaves,including 232 flavonoids,131 phenolic acids,119 lipids,82 alkaloids,72organic acids,56 amino acids,50 nucleotides,49 lignans and coumarins,49 sugar alcohols,24 tannins and 16 terpenoids.Drought increased the contents of most amino acids and flavonoids in Fengjiao;as to Hanjiao,drought mainly promoted the accumulation of amino acids and alkaloids.In addition,drought stress aggravated the degradation of organic acids in both two Z.bungeanum.The key drought-resistant genes in Z.bungeanum were screened by the intergrative analysis,and the drought response mechanism of Z.bungeanum was revealed:drought stress caused ROS accumulation in Z.bungeanum plants;ROS,as a secondary signal,activated transcription factors responding to drought stress through hormone signal transduction and MAPK signal transduction;transcription factors further activate the expression of downstream genes that contribute to drought response.3.In this study,we cloned the full-length sequence of ZbCPD（constitutive photomorphogenesis and dwarfism）in Z.bungeanum.The open reading frame（ORF）length of ZbCPD was 1416bp and encoded 471 amino acids.ZbCPD protein was localized in the endoplasmic reticulum.Two ZbCPD transgenic Arabidopsis lines and two CPD mutant lines were obtained by genetic transformation and CRISPR,respectively.The absence of CPD significantly reduced the content of BRs in Arabidopsis and seriously influenced the morphology;the overexpression of ZbCPD increased the content of BRs and had no influence to morphology.Under drought conditions,ZbCPD enhanced the activities of CAT,POD and SOD,increased the contents of proline,soluble sugar and soluble protein,decreased the contents of H₂O₂ and MDA,reduced stomatal opening and stomatal density,and increased the water content of leaves and the survival rate of plants.4.The ZbROT3（rotundifolia 3）ORF is 1476bp and encodes 491 amino acids.Subcellular localization showed that the ZbROT3 protein functioned in endoplasmic reticulum.Two ZbROT3 transgenic Arabidopsis lines were obtained by gene transformation technique;two ROT3 mutant lines were obtained by CRISPR.The absence of ROT3decreased BRs content in Arabidopsis thaliana,caused shorter petiole and wider leaf shape,and weakened its drought resistance.Overexpression of ZbROT3 increased BRs content in Arabidopsis thaliana.Under drought conditions,ZbROT3 inhibited water loss by increasing the contents of proline and soluble protein,and decreasing stomatal opening;reduced oxidative damage of ROS to plant cells by enhancing POD and CAT activities.Overexpression of ZbROT3 significantly increased the relative water content of leaves under drought and the survival rate after rehydration,indicating that ZbROT3 enhanced the drought-resistance ability of plants.