Insight Into The Molecular Basis Of Ocimene-primed Plant Defense

In response to biotic stresses,the plants have evolved a complexed networking system fine-tuning their defense responses.To date,the defense networking is constructed by signaling pathways of phytohormones including salicylic acid,jasmontae,ethylene,abscisic acid and brassinosteroids,etc.However,hyperactivation of these pathways often results in strong induction of defense genes,mounting up excessive defense responses tends to be wasteful of carbon resources,which further impedes the growth and development of plants.Moreover,the antagonism among different hormones imposes mutual inhibitive effects and makes it impossible to simultaneously activate multiple pathways to counteract a combinational invasion by multiple stresses.In the present study,we demonstrated that ocimene,a common signal molecule in the volatile blends emitted by herbivore-attacked plants,imparted wide-spectrum resistance against pathogens and herbivores in many plant species.The induced defense patterns were distinguished from the reported defense patterns induced by phytohormones.Ocimene primarily primes plant defense without sacrificing plant growth.Genetic analyses suggested the priming by ocimene was independent of signaling of plant hormones while the full-scale activation upon subsequent attacks required the intact signaling pathways of hormones.Using methodologies of biochemistry,molecular biology,genetics and plant pathology,our study presents several major points as follows.1)Using Arabidopsis thaliana,Brassica napus,Nicotiana benthamiana,Solanum lycopersicum,Solanum tuberosum,Cucumis sativus,Artemisia annua and Oryza sativa as models,application of exogenous ocimene enhanced the plant defense against fungi,bacteria and phytophagous insects.2)Phenotyping,histochemical analysis,bioassay and gene expression analysis in ocimene-primed Arabidopsis thaliana suggested biomass,auxin signaling,flowering time,seed yield as well as ROS homeostasis was not affected by ocimene treatment.Moreover,the enhance defense was possibly not due to strengthend physical barriers such as callose depositon and trichome density.Compare to the non-treated plants,ocimene primed Arabidospis thaliana against biotic stresses with stronger responses,which was indicated by more robust expression of defense-related genes under stress.3)Transcriptomic analysis revealed that a plethora of differentially expressed genes in ocimene-treated Arabidopsis were categorized as SA-,JA-or ET-responsive genes,suggesting a simultaneous activation of multiple hormone pathways primed plant defense.Moreover,there were many unreported genes with no functional annotations existed in the DEGs,indicating the difference between ocimene with well-documented hormones.Analyses in the mutants with defective signaling of hormones,we demonstrated that ocimene enhanced defense responses against Pseudomonas syringae in the absence of NPR1 and SID2,which was deprived of SA receptor and blocked in SA accumulation respectively.The defense priming was not obvious impaired in the absence of COI1,AOS or MYC2.Both primed etr1 and Col-0 showed enhanced defense agaisnts Plutella xylostella larvae,and ocimene primed the expression of PDF1.2 even in etr1.4)Using RT-qPCR to investigate expression patterns induced by ocimene,we have found that expression of FMO and SUR1 was constantly increased in 6 hours in a time-logging pattern,which reached the peak after 4 to 5 hours.Biochemical measurements revealed a significant increase of total glucosinolates in leaves of Col-o,npr1 or coi1 plants.Non-targeted metabolomic analysis of ocimene-conferred priming suggested a set of secondary metabolites and their precursors might be involed in the modulation of plant defense,as intrinsic signals or chemical weapons.5)Histochemical results showed expression of FIBO,which was quickly expressed in response to ocimene,was upregulated by SA,JA,ACC,wounding and ocimene,suggesting a possible role involved in ocimene-conferred resistance.Through analyzing the promoter of SDG8 encoding a histone methyltransferase,we demonstrated that the activity of SDG8 was increased by ocimene.Most interestingly,ocimene enhanced plant resistance against Pseudomonas syringae though the induction of PR1 gene in sdg8 was impaired.The results suggested the defense priming by ocimene was indicated by PR1 expression,which was not the primary causative mechanism underlying defense against bacterial pathogens.6)To further support the independence of ocimene signaling from hormonal signaling and obtain mutants impaired in ocimene signaling via EMS-based forward genetics,three ocimene-responsive reporter systems including proFIBO-GUS,proPR1-LUC and proPDF1.2-LUC were introduced into backgrounds with blocked SA,JA/ET signaling respectively.Our results are fundamental to further identification of mutations abolishing the unique signaling pathway of ocimene.