Regulation Of Ethylene,Ethylene And Nitric Oxide Crosstalk In Response To Cadmium Stress In Lotus

Lotus（Nelumbo nucifera）is one of the ten most famous traditional flowers in China,which is usually used in gardening and landscaping with highly ornamental value.Recently,researches have provend that lotus could enrich heavy metal Cd,for instance.However,Cd is an unnecessary element,which can be poisonous when it reaches to a high concentration.This study choose ’Weishanhu honglian’ as experimental materials.Based on the selection of Cd treated concentration,we studyed the mechanism of ethylene on lotus response to Cd stress.Furthermore,we cloned and analyzed the NnACS and verified its function on tobacco leaves by instantaneous conversion.Moreover,the effects of ethylene and nitric oxide on lotus under Cd stress were studied.This thesis provided a physiological and molecular mechanism of lotus resisting to Cd,and lay a foundation of Cd-resistant lotus for further molecular breeding,and also provided a theoretical support for the purification of water bodies by phytoremediation materials.1 The effect of Cd on lotusThe damage degree was high when lotus were treated with different concentrations of CdC12 for 48 hours,and the damage area was increased with the increase of Cd concentration,especially the damage on the leaves which became chlorotic brown completely at the concentration of 400 μM,but there was no significant difference between the number of roots and length of roots or petioles.Further experiment showed the relative conductivity,MDA and H2O2 contents were positively correlated with different Cd concentrations.The results showed that 50 μM was the proper Cd concentration on lotus.According to this concentration,Cd was found in all of the three organizations mentioned,and this indicated Cd was responsible for the damage of lotus leaves.2 The mechanism of Ethylene-regulated Lotus Response to Cd stressBased on the results above,the effect of ethylene on lotus response to Cd was investigated.Study found that ACC can aggravate the injury of Cd to lotus leaf,while STS has obvious mitigation effect.The further study showed that ACC could increase MDA and H2O2 contents in lotus leaves under Cd treatment.By measuring the amount of ethylene released from lotus flowers,it was found that the ethylene release of Lotus rose significantly due to Cd stress,and ACC further promoted the increase of its ethylene release,while STS could significantly reduce the ethylene release of lotus under Cd stress.As for the antioxidant system,Cd couldn’t change the activity of SOD and POD in lotus leaves,but significantly reduce the activity of CAT,APX and GR,and increase the content of AsA and GSH.Ethylene didn’t not participate in the regulation of SOD and POD activities,but it reduced the activity of CAT,APX and GR under Cd stress and increased the content of AsA and GSH,while STS played an opposite role.As for petiole,Cd declined the activities of APX and GR,but increased AsA and GSH contents.ACC increased SOD activity and GSH contents,but decreased CAT activity either.STS could increase POD and CAT activities but also decrease the GSH content;in root,SOD activity,AsA and GSH contents were repressed by Cd.ACC decreased the activity of SOD and the content of GSH,but increased AsA content.STS could increase SOD activity,AsA and GSH contents.According to qRT-PCR analysis,NnGPP1（XM₀10254376）,NnGalDH（XM₀10273240）,NnECS（XM₀10257096）,NnGSHS（XM₀19197634）,NnAPX2（XM₀119197334）and GR2（XM019198752）played important roles in lotus antioxidant system.Therefore,ethylene is involved in the regulation of the response of lotus to Cd stress,which affects the antioxidant system of the plant by affecting the release of endogenous ethylene from lotus,and then affects the membrane lipid peroxidation and H2O2 content of the lotus seedling leaves,and finally regulates the damage of lotus.3 The cloning and analysis of NnACSACS is a limit enzyme in plant ethylene synthesis pathway.We cloned lotus NnACS（XMe 010263050）whose ORF area size is 1479 bp which encode 492 amino acids.According to the secondary structure and structural domain analysis,this protein had the function of ACC synthesis and aspartate aminotransferase dependent on pyridoxal phosphate.Its homology with other species such as Arabidopsis is not high,but it is relatively conservative at key binding positions of pyridoxal phosphate and dimer,and closely related to the ACS6 in Arabidopsis thaliana.qRT-PCR data showed that the expression of NnACS was increased significantly under the stress of Cd,and ACC could further increase its expression level,while STS could repress its expression level,which was similar with the results of ethylene release content.According to the instantaneous transformation in tobacco leaves,it was confirmed that NnACS had the ability to synthesize ethylene,and increase the damage leaves of Cd.Therefore,NnACS indeed has the ability to promote the synthesis of ethylene in plants,and by increasing the release of endogenous ethylene from plants,it will increase the damage to plants under Cd stress.4 Effects of ethylene and NO crosstalk on lotus response to cadmium stress.Ethylene and NO played important roles in lotus response to Cd stress,but the relationship between them was complex.we found that reducing the content of endogenous NO on the basis of combined treatment of Cd and ACC can alleviate the damage of Cd to lotus leaves to some extent.Among them,PTIO has a significant alleviation effect on APX,GR,MDHAR and DHAR activity in lotus leaf,which reduces the oxidative damage.The effect of L-NNA and sodium tungstate is not as obvious as that of PTIO.The application of SNP on the basis of Cd can further reduce the activity of two enzymes,MDHAR and GR,to make the AsA-GSH cycle obstructed,and thus to some extent increase the toxicity of lotus leaf under Cd stress.Applying STS on this basis can significantly increase the activities of four antioxidant enzymes in lotus leaves,then AsA and GSH were used to reduce the damage of lotus leaves.Therefore,there is an interaction between ethylene and NO.They regulate the AsA-GSH circulatory system together,which affects the degree of membrane lipid peroxidation of lotus leaf,and then regulates the oxidative stress level of lotus seedlings.Finally,it participates in the regulation of lotus to Cd stress.