CALMODULIN1 And WRKY53 Function In Linalool-mediated Defense To Plutella Xylostella By Regulating The Jasmonate Biosynthesis Pathway In Arabidopsis

Linalool,a volatile with fragrance,belongs to terpene which could be induced when plants are in biotic stresses.Previous study has shown that a lot of volatiles including linalool were emitted by insect injured.And other studies illustrated another point,linalool which induced by herbivory insect injury can also increase plant resistance.In this study,we test H₂O₂ levels and calcium concentrations in Arabidopsis mesophyll cells by performing confocal laser scanning microscopy,and calcium flux by performing non-invasive micro-test technology（NMT）.We identify interactions between Calmodulin1（CAM1）and WRKY53,Calmodulin3（CAM3）and Ca²⁺-ATPase isoform 8（ACA8）through yeast two-hybrid,luciferase complementation imaging,and pull-down assays.And we use EMSA and Luciferase activity assay to explore interactions between WRKY53 and genes’promoters of lipoxygenases,LOX3 and LOX4.In addition,we detect defense gene and jasmonate biosynthesis pathway gene expressions by performing q-PCR,and JAs concentration by performing LC-MS.The purpose of this study is to explore the process of linalool-induced insect resistance from early signal to jasmonate biosynthesis in Arabidopsis.The main conclusions are as follows:1.To confirm that linalool induces resistance to Plutella xylostella in Arabidopsis,we inoculated WT plants with Plutella xylostella larvae after 24 h of linalool treatment;the control group was treated with ethyl alcohol.After 7 days,we measured the length and weight of Plutella xylostella larvae.Both growth indicators were significantly lower in linalool-treated plants than in control plants.In addition,the plant leaf damage was also more severe in linalool group than control group.These results indicate that linalool induces plant defense against Plutella xylostella.In addition,we measured the expression of defense genes,PDF1.2,THI2.1,VSP1,and VSP2 in WT plants following linalool treatment for 0.5 h,2 h and 8 h.Almost all genes expressed in the WT.2.When Arabidopsis recognizes linalool,H₂O₂ levels in leaf mesophyll cells rapidly increase.We found that this H₂O₂ burst was inhibited in leaves of WT plants pretreated with the NADPH oxidase inhibitor diphenyleneiodonium chloride（DPI）and in leaves of the NADPH oxidase mutant rbohd.Then intracellular calcium store channel,TPC1,activated and Ca²⁺concentration increased.3.CAM1 interacts with WRKY53,WRKY53 affects the transcription of LOX3 and LOX4 by activating LOX3 and LOX4 promoters,then the jasmonate（JA）biosynthesis genes（LOX2,LOX3,LOX4,OPR3（OPDA reductase 3）and JMT（Jasmonate carboxyl methyl transferase））,JA and OPDA（cis（+）-12-oxophytodienoic acid）levels increased more in the linalool-treated group than in control group.4.By examining Ca²⁺flux,we showed that linalool-treated mesophyll cells exhibit strong Ca²⁺efflux.Therefore,we investigated whether the Ca²⁺-ATPase inhibitor Eosin B would inhibit this Ca²⁺efflux.Our findings indicate that Ca²⁺-ATPase participates in linalool-induced Ca²⁺efflux in mesophyll cells and that Ca²⁺efflux is inhibited in cam3 plants.And CAM3 binds to the N-terminus of ACA8 and functions in the Ca²⁺efflux process.Previous studies have indicated that linalool can enhance plants’resistance,but few researches about how to increase the resistance was reported,which was the aim of this work.In this study,we confirmed that linalool can enhance the intracellular calcium concentration in leaves,then CAM1 combined with WRKY53 to active JA biosynthesis pathway to increases JAs concentration,naturally enhancing the insect resistance of Arabidopsis,then CAM3 combined with ACA8 to transfer Ca²⁺out of cytoplasm.This result indicated that JA biosynthesis pathway plays an important role in linalool-induced resistance in Arabidopsis.it also provides a new idea on how plant volatiles induce plant defense response.