Cadmium Tolerance Gene Mining And Molecular Mechanism Of Solanum Tuberosum Varieties With Low Cadmium Accumulation

Because of the excessive Cadmium（Cd）content in the soil in Guizhou due to the special parent material of soil in Guizhou,which will affect the safety,quality and yield of potatoes in Guizhou（the main potato producing area in Southwest China）,the study on the accumulation mechanism of heavy metals in potatoes is one of the basic for the formulation of countermeasures.In this study,Cd tolerance and accumulation mechanism of low-accumulation Cd type potato were explored.On the basis of transcriptome and whole genome,related genes that may cause differences in Cd accumulation mechanism among potato varieties were excavated.Through systematic and comparative omics,q RT-PCR,tobacco leaf subcellular localization,molecular cloning,yeast functional complementarity,plant overexpression,ion scanning selection and other techniques,the functions of candidate genes were elucidated,and the mechanism of candidate genes in the transport of Cd from root to aboveground in low-accumulation potato（Yunshu 505）was discussed.The main findings are as follows:（1）Cluster analysis was conducted to compare the cadmium contamination and enrichment degree in different tissue parts of 20 potato varieties and the health risk index of human consumption of these potato varieties.Yunshu 505（low accumulation variety）and Weiyu 7（high accumulation variety）were selected as the materials for subsequent research.The tissue contamination index of different varieties was:root>stem>leaf>tuber,or root>leaf>stem>root tubers,the classification of interval（grade 4 or 5）belongs to the high level of pollution,large difference between two different varieties of stem and leaves of grading range（grade 1 to grade 5）,in addition to the green potato tuber of 9 class interval of 2 alert level,the rest all varieties of tuber for level 1 level of security,the whole plant pollution range（grade 3 to 4）,the pollution level for the mild;The enrichment coefficient from large to small is as follows:root>stem>leaf>20 potato varieties were divided into low enrichment group,medium enrichment group and high accumulation group.The food risk assessment of potato showed that THQ values of different varieties were far less than1 and the highest was only 0.14,indicating that long-term consumption of potatoes growing in the soil with high cadmium background would not affect human health.（2）The changes of soil properties and different Cd forms before and after planting different potato varieties were studied by Bayesian network and random forest.It was preliminarily clarified that organic matter（SOM）and p H were the important factors affecting the available Cd in soil,and thus affecting the accumulation and toxicity of Cd in potato.（3）The study design method based on the transcriptome data sets,setting up the differences and expressed genes based on analysis of enrichment of GSEA gene set,the results showed that the differences in gene sets（G2＿up）main enrichment in ion transport across the membrane,amino acids across the plasma membrane,carboxylic acids across the plasma membrane process,including ABC transporters,heavy metal related isoamyl alkenyl plant protein or HMA,ion binding protein,Fe²⁺transporters,etc.,and a total of expressed genes set（G1＿up）enrichment in secondary metabolism,regulation and control of catalytic activity of negative negative regulation,molecular function,biological process of negative regulation,These results indicated that the expression patterns of transmembrane transporters in the two cultivars might be different.Cluster analysis of the expression patterns of ABC and HMA family members showed that the two cultivars had obvious differentially expressed genes,which may be the key reason for the high/low accumulation of cadmium between the two cultivars due to the specific differentiation of St ABCs and St HMAs.The co-expression gene set found that WRKY had the highest number of differentially expressed genes（82）compared with other members of the transcription factor family,so St ABC,St HMA and St WRKY family were selected for subsequent studies.（4）Based on plant genome data,and by using the HMMER localization retrieval commands,according to ABC,HMA,classic WRKY family structure domain（PF03106,PF00403,PF00005）of hidden markov model（HMM）,whole genome database identified in many species,including potato and model plant（arabidopsis,rice）,and other six species of ABC,HMA,WRKY family members.84 ABCs,36HMAs and 71 WRKYs were identified in potato,respectively.They were named St ABC1-84,St HMA1-St HMA36,and St WRKY1-71.According to the family gene structure,motifs structure and phylogenetic tree between species,St ABC family was divided into 6 subfamilies,which were named St ABCA,St ABCB,St ABCC,St ABCD,St ABCE and St ABCF subfamilies.The St HMA family is divided into six subfamilies,which are named as St HIPPa,St HIPPb,St HIPPc,St CCH,St PAA1,and St RAN1subfamily according to the functional annotations and names of model plants（Arabidopsis thaliana and Oryza sativa）in the same evolutionary branch.The St WRKY family is divided into 6 subfamilies,which are named as:St WRKYa,St WRKYb,St WRKYc,St WRKYd,St WRKYe,and St WRKYf subfamily.（5）The functional specific or conserved members of each family were analyzed and counted by systematic omics and comparative omics,which provided genetic resources for subsequent molecular studies;Real-time quantitative fluorescence PCR（q RT-PCR）was used to establish an important framework for tissue spatiotemporal specific expression under heavy metal stress（St ABCs,St HMAs,and St WRKYs）.Functional genes that may be actively involved in cadmium stress were further mined,and representative or specific genes were selected from each family for subsequent verification experiments.Conservative members included intraspecie replication template,interspecific co-replication template,non-intron,co-expression,and up-regulated positive response.Specific members include subcellular localised variants,transmembrane domains,beneficial mutations,promoters,and tissue site specific expressions.In this study,functional genes that may be actively involved in cadmium stress were further selected for subsequent validation experiments（St ABCs4/15/27/39,St HMAs 8/22,and St WRKY29）.（6）Through yeast functional complementarity and cadmium accumulation experiments,it was found that candidate gene expression increased the tolerance of cadmium sensitive strain△ycf1,St WRKY29 and St ABC4 conferred higher tolerance of△ycf1,p St WRKY29>p St ABC4>p St ABC39>p St HMA22>p St ABC27>p St ABC15>PSt HMA8;St HMA22,St ABC27,St ABC15,St HMA8,and St ABC39 are genes with high cadmium accumulation.St ABC4 and St WRKY29 are genes with low cadmium accumulation.Among them,St HMA8 has the highest cadmium accumulation,which is 16.47 times higher than that of St ABC4.Transcriptional factor WRKY39 may regulate the accumulation of cadmium by the strain by regulating downstream related kinases.Interestingly,St ABC4,which has no transmembrane domains（TMDs）and has nucleotide structure（NBDs）mutations,seems to have a special mechanism to respond to cadmium stress and may interact with other transporters to regulate cadmium accumulation.These mechanisms need to be further studied.（7）According to the phenotypes of the overexpressed plants,non-invasive scanning ion-selective electrode（SIET）and physiological index analysis results,the overexpression of St WRKY29 conferred high tolerance to the plants,while St HMAs8/22 conferred moderate tolerance to the plants.Specifically,Arabidopsis thaliana overexpression of St WRKY29 showed advantages in lateral root density and root length,and pollen tube elongation occurred under long-term high concentration stress,the reasons for which remain to be explored.（8）St WRKY29 and St HMAs 8/22 were transient expressed in tobacco leaf tissue cells.The results showed that St WRKY29 was localized in the nucleus.St HMAs 8/22was found in the cell membrane and cytoplasm.The overexpression of St WRKY29and St HMA22 in Arabidopsis thaliana and St HMA8 in tobacco indicated that St WRKY29 and St HMA8 caused differences in cadmium tolerance and accumulation of tissue sites in plants.We defined three types:low accumulation of St WRKY29,root-stem accumulation of St HMA22,and stem-xylem accumulation of St HMA8.The overexpression of St WRKY29 plants only accumulated Cd in the root,which greatly reduced the Cd content in the stem and xylem,while St HMAs 8/22 increased the Cd content in the root and lower part of the stem,especially St HMA8 resulted in significant Cd accumulation in the xylem of the stem,indicating that St HMAs 8/22can transport Cd from root to stem,and combine Cd ions to form a less toxic Cd-complex,which is fixed in the xylem of the stem.This process seems to increase the content of Cd,but actually reduces the toxicity of Cd,thus increasing the tolerance of the plant to Cd.In summary,The candidate genes differentially upregulated in Yunshu 505（low accumulation type potato）may be important genes leading to low accumulation of cadmium in tubers of Yunshu 505.Plant overexpression of candidate genes showed high tolerance to cadmium-high accumulation in root-low accumulation in stem and xylem（St WRKY29）,medium tolerance-root-stem accumulation（St HMA22）,and medium tolerance-stem-xylem accumulation（St HMA8）.The expression of the candidate gene St WRKY29 prevented the transport of cadmium to the aboveground parts.St HMA22 binds most Cd ions and binds them to roots and lower parts of stems,while St HMA8,on the other hand,accumulates cadmium in the xylem between the root and the stem,thus reducing cadmium content in the aboveground part of the plant.It is hypothesized that the mechanism of cadmium transfer from the aboveground part down to the tuber in potato cultivars with high and low cadmium accumulation is the same.Therefore,the significantly up-regulated expression of candidate genes in Yunshu 505 was the main reason for the different accumulation of cadmium in tubers among potato varieties.This finding provides a new perspective for the study of the mechanism of plant blocking Cd transport to above-ground parts,provides an excellent genetic resource for potato germplasm innovation,and lays a certain foundation for the further study of the molecular mechanism of Cd accumulation.