Lignin Mediated Defensive Responses Of Tea Plant (Camellia Sinensis) Against Empoasca Onukii (Matsuda) Attack

The small green leafhopper（Empoasca onukii（Matsuda））is one of the main pests of tea（Camellia sinensis（L.）O.Kuntze）in China.Its damage seriously affects the yield and quality of tea.The high-frequency application of pesticides to E.onukii in production will also cause tea quality and safety problems.Using resistant tea cultivars to control the population of E.onukii is an important content of comprehensive pest management in tea garden.At present,although the research on the resistance of tea plant to E.onukii has been widely carried out,it has not been in-depth.The research on the mechanism of lignin that participates in tea resistance against to insect is still blank.In this study,the resistant tea cultivar Juyan（JY）and the susceptible tea cultivar Enbiao（EB）selected before were used as experimental materials to compare and analyze the lignin content of fresh leaves,differentially expressed genes（DEGs）in lignin synthesis pathway,differential metabolites and related enzyme activity levels,micro distribution and deposition of lignin in cells in tea samples at different feeding time points,to reveal the mechanism of lignin that participates in tea resistance,which induced by feeding of E.onukii.The main results are as follows:1.Study on metabolism and transcriptome of resistant and susceptible tea cultivars in response to the damage of E.onukii.The transcriptome sequencing and metabolome determination of fresh leaf samples from different time points（0 h,48 h and 72 h）of tea varieties induced by continuous feeding of E.onukii were carried out.The following results were obtained:after continuous feeding of E.onukii,the number of DEGs of EB was higher than that of JY,and the number of DEGs of the two tea cultivars reached the highest level at 48 h;After E.onukii feeding,the metabolic pathways with significant enrichment of DEGs between JY and EB mainly include metabolic and secondary metabolic biosynthesis,flavonoid and phenylpropane biosynthesis,plant pathogen interaction,diterpene biosynthesis and plant hormone signal transduction.The expression levels of L-phenylalanine,4-coumaric acid,caffeic acid,coniferol,mustard aldehyde,ferulic acid,coumaric aldehyde,p-coumaric alcohol,coniferol,cinnamic acid and caffeic aldehyde in fresh leaves of JY and EB before and after E.onukii induction changed significantly.The expression levels of coniferol and L-phenylalanine increased in both tea cultivars,reaching the highest at 48 h,and the increase range of L-phenylalanine in JY was greater than that of EB;The expression of caffeialdehyde in JY and EB fresh leaves decreased significantly;The expression of erucic aldehyde,ferulic acid and coumarin aldehyde in JY decreased significantly,but increased significantly in EB.The results showed that the expression of L-alanine was the highest when it was combined with L-alanine biosynthesis in tea plants;After pest induction,the expression of POD in JY was higher than that in EB,but there was no significant difference between them;C4H positively regulated the production of ferulic acid.When JY was induced by E.onukii for 48 hours,the expression of C4H and ferulic acid decreased,and the expression of C4H and ferulic acid increased in EB for72 hours.CAD positively regulated the expression of 4-coumaric acid.When the expression of 4-coumaric acid in JY decreased significantly at48 hours of E.onukii induction and increased significantly after 72 hours of E.onukii induction,but the opposite trend appeared in EB.In addition,in EB,the expression level of CAD gene decreased with the increase of feeding time,but its expression in JY increased with the increase of feeding time.2.Changes of total lignin and related enzyme activities in the synthesis pathway of resistant and susceptible tea cultivars in response to the damage of E.onukii.The total lignin content and related enzyme activities of fresh leaf samples from different feeding time points（0 h,48 h and 72 h）of resistant and susceptible tea cultivars were measured.The results showed that after pest induction,the lignin content in JY and EB leaves increased,and the growth rate of JY was significantly higher than that of EB.It is speculated that the insect resistant varieties can regulate lignin synthesis faster in response to external pest stress;After pest induction,the activities of PAL,4CL,CAD,CCR,CSE,C4H,F5H,POD and HCT in fresh leaves of JY and EB were significantly increased.Whether in healthy tea plants or tea plants in pest treatment group,the activities of PAL,CAD,C4H and 4CL in JY were higher than those in EB,while the activities of CCR,CSE and HCT in EB were higher than those in JY.The total lignin content and related enzyme activities of fresh leaf samples from different time points（0 h,48 h and72 h）of resistant and susceptible tea cultivars induced by continuous feeding of E.onukii were measured.The results showed that after pest induction,the lignin content in JY and EB leaves increased,and the growth rate of JY was significantly higher than that of EB.It is speculated that the insect resistant varieties can regulate lignin synthesis faster in response to external pest stress;After pest induction,the activities of PAL,4CL,CAD,CCR,CSE,C4H,F5H,POD and HCT in fresh leaves of JY and EB were significantly increased.Whether in healthy tea plants or tea plants in pest treatment group,the activities of PAL,CAD,C4H and 4CL in JY were higher than those in EB,while the activities of CCR,CSE and HCT in EB were higher than those in JY.3.Distribution and deposition of lignin in resistant and susceptible tea cultivars induced by the damage of E.onukii.The distribution and deposition of lignin in the leaves of resistant and susceptible tea cultivars were compared and analyzed by using colloidal gold immunolabeling technology and Transmission Electron Microscopy（TEM）.It was found that the cell walls of JY and EB leaves without pest treatment were full and stretched,there was no separation of qualitative wall,no fillers and electronic compacts in the thick wall tissue,the internal structure of the cells was clear,and the chloroplasts were elliptical and arranged close to the cell wall.After the induction of pests,the cell wall structures of JY and EB leaves were damaged to varying degrees or severely distorted and deformed,the cytoplasm was thin and uneven,and the plasmodesm was severely separated,the cell membrane was damaged or even disappeared,the chloroplasts disintegrated in large numbers.The degree of damage to the cell wall of EB was more serious than that of JY.both JY and EB induced different degrees of uneven thickening on the parenchyma cell wall,and the range of uneven thickening in JY was higher than that of EB.A large number of osmophilic electron-dense substances were accumulated in the cells and spaces of the leaves of the two tea plants,and the electron-dense substances in the posterior wall tissue of JY were significantly more than those of EB;healthy and pest-induced tea plants were observed by colloidal gold immunoelectron microscopy.The deposition of lignin in leaves showed that a large number of dense colloidal gold particles were aggregated in the middle xylem and secondary xylem of the leaf cell wall of JY and EB after insect pest induction.Among them,the distribution of colloidal gold particles in the leaves of JY and EB was extremely uneven,and the lignin in the leaves of the two tea cultivars was distributed in an uneven pattern;among the CML layers,S₁layers and S₂layers of the parenchyma cell walls of JY and EB without pest treatment.A small amount of colloidal gold particles appeared in all of them,and the density of colloidal gold black particles aggregated in JY was higher than that in EB;after pest-induction,the colloidal gold particles aggregated in JY leaves showed a highly irregular deposition pattern,among which the S₁layer and the S₂layer.There was a large amount of dense deposition,that means a large amount of lignin is deposited in the S₁and S₂layers in JY,while the colloidal gold particles in EB appear more in the S₂and S₃layers,and less in the S₁layer,that means a large amount of lignin in EB is deposited in the S₂and S₃layers.