Mechanism Of Exogenous Nitric Oxide Mediating The Enhancement Of Tomato Defense Against Spodoptera Litura

Nitric oxide(NO)plays a key role in promoting seed germination,root growth,regulating stomatal opening and closing,alleviating leaf senescence,and enhancing plant resistance to biotic and abiotic stresses.Exogenous NO donor sodium nitroprusside(SNP)could be degraded into nitrite and release NO in the plant.In the natural environment,SNP is easy to dissolve in water and easily degrade when exposed to the light.In addition,quick action,fast residue degradation,and low price of SNP could meet the requirements of green and safe agriculture.However,few report investigates the mechanism of NO mediated plant defense against herbivorous insects.In this study,exogenous nitric oxide donor SNP was used to pretreat tomato(Solanum lycopersicum),which were then inoculated with Spodoptera litura larvae.The suitable SNP concentration and treatment time were selected by observing the feeding phenotype of tomato leaves and the weight increment of caterpillars.Phytohormone content,defense-related enzyme activities and related gene expression were measured in both wild type tomato(cv.Castlemart,CM)and LOXD-mutated jasmonic acid synthesis defective mutant tomato Suppressor of prosystemin-mediated responses 8(spr8).The results showed that tomato resistance to the larvae of S.litura was enhanced by pre-treatment with SNP.Finally,the hormone signaling pathways induced by exogenous NO were analyzed through transcriptome analysis.The detailed results are as follows:(1)Pretreatment with SNP at a concentration of 100 μM for 12 h enhanced tomato anti-herbivore resistance without affecting tomato growth.To screen suitable concentration of SNP,SNP concentrations of0 μM,100 μM,200 μM,400 μM and 800 μM were set up and used to treat tomato seedlings,respectively.After pretreatment with SNP for 12 h,the tomato plants were inoculated with S.litura larvae for 24 h.Based on the larval body weight gain and the phenotype of tomato leaves treated with SNP,100 μM was the desired SNP concentration to inhibit larval growth without affecting the growth of tomato.For three time points including 3 h,12 h and 24 h,tomato treated with SNP for 12 h was enough to enhance toamto defenses aganist S.litura larvae..(2)Exogenous NO donor SNP improved the resistance of tomato to S.litura larvae.After feeding on SNP-treated tomato,the weight gain of the third instar larvae was decreased by 41.1% compared with that of the controls.Compared with the control group,the activities of PPO,POD and PAL were increased by 1.26 times,1.29 times and 1.29 times,respectively in the SNP-treated group.The transcript levels of jasmonic acid(JA)synthesis genes AOC and LOXD,protease inhibitor genes(PI-1and PI-2),genes encoding threonine dehydrogenase(TD)and leucine ammonia-lyase(Lap A)were significantly increased at 6 h and 12 h after SNP application.The contents of JA and jasmonic acid-isoleucine(JA-Ile)were significantly increased from 6 h to 24 h.(3)Synthesis of defective mutant tomato spr8 by JA was used to analyze the mechanism of SNP in tomato insect resistance.The mutant spr8 also showed resistance to insects under SNP treatment,and the body weight gain of the third instar larvae decreased by 31.2% compared with the control group.The enzyme activities of PPO and POD were increased by 1.23 times and 1.27 times,respectively.The hormone contents of JA and JA-Ile were increased by 3.95 times and 1.76 times,but not as much as the upregulation factor of wild-type tomato CM(10.75 times and 7.7times).Compared with wild-type tomato plants,spr8,the change trend of insect resistance index and related gene expression was the same,but the change amplitude of spr8 was not as significant as CM.Further transcriptomic analysis of tomato samples including control group(CK),SNP treatment group(SNP),CK＿SL-tomato plants only damaged by S.litura larvae,SNP＿SL-tomato plants treated with SNP and damaged by S.litura larvae.The results showed that SNP treatment not only induced genes associated with the JA signaling pathway(LOXD,AOC,AOS,OPR3,MFP2,ACXX gene family,JAR1,COI,JAZ,MYC2),but also activated genes associated with the ethylene(ET)pathway(SAM,ACO and ACS,ETR,SIMKK,EBF,EIL and ERF)and glutathione synthesis and most gene expressions of its signal transduction pathway(GSH,GST and GR gene families).(4)In the follow-up verification assay by q RT-PCR,nine differential genes(LOXD,AOC,AOS,JAZ,MYC2,ACS,ACO,ETR4 and ERF1)were selected.It was confirmed that exogenous NO enhanced tomato resistance to S.litura larvae through JA and ET signaling pathways.We also investigated the content of S-nitroso-L-glutathione(GSH),the synthetic precursor of S-nitroso-L-glutathione(GSNO),the glutathione S-transferase(GST)and S-nitroso-L-glutathione reductase(GSNOR).It was found that SNP-treated GSH,GST and GSNOR enzyme activities were significantly increased(1.17 times,1.12 times and 1.10 times),which suggested that S-nitroso protein modification might exist.In summary,this study found that exogenous NO enhanced tomato resistance against S.litura larvae by activation of JA and ET signaling pathways,which may provide a novel method for integrated pest control.