Physiological And Molecular Response Mechanism Of Zanthoxylum Bungeanum Seedling Under Low Temperature Stress

Zanthoxylum bungeanum Maxim.belongs to Rutaceae,which is an important economic tree species with both edible,medicinal and ornamental value.There are about250 species of Zanthoxylum all over the world.Z.bungeanum is rich in germplasm resources in China,which is about 41 species.China is a big country in the production and trade of Z.bungeanum.In recent years,as an important tree species to promote the development of agricultural economy,improve the ecological environment and increase farmers' income,Z.bungeanum has become the preferred choice.Low temperature is the main factor that limits the natural distribution and cultivation area of plants.Z.bungeanum is a kind of economic tree with low cold tolerance.Low temperature will affect the growth and development of Z.bungeanum,which is one of the important factors restricting the expansion of planting scope and industrialization development of Z.bungeanum.Therefore,cultivating varieties with strong cold tolerance is an important way to reduce the harm of low temperature,maintain the high and stable yield of Z.bungeanum and promote the development of industrialization.In this study,two varieties of Z.bungeanum: Fuguhuajiao and Fengxiandahongpao were selected,and the physiological phenotypes,transcriptional regulation and metabolites under low temperature stress were comprehensively explored and analyzed by means of transcriptome and metabolic technology.The purpose of this study is to provide a theoretical basis for revealing the physiological and molecular regulation mechanism of cold response of Z.bungeanum,and lay a foundation for promoting the breeding of resistant varieties.The main results are as follows:1.The physiological response mechanism of Z.bungeanum.Cell ultrastructure observation showed that the damage degree of organelle in Fengxiandahongpao was more serious than that in Fuguhuajiao.Under low temperature stress,POD enzyme activity and soluble protein of Fuguhuajiao were significantly higher than those of Fengxiandahongpao.The results of OPLS-DA analysis showed that POD and soluble protein VIP values were both greater than 1,which could be used as physiological substances to distinguish the two varieties.In addition,Fuguhuajiao and Fengxiandahongpao have different physiological substances in response to low temperature respectively.POD,MDA and free proline are important physiological regulatory substances in Fuguhuajiao.The important differential physiological regulatory substances played roles in different time periods during low temperature stress in Fengxiandahongpao.The antioxidant system and osmotic regulatory substances in Z.bungeanum play important roles in cold response.2.Based on the physiological response indexes and transcriptome data of the two Z.bungeanum varieties under cold stress,the co-expression network and cold response model were constructed.The core genes related to cold response in Z.bungeanum were identified.There were 3513 differentially expressed genes in Fuguhuajiao and 25157 differentially expressed genes in Fengxiandahongpao under low temperature stress.The weighted co-expression network(WGCNA)analysis showed that MEfirebrick4 module,which had a high correlation with physiological index POD,was enriched with multiple cold response genes and pathways.Under low temperature stress,Z.bungeanum can respond to cold through signal transduction,plant hormones,lipid metabolism and carbohydrate metabolism.Furthermore,endocytosis related gene ERD7,abscisic acid pathway related gene PP2 C,late embryonic development rich protein gene LEAD-29 and two plasma membrane/lipid raft related genes ZB01477 and PP34 were located at the core of the network,which played key roles in the cold response of Z.bungeanum.3.Analysis of hormone components and response profile of two Z.bungeanum varieties under cold stress.A total of 45 hormone components were detected in the leaves of two Z.bungeanum varieties.The abscisic acid,abscisic acid glucose ester,indole-3-formaldehyde,L-tryptophan,1-aminocyclopropane carboxylic acid,salicylic acid and salicylic acid-2-O-?-glucoside in were main hormone components in both two varieties.OPLS-DA results showed that the important hormone components varied in Fuguhuajiao and Fengxiandahongpao.There were intersections and independent response pathways in hormone response.The hormone response for Fengxiandahongpao responsing to cold stress at 6 h treatment was the strongest.WGCNA analysis showed that hormones,as upstream signals of low temperature stress,were associated with other response pathways in plants,including carbon metabolism,fatty acid metabolism,sugar metabolism,amino acid metabolism and other signal transduction pathways.Two hormone-related core genes ZB43608(a TGA transcription factor in the salicylate hormone signaling pathway)and ZB56627(a two-component response regulator ARR11 in the cytokinin signaling pathway)were identified.4.Effects in fatty acids profiling of Z.bungeanum by low temperature.Twelve main fatty acids were detected in the leaves of two Z.bungeanum varieties.Fuguhuajiao and Fengxiandahongpao contain more palmitic acid and linolenic acid.OPLS-DA results showed that the important fatty acids in two varieties were inconsistent.Fuguhuajiao preferred to synthesize unsaturated fatty acids,while Fiandahongpao preferred to synthesis saturated fatty acids under cold stress.Fatty acid synthesis pathway genes FATA,SLD2,CER3 and FAD3 were identified as the key genes affecting the fatty acid composition in cold tolerant variety Fuguhuajiao,while SLD2,KAS I,FATB and FAD5 were key genes in cold sensitive variety Fengxiandahongpao under low temperature stress.5.Whole genome analysis of type 2C protein phosphatase PP2 C family.A total of 198 PP2 C family members with conserved domains were annotated in the genome of Z.bungeanum,and the protein encoded by this family gene was 155?1083aa in length and 17.58?121.99 KD in molecular weight.Phylogenetic analysis showed that the PP2 C family genes could be divided into 13 subfamilies.The genetic structure of the same subfamily is similar.The PP2 C family genes were distributed on 36 chromosomes,and there were multiple gene clusters.The gene expression patterns of PP2 C family varied in the two varieties of Z.bungeanum under low temperature stress.