Eugenol Confers Resistance To Cadmium Stress By Triggering H₂S Immune Signaling In Brassica Rapa

Eugenol,a small natural plant-derived compound,has been widely used in food protection and medical studies.Our laboratory firstly reported that eugenol was capable of regulating plant innate immunity against virus infection.Given the comprehensive important role of plant immunity in the fight against biotic and abiotic stress,the present study investigates the regulatory role of eugenol against cadmium?Cd?toxicity belonging to abiotic stress.Hydrogen sulfide?H₂S?acts as a pivotal gaseous signal molecule to be involved in the regulation of multiple important physiological processes in both animal and plant cells.The present study reveals that eugenol confers Cd tolerance by regulating plant immune responses modulated by endogenous H₂S.Basically,the following results are obtained.1.The root of Brassica rapa seedling was selected in this study.Compared to the control,treatment with CdCl2?10 ?mol/L?resulted in the significant decrease in root elongation by about 50%.However,simultaneous treatment or pre-treatment with eugenol?20 ?mol/L?significantly alleviated root growth stunt induced by Cd stress.2.The total reactive oxygen species?ROS?,hydrogen peroxide?H2O2?,and cell death in root tip were detected in situ by using DCFH-DA,HPF,and PI fluorescent probes,respectively.In addition,Schiff's reagent,Evans blue,and Trypan blue were applied to detect lipid peroxidation,loss of membrane integrity,and cell death in root tip,respectively.The results showed that eugenol remarkably ameliorate Cd-induced over-generation of ROS and H2O2,lipid oxidative injury,and cell death,suggesting that eugenol may trigger the endogenous immune signals to resist Cd stress efficiently.3.Cd stress led to the significant increase in the content of endogenous H₂S in root tip,which can be selectively detected by using specific fluorescent probe WSP-1.Exogenous treatment with NaHS?a H₂S donor?significantly attenuated Cd toxicity,suggesting that H₂S played important role in the regulation of Cd tolerance.Compared to Cd treatment alone,simultaneous treatment or pre-treatment with eugenol induced further significant increase in the endogenous H₂S in root tip.However,the addition of PAG?H₂S synthesis inhibitor?or HT?H₂S scavenger?resulted in the decrease in endogenous H₂S level under Cd or eugenol treatment,which further blocked the alleviated effect of eugenol on Cd toxicity?e.g.root inhibition,oxidative stress,and cell death?.4.The free Cd²⁺ in root tip was detected in situ by using specific fluorescent probe Leadmium Green.It was found that eugenol pronouncedly decreased free Cd²⁺ content in Cd-treated root by regulating endogenous H₂S.Glutathione?GSH?in root tip was measure by using biochemical assay and GS-bimane fluorescent detection.Eugenol significantly enhanced GSH content in Cd-treated root by regulating endogenous H₂S.These results indicated that GSH was an important downstream of H₂S in eugenol-activated immune pathway against Cd stress because GSH not only chelates free Cd²⁺ but also help scavenging the accumulated ROS.5.D-cysteine desulfhydrase?DCD?is one of the important enzymatic sources for the generation of endogenous H₂S in plants.In the present study,the full length cDNA of Br_DCD1?1146 bp?was cloned from B.rapa.The typical DCD catalytic structure was revealed by the analysis of amino acid sequence and protein structure of Br_DCD1.The promoter region of Br_DCD1 gene was full of stress-responsive cis-elements?including salicylic acid?SA?-responsiveness?,indicating the important role of Br_DCD1 in response to environmental stresses.Additionally,there were many kinds of phosphorylated modification sites in Br_DCD1,suggesting that the activity of Br DCD1 can be regulated in post-translational level.Gene expression analysis suggested that eugenol significantly stimulated the expression of Br DCD1 in Cd-treated root.This is consistent with the changes of endogenous H₂S level in root tip.Combined with the previous study that eugenol regulated SA against virus infection,it can be speculated that SA may be the common regulatory target in eugenol-conferred resistance to both biotic and abiotic stresses.In sum,this study firstly identified the pivotal role of eugenol in the regulation of Cd tolerance,which was closely associated with H₂S-meidated immune signaling pathway.This study provides important reference information for the development of eugenol as a broad-spectrum plant immune regulator.