Analysis Of SaHsf Family In Hyperaccumulator Sedum Alfredii Hance And Cadium Tolerence Function Of SaHsfA4a/c

Sedum alfredii Hance,a cadmium?Cd?/zinc?Zn?/lead?Pb?co-hyperaccumulating species,is considered to be as a promising phytoremediation candidate accumulating substantial heavy metal ions without obvious signs of poisoning.Therefore,it is very significant to study the mechanism of heavy metal tolerance and hyperaccumulation in this species.In our previous transcriptome sequencing of S.alfredii,heat shock transcription factor family?Hsf?was identified to be regulated by Cd stress.Although many studies have reported the role of this family in abiotic stresses,the mechanism of Hsfs is still unclear on heavy metal stress response.In this study,a comprehensive analysis of Hsf gene family was presented in S.alfredii based on transcriptome database.Subsequently,we analyzed the conserved domains,phylogenetic relationships,tissue-specific expression and response to abiotic stresses in Hsf family members,and two Cd stress related genes?SaHsfA4a and SaHsfA4c?were screened.Taking non-accumulating ecotype?NHE?as a control,hyperaccumulating ecotype?HE?as the object were studied to explore the role and mechanism of SaHsfA4a and SaHsfA4c genes in Cd tolerance.The main results were as follows:1.There were 22 members in hyperaccumulating ecotype Hsf?SaHsf?gene family,which could be divided into three classes based on the oligomerisation domains,class A,B and C;among them,the number of class A members was largest;Furthermore,the members of SaHsf shared a highly conserved structure.Different members of SaHsf family responded to different abiotic stresses such as cold,heat,drought,salt,abscisic acid and heavy metal.The expression patterns of these genes were tissue-specific.Besides,there were three similar expression patterns of SaHsfs gene and SaHsps gene in roots,stems and leaves under Cd stress.2.Two members of the A4 subgroup,SaHsfA4a and SaHsfA4c,were isolated from HE and NHE respectively.A completely different expression pattern was found between these two genes.They were both located in the nucleus and had transcriptional activity.Compared with SaHsfA4an and SaHsfA4cn from NHE,overexpression of the SaHsfA4ah or SaHsfA4ch gene improved the tolerance to CdCl₂ stress in transgenic yeast.The transgenic Arabidopsis thaliana with overexpression of the SaHsfA4ah or SaHsfA4ch gene showed a lighter degree of withered and yellow.In addition,the overexpressions of SaHsfA4ah or SaHsfA4ch gene were able to enhance the Cd tolerance of transgenic plants,through active ROS scavenging enzyme activities.SaHsfA4ah or SaHsfA4ch could regulate ROS content and the expression of related genes,including heat shock proteins?Hsps?,peroxidase gene?POD?,catalase?CAT?and ascorbate oxidase gene?APX?in transgenic lines.The promoters of SaHsfA4ah and SaHsfA4ch genes were mainly expressed in vascular tissue of root,stem and leaf,stigma and stamens of A.thaliana and also induced by Cd stress.The results of western-blot and immunohistochemistry showed that the relative expression level of SaHsfA4ch protein was the highest in the stem,followed by the root.SaHsfA4ch was detected specifically in parenchyma cells.These results indicated that the SaHsfA4ah and SaHsfA4ch from HE may involve in the mechanism of Cd stress response.3.The phenomenon of alternative splicing was detected in SaHsfA4ah and SaHsfA4ch from HE.The genes,InSaHsfA4ah and InSaHsfA4ch,retaining 79 bp and 201 bp intron,respectively,only encoded 103 and 109 amino acids,and contained incomplete DBD domain.The InSaHsfA4ah and InSaHsfA4ch gene revealed that introns were retained without Cd treatment,and then cleaved in root,stem and leave of HE with Cd treatment;InSaHsfA4ah and InSaHsfA4ch could not enhance Cd tolerance in transgenic yeast and A.thaliana.Thus,alternative splicing of SaHsfA4ah and SaHsfA4ch in HE may have the functions through the precise splicing of introns.4.Natural Resistance-Associated Macrophage Proteins?NRAMP?are a kind of Fe,Zn,Mn,Cd transporters that widely present in animals,plants and microbes and encodes a class of transporters that transport heavy metals such as iron?Fe?,zinc?Zn?,manganese?Mn?,and cadmium?Cd?.In the present study,SaNramp6 was identified from S.alfredii based on the transcriptome sequencing.SaNramp6 was detected to contain heat-shock binding element?HSE?in the promoter region,and its expression pattern was similar to the SaHsfs gene.The relative expression level of AtNramp6 was up-regulated in the transgenic A.thaliana lines with overexpression of SaHsfA4ah or SaHsfA4ch.Thus,it was predicted that SaNramp6 may be the downstream target gene of Hsf genes;Furthermore,we found SaNramp6 may involve in Cd uptake.The results showed that SaNramp6 gene localized in the cell membrane,and could improve the Cd accumulation,Cd translocation factor and Cd²⁺influx in transgenic A.thaliana.Additionally,overexpression of SaNramp6 can also enhance Cd accumulation and translocation factor in transgenic poplar.In conclusion,SaHsf may play an important role through activating downstream gene expression by binding to HSE elements of downstream genes in Cd accumulation.