Physiological Response Of Trollius Chinensis Seedlings To Cu And Cd Stress And The Transcriptomic Analysis Under Cd Stress

Trollius chinensis is a kind of perennial herb with high ornamental value,which can be an important landscape plant in the north.In recent years,with the accelerated industrialization and urbanization,copper and cadmium contamination in the soil has become increasingly serious,causing adverse effects on the natural environment and human life.Therefore,in the study,T.chinensis seedlings were used as the experimental materials and stressed by different concentrations of Cu and Cd solutions for 6 days.The growth and physiological indicators of seedlings were analyzed after the stress,exploring the physiological response to Cu and Cd.Meanwhile,the transcriptome analysis was combined with Cd stress to further elucidate the response mechanisms of Cd stress from the morphological-physiological-molecular levels.The main findings are as follows:（1）The increased level of Cu stress led to a more severe damage to T.chinensis.The electrolyte leakage（EL）,minimal fluorescence（Fo）,coefficient of nonphotochemical quenching（q N）,and contents of malondialdehyde（MDA）,soluble protein（SP）,and reactive oxygen species（ROS）and glutathione（GSH）already increased at 1.5 m M Cu treatment,showing sensitivity to Cu toxicity.When the treatment concentration reached 9.0 m M,the growth and physiological activities of T.chinensis were severely inhibited,the biomass was reduced by more than 50%and the plants reached a semi-lethal state;the SP content increased the most among the osmoregulatory substances under Cu stress,1.6 times of that in the control.SOD,monodehydroascorbate reductase（MDHAR）and ascorbate oxidase（AAO）reached their maximum value;the above-ground Cu²⁺content was 20.5times higher than the control.At Cu concentrations up to12 m M,the root-to-shoot ratio,the biomass of T.chinensis seedlings reached their lowest values and photosynthetic pigment content,photosynthetic rate（P_n）,transpiration rate（T_r）and maximum photochemical efficiency（Fv/Fm）all reached their lowest values;the content of osmoregulatory substances Soluble protein（SP）,soluble sugar（SS）,proline（Pro）and antioxidant enzymes such as SOD,MDHAR,GSH and AAO showed a decreasing trend,which means that the maximum stress concentration is beyond what the plant can tolerate and causes extremely serious toxicity to the plant.（2）The effect of low concentration of Cd stress on T.chinensis was not significant,while the higher the concentration the greater the toxic effect.Under 5 and 10μM Cd treatment,plant growth phenotype,ROS and ion content were not significantly different from the control.The root-to-shoot ratio Chlb,P_n,T_r,Fv/Fm and Y（II）decreased significantly,while GSH and MDHAR increased significantly.The plant growth,root length and activity,photosynthetic indicator parameters were significantly decreased by increasing Cd treatment concentration.The Cd content in the root was always higher than that in the upper part,while the enrichment and transportation factors were continuously decreased.The activities of osmoregulatory substances,SOD,POD,ascorbic acid correlation,indicating that plants tolerated Cd toxicity by promoting the synthesis of osmoregulatory substances such as Pro and enhancing the activities of antioxidant enzymes like SOD.（3）The correlation and principal component analysis between Cu and Cd indicators showed that the representative Cu comprehensive evaluation indicators were plant height growth,root length,root-shoot ratio,MDA,GSH,Chl（a+b）,intercellular CO₂ concentration（C_i）,Y（II）,q P and q N.The comprehensive evaluation indicators of Cd tolerance in T.chinensis seedlings are MDA,H₂O₂,POD,GSH,AAO,MDHAR,stomatal conductance（G_s）,T_r,Fo and q N.Meanwhile,MDA and GSH can be regarded as potential Cu and Cd biomarkers.A comprehensive analysis of the affiliation functions showed that T.chinensis seedlings were overall more tolerant to Cd stress than Cu stress,indicating the seedlings showed greater adaptability under Cd stress conditions.（4）The RNA-seq results showed that a total of 308 significantly differentially expressed genes were got under Cd treatment at 10μM,of which 219 genes were up-regulated and 89 genes were down-regulated.The GO and KEGG enrichment analysis showed that the differentially expressed genes under cadmium stress of T.chinensis were significantly enriched in the catalytic activity of molecular functions and carotenoid biosynthesis pathways in the metabolic pathway of T.chinensis.Also,transcription factors such as WRKY,MYB and MATE showed significant responses,indicating that plants respond to Cd toxicity at the mole铜lar level by inducing the synthesis of secondary metabolites and transcription factor regulation.Six randomly selected co-expressed genes were validated using q RT-PCR and the results showed that the expression of six genes was consistent with the transcriptome sequencing results.