Cadmium Stress Response In Abelmoschus Manihot And Its Underlying Mechanism Based On Transcriptomics And Metabolomics

Among the types of heavy metal soil pollution in China,cadmium（Cd）pollution is particularly prominent.Ornamental plants with high heavy metal tolerance or accumulation are more feasible for practical applications than traditional heavy metal accumulator/hyperaccumulators.Abelmoschus manihot is a type of multifunctional ornamental plant with a wide distribution area,strong vitality and survival and beautiful flowers.This species is not only used as an ornamental plant in urban gardens but also contains abundant biologically active ingredients and metabolites.In this experiment,the physiological,biochemical,molecular transcriptomic and metabolic differences in A.manihot under Cd stress were studied to understand the response mechanism of Cd absorption,transport and detoxification from multiple levels.The main results were as follows:（1）Using a soil-pot experiment with Cd concentration gradients of 0（control）,5,15,30,60 and 100 mg/kg,the Cd tolerance,absorption and accumulation ability of A.manihot were evaluated.A.manihot plants did not show any obvious symptoms,including chlorosis or necrosis,under Cd stress.In addition,under the 5-100 mg/kg Cd treatment,the dry matter quality of the aboveground and underground parts of A.manihot increased by 30-76%and 18.5-70%,respectively,compared with the control group,showing an excitotoxic effect.The accumulation of Cd in all parts of A.manihot increased with increasing treatment concentration,and the maximum accumulation in leaves and roots reached 185 and 210.24 mg/kg,respectively.At the same time,the enrichment factors（BCFs）of the aerial parts of A.manihot were greater than 1 in the treatment group,and the transfer factor（TF）in the Cd<100 mg/kg group was greater than 1.The results showed that A.manihot can steadily absorb and accumulate a large number of Cd ions,and a large amount of Cd can be transported to the aerial parts of plant.Thus,A.manihot is a potential ornamental Cd accumulator/hyperaccumulator that can be used in the study of heavy metal phytoremediation of Cd-contaminated soil.（2）Hydroponic experiments with a Cd concentration gradient of 0（control）,50,100,200and 400μM were used to study the influence of Cd on the biomass,physiological and biochemical characteristics of A.manihot,including the mineral element uptake,photosynthetic parameters,root morphology,root activity,root oxidative stress and antioxidant stress,and root tip ultrastructure.It was found that when the concentration of Cd was less than 200μM,the A.manihot had no obvious symptoms of Cd toxicity.With the increase in Cd concentration,the dry matter mass of A.manihot increased first and then decreased.Under the 100μM Cd treatment,the dry matter mass of roots,stems and leaves of A.manihot reached maximum values,which were 9.81%,25.01%and 11.29%higher than those of the control group,respectively,showing an excitotoxic effect.However,when the concentration of Cd was greater than 200μM,the apical leaves of A.manihot showed chlorosis,and growth was significantly inhibited,with a significant decrease in biomass.The accumulation of Cd in the aboveground parts of A.manihot reached a peak value of 178.17 mg/kg under the 200μM Cd treatment,while the accumulation of Cd in the belowground parts reached a maximum value of 289.01 mg/kg under the 400μM Cd treatment.Studies on the absorption and accumulation of other mineral elements of A.manihot showed that under low concentration Cd treatment（Cd≤100μM）,Cd caused fewer disturbances to N,Ca,Zn and Mn,which suggested that A.manihot has strong Cd tolerance.Under Cd concentrations≥200μM,Cd disrupted the mineral nutrient balance of A.manihot.Compared with the control group,the contents of N,P,Ca,Fe,Mg,Zn,Cu and Mn in the aboveground parts significantly decreased under Cd stress,and the content of K significantly increased.In addition,the corresponding contents of N,Ca,Mg,Zn,Cu and Mn in the roots significantly decreased,and the content of P significantly increased（p<0.05）.Studies on A.manihot photosynthesis showed that P_n,T_r and WUE increased first and then decreased under different concentrations of Cd.Among them,P_n reached the maximum value under the 100μM Cd treatment,increasing by 19.82%compared with the control group,and there was a linear relationship between P_n and the dry matter of the aboveground parts（R²=0.806,p<0.05）.The result suggested that the increase in P_n may be closely related to the increase in the biomass of A.manihot.When Cd≥200μM,P_n,T_r and WUE were significantly inhibited.Although G_S and SPAD decreased with increasing Cd concentration,these parameters did not change significantly under low Cd stress（p>0.05）.C_i did not change significantly under the different Cd treatments.The results suggested that a decrease in P_n was mainly caused by non-stomatal limiting factors.In addition,the decrease in F_v/F_o,F_v/F_m,Φ_PSⅡ,ETR and q P,and the increase in NPQ in plants exposed to Cd≥200μM further indicated that high doses of Cd strongly inhibit the photosynthesis of A.manihot.Studies on the physiological and biochemical indexes of A.manihot roots showed that when Cd≤100μM,there was no significant difference in total root length,average root diameter,total volume and root activity compared with those of the control group（p>0.05）.The total number of root tips of A.manihot increased significantly under the 50-100μM Cd treatment（p>0.05）and reached maximum values at 100μM.This is most likely due to Cd-stimulated lateral root growth.When the Cd concentration exceeded the tolerance threshold of 200μM,these root morphological parameters showed significantly lower values than the control group（p<0.05）.The results suggested that high-dose Cd treatment significantly inhibited the growth and development of roots.Under Cd stress,the content of H₂O₂,O₂^-and MDA in the roots of A.manihot increased gradually with increasing Cd concentration（p<0.05）.In addition,ROS activated antioxidant defense in vivo.The activity of SOD and POD in roots increased with increasing Cd concentration,and the activity of CAT increased first and then decreased.Studies on the ultrastructure of the root tip of A.manihot showed that when exposed to100μM Cd,a large number of vacuoles were found in the root tip ultrastructure of A.manihot,which coalesced with each other to form larger vacuoles.Moreover,high electron density particles were observed in some vacuoles.In addition,under Cd stress,the membrane system of A.manihot was obviously destroyed,and the cell membrane,Golgi body and mitochondria became unclear.（3）Transcriptome sequencing（RNA-seq）was used to analyze the molecular mechanisms of gene expression and regulation related to Cd absorption,transport and stress response in the root system of A.manihot.After sequencing and quality control,40.61-61.56 million high-quality clean reads were obtained,and 73122 unigenes were obtained after de novo assembly,with an average length of 873 bp and N50 of 1412 bp.By comparing changes in the quantities of differentially expressed genes（DEGs）between the Cd treatment（100μM）and the CK at four time points（12 h,36 h,72 h and 7 d）,it was found that the number of downregulated genes was more than that of upregulated genes.This indicated that the roots of A.manihot responded to Cd stress mainly through the negative regulation of gene expression.STEM was used to analyze the trend of all the DEGs of A.manihot roots at different time points under Cd stress.There were 20 profiles,among which the significant profiles were 1,5,7,8 and 11（p<0.05）.Through the analysis of GO functional enrichment of DEGs in the significant profiles,it was found that starch and sucrose metabolism,phenylpropane biosynthesis,plant hormone signal transduction,glutathione metabolism,membrane lipid metabolites and their signal molecule-related metabolic pathways played a key role under Cd stress.In addition,the DEGs associated with Cd transport in A.manihot were mainly concentrated in the ZIP family and the ABC family and were significantly upregulated at 72 h.This result indicated that a large number of transporter-related genes were activated at 72 h under Cd stress.（4）A total of 627 metabolites in the roots of A.manihot were detected by LC/MS/MS technology through a wide range of targeted analyses.Principal component analysis（PCA）showed that,compared with the early stage（12 h,36 h）of Cd treatment,the metabolites in the late stage（72 h,7 D）of Cd treatment were significantly different from those in the control,indicating that the root metabolites of A.manihot changed greatly in the late stage of Cd treatment.By comparing differential metabolites（p<0.05,|log2FC|>1.5）between the Cd treatment and control groups at different time points,differential metabolites associated with the RNA-seq enriched metabolic pathway were further extracted.It was found that the pathway of lignin synthesis in roots of A.manihot responded to Cd stress,including the expression of PAl-,4CL-,F5H-and CAD-related genes,and the contents of sinapadehyde and coniferaldehyde were significantly reduced.Moreover,the contents of some flavonoids,GSH and GR,and GPX-and GST-related gene expression in the roots of A.manihot increased under Cd stress,playing an important role in the resistance to Cd.In addition,the changes in membrane lipid metabolites and the increase in metabolites in the linolenic acid metabolism pathway and a significant upregulation of the expression of related unigenes in the JA pathway indicate that the JA pathway is important in the A.manihot response to Cd stress.