The Defense Response Of Tea Plants To Green Leafhopper Infestation: A Multi-omics Study

Green leafhoppers(Empoasca(Matsumurasca)onukii Matsuda),as one of the main pests in the tea garden,greatly impact the yield and quality of tea products.On the other hand,Oriental Beauty tea,which wins warm praise from customers for its ripe fruity aroma and honey taste,is made of fresh tea leaves infested by green leafhoppers.Therefore,the interaction between tea plants and green leafhoppers is unique and complex.The defense responses and resistance mechanisms of tea plants to chewing insects have been thoroughly studied.However,relatively few studies have focused on the stress response of tea plants to piercingsucking insects.In recent years,the use of omics technology has inspired the understanding of tea-insect interactionsin in the gene and metabolite levels,and has improved the understanding of the molecular mechanisms behind the various resistance strategies.In this study,mechanicallydamaged(MD),leafhopper-damaged(LD)and untreated tea leaves(CK)were harvested for metabolomics analysis and transcriptome sequencing.We studied the changes of a series of metabolites and phytohormones in tea plants infested by green leafhoppers,and explored the differences in gene expression during the interactions between the tea plant and the green leafhoppers,in order to unravel the defense responses of tea plants against green leafhopper feeding from different aspects of genes,metabolites,signal transduction and regulation.The current study will provide a theoretical basis to further research on insect resistance of tea plants.The results are as follows:1.Effects of green leafhopper infestation on the metabolic profiles of fresh tea leavesNon-targeted metabolomics analysis of tea samples were performed by ultra-high performance liquid chromatography-time-of-flight mass spectrometry(UPLC-QTOF MS).After screening,a total of 23 differential metabolites were identified,including flavanol,flavoneglycosides,flavanone glycosides,flavonol glycosides,amino acids,peptide,phenolic acids,hydrolyzable tannins,glycosidically bound volatiles and proanthocyanidins.The flavanols(EGCG,EGC,ECG,EC,EGCG3"Me and ECG3"Me)showed a significant increase in the LD group.Levels of three proanthocyanidins(EGC-ECG dimer,EC-ECG dimer and prodelphinidin A2 3?-gallate),one flavone(tricetin)and one flavone glycoside(C-hexosyl-apigenin O-rhamnoside),one flavonol glycoside(kaempferol 3-O-glucosylrutinoside)and one hydrolysable tannin(methyl 6-O-galloyl-?-D-glucose)were also increased in the LD group.The results indicated that flavonoids played an important role in the stress defense of tea plants against green leafhoppers.On the contrary,the content of glutathione and theanine showed a downward trend after the infestation of green leafhoppers.Amino acids and catechins in tea samples were quantified by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLCQqQ MS).The results showed that the total catechin content in different samples was: LD>MD>CK.Compared with CK,catechins including EGC and ECG significantly increased in concentrations in the MD group,while EGCG,EGC,ECG,EC and EGCG3"Me significantly increased in the LD group.The total amino acid content in different samples was: CK>MD>LD.The content of amino acids such as glutamate and theanine decreased after green leafhoppers infestation.Caffeine presented no dramatic change in response to herbivore attack and mechanical attack.2.Effects of green leafhopper infestation on the phytohormones of fresh tea leavesPhytohormones in tea samples were quantified by UPLC-QqQ MS.Compared with CK and MD,the content of salicylic acid(SA)increased significantly in LD.Compared with CK,the content of jasmonic acid(JA)showed an upward trend after green leafhopper infestation.The content of abscisic acid(ABA)and indoleacetic acid(IAA)among different treatments was relatively stable.SA and JA are important phytohormones for inducing defense response in plants.This study showed that the signaling pathways of SA and JA were simultaneously activated in tea plants after green leafhoppers feeding.3.Effects of green leafhopper infestation on the gene expression level of fresh tea leavesThe RNA-Seq technology was used to analyze transcriptome reprogramming in tea plants undergoing different treatments.In comparison to the undamaged control,2,876(1,826 up-regulated and 1,050 down-regulated)and 588(384 up-regulated and 204 downregulated)genes were found to be differentially expressed in LD and MD groups.Compared with the MD group,the LD group contained 2,019 DEGs,of which 1,395 were up-regulated and 624 were down-regulated.Among three pairwise comparisons,only 86 genes were commonly regulated,whereas 45.5%(1,309/2,876)and 26.0%(153/588)of DEGs were exclusively modulated by green leafhoppers and mechanical damage,respectively.The differentially expressed genes were classified and enriched by KEGG.We found that the up-regulated genes induced by green leafhoppers were significantly enriched in “phenylpropanoid/flavonoid biosynthesis”,“plant-pathogen interaction”,“plant hormone signal transduction” and “cutin,suberine and wax biosynthesis”.In the catechin biosynthetic pathway,the key genes such as phenylalanine ammonia-lyase gene(PAL),4-coumarate-CoA ligase gene(4CL),chalcone isomerase gene(CHI),flavonoid 3?-hydroxylase gene(F3?H),flavanone 3-hydroxylase gene(F3H),flavonol synthase gene(FLS),dihydro flavonol 4-reductase gene(DFR)and leucoanthocyanidin reductase gene(LAR)all dramatically increased in LD.The result was consistent with patterns of catechin variations in metabolomics analysis.Genes involved in the biosynthesis of SA(e.g.,PAL)and JA(e.g.,LOX,AOS,OPR and ACX)were up-regulated on green leafhopper-infested tea plants.It indicates that the infestation of green leafhopper activated SA and JA biosynthetic pathways.This result was consistent with the changes in SA and JA contents.Interestingly,the transcript TEA008365 in LD increased by 21.31-and 41.20-fold compared with CK and MD.This gene encodes a fatty aldehyde decarbonylase(CER1 protein),which is involved in the synthesis of wax alkane and plays an important role in the resistance of many plants to biotic and abiotic stresses.The response of plants to environmental stress is mainly regulated by transcription factors(TFs)and as a result.we further analyzed the changes in transcription factor expression.The results showed that 228 differentially expressed TFs were found in comparison of LD with CK,in which 153 were up-regulated and 75 were down-regulated.Comparisons of LD with MD identified 213 differentially expressed TFs,in which 138 were upregulated and 75 were down-regulated.The up-regulated TF families in LD included WRKY(24),AR2/ERF(24),bHLH(18),NAC(10)and GRAS(10).WRKY,AR2/ERF and MYC2(belonging to bHLH family)were closely related to JA and SA defense signalling.NAC90,NAC2,SCL21 and SCL33,members of NAC and GRAS TF families,were substantially up-regulated in LD.These TFs could be regarded as potential candidates for regulating the defense against green leafhoppers.Finally,Thirteen DEGs were selected for qRT-PCR,which showed consistent results with RNA-Seq analysis.Therefore,the RNA-Seq results were reliable.In summary,this study comprehensively analyzed the changes of metabolites,genes,transcription factors and hormones in tea plants under infestation of green leafhoppers.There were a series of changes in the gene expression and metabolic profile of tea plants after green leafhoppers feeding: the genes of phenylpropane and flavonoid metabolic pathways were significantly up-regulated,promoting the synthesis and accumulation of various flavonoids;The multiple protein-coding genes interacting with pathogens were significantly up-regulated,which may increase the resistance of tea plants to biotic stress;the genes involved in leaf wax alkane synthesis were significantly up-regulated,enhancing the ability of tea plant physical defense.In addition,SA and JA signaling pathways involved in plant defense were simultaneously activated,which were signaling molecules that played a leading role in tea plants defense response to green leafhoppers.This study provided a theoretical basis for controlling green leafhoppers in tea garden and help to improve the utilization of tea fresh leaf resources and the pre-harvest treatment technology of oolong tea by regulating the interaction between insects and tea plants.