Regulatory Mechanism Of MAPKs On Channel Proteins Under Cadmium Stress

Cadmium(Cd)is a common environmental pollutant,and accumulated in farmland,river and plant.Cadmium assimilated in tissue and organs through the food chain,which led to various disease and even cancer.Cadmium in vivo would break the balance of ROS and Calcium homeostasis and activate the mitogen-activated protein kinase(MAPK)pathway,mainly the phosphorylation of Hog1 p and Slt2 p in yeast.But the downstream components of MAPKs and the regulatory mechanism are still unclear.The eukaryotic cell model Saccharomyces cerevisiae(including BY4741,hog1 and slt2)was employed by RNA-sequencing(RNA-seq).The RNA-seq data was performed to predict and screen the signal transduction pathway and the potential effect molecules regulated by MAPKs at cellular and molecular level.The main results were listed as follows:(1)A total of 1545 differential expressed genes(DEGs),including 790 Cdinducible and 755 Cd-represses genes were identified the differences between cadmium-free and cadmium-stressed conditions in BY4741.The main functions were related to oxidative damage,material metabolism,DNA repair and endoplasmic reticulum stress according to GO,KEGG and COG databases.(2)By comparison with BY4741,103 DEGs were induced in HOG1 deletion mutants,and 9 DEGs were identified in SLT2 deletion strains under the cadmium stress.The functional annotation of these DEGs was similar to wild type group.Furthermore,31 genes regulated by Hog1 p and 2 genes induced by Slt2 p,which related to oxidative damage,DNA replication stress and detoxification,were screened under cadmium exposure by Venn diagrams.(3)A cysteine-specific transporter(Yct1p)modulated by Hog1 p and its transcription factor was confirmed via RNA-seq result.Firstly,8 DEGs related to transport,DNA replication and detoxification were selected to construct double deletion strains under the background of hog1 or slt2 strain.Meanwhile,we tested the cadmium sensitivity and intracellular cadmium concentrations of double-gene deletion mutants,and the results indicated that the hypersensitivity of the hog1 to Cd was partly abrogated in YCT1 gene deletion.Secondly,we generated pRS316-Yct1p-LacZ,pHAC111-Yct1 p and pGFP33-Yct1 p plasmid and transformend into yct1,hog1 and transcripton factors deletion mutants,in order to determine the transcriptional and translational regulation by ?-Galactosidase assay,Western Blot and confocal imaging.The results suggested that Yct1 p was epistatic to the Hog1 p in Cd tolerance.YCT1 gene expression at the transcriptional and translational levels was positively regulated by Hog1 p,while transcription factors Msn2 p,Msn4p and Hot1 p negatively influenced the expression of Yct1 p.In conclusion,we confirmed that Hog1 p maintain the cell homeostasis by regulating the carbohydrate metabolism,oxidative damage,transporters and DNA damage.The cysteine-specific transporter Yct1 p was downstream effector of Hog1 p and played a complementary role in cell detoxication.The investigation of the transcriptome of MAPKs under Cd stress provided valuable information for future molecular studies of cadmium tolerance.