Mechanism Research On Thymol And--Cinnamaldehyde Alleviate Cadmium Stress In Tobacco Seedlings

Thymol and Cinnamaldehyde?CA?both are belong to small natural plant-derived compound,which have been widely used in food protection and medical studies,and other fields.It has been reported that thymol and cinnamaldehyde were capable of regulating plant innate immunity against pathogen 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 thymol and cinnamaldehyde against cadmium?Cd?toxicity belonging to abiotic stress.Among the resistant strategies developed by plants,glutathione?GSH?palys a dual functions in combating metal toxicity,GSH-dependent PC biosynthesis is one of the key mechanisms of plantto combat Cd toxicity.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 thymol activated GSH-PCs pathway in tobacco seedlings,which could arrested and further limit the transportation of free Cd²⁺in cells to alleviate Cd-induced toxicity.Cinnamaldehyde confers Cd tolerance by regulating immune system in Cd-treated tobacco seedlings via modulating endogenous H₂S.Basically,the following results are obtained.1.The tobacco?Nicotiana tabacum?seedling was selected in this study.Compared to the control,treatment with CdCl₂?20?mol/L?resulted in the significant decrease in root elongation by about 50%and fresh weight.However,treatment with thymol?100?mol/L?and CA?20?mol/L?could significantly alleviate root growth and fresh weight induced by Cd stress.The addition of thymol at 100?mol/L and CA at 20?mol/L induced significant increase in root elongation by 23.23%and 42.58%as compared to Cd treatment alone,respectively.2.The toxicity indexes of tobacco seedlings were detected in situ by using fluorescent probes and chemical tissue staining.The results showed that thymol and CA remarkably ameliorate Cd-induced over-generation of ROS and Cd²⁺,lipid oxidative injury,and cell death.Thymol and CA trigger the endogenous immune signals to resist Cd stress efficiently in Cd-treated tobacco seedlings.3.GSH in different treatment was measured by biochemical assay and GS-bimane fluorescent detection.Cd stress led to the significant increase in the content of GSH in Cd-treated tobacco seedlings,and the addition of thymol could further stimulate the production of GSH.The results from fluorescent microscope suggested that the increased GSH accumulation shared the same region with the decreased Cd²⁺in roots.Moreover,Gene expression analysis suggested that Cd treatment significantly stimulated the expression of GSH1 and PCS1 in Cd-treated root,and the addition of thymol still upregulation the expression of GSH1 and PCS1.These results indicated that thymol trigger the GSH-PCs pathway in Cd-treated tobacco seedlings mainly through the role of GSH in scavenging over-generation of ROS and the function of PCs in chelating free Cd²⁺,which strengthen the resistance to combat Cd toxicity in tobacco seedlings.4.Cd treatment increased the content of H₂S in the root tip of Cd-treated tobacco seedlings,which can be detected by using specific fluorescent probe WSP-1.Compared to Cd treatment alone,the addition of CA made a significant decrease in the content of H₂S,and exogenous treatment with NaHS?a H₂S donor?significantly increased the content of H₂S and aggravated cell death.However,the addition of PAG?H₂S synthesis inhibitor?or HT?H₂S scavenger?resulted in the significant decrease in endogenous H₂S level under Cd treatment alone,which further promoted the alleviated effect of CA on Cd toxicity?e.g.root inhibition and cell death?.Gene expression analysis suggested that the expression level of DCD significantly increased in Cd-treated roots compared to control,however the addition of CA resulted in the dramatic decrease in the expression of DCD,which were consistent with the changes of endogenous H₂S level in root tip.These results indicated that CA trigger the H₂S-meidated immune signaling pathway in Cd-treated tobacco seedlings mainly through the role of CA in decreasing endogenous H₂S production,which confer tobacco seedlings tolerance to Cd stress.In sum,this study found the pivotal role of thymol and CA in the regulation o f Cd tolerance in tobacco seedlings,which was closely associated with GSH-PCs pa thway and H₂S-meidated immune signaling pathway.This study provides important r eference foundation for tobacco resistance to heavy metal stress.