Expression Characteristics And Functions Analysis Of Glutathione S-Transferase And Germin-like Protein In Rice Under Heavy Metal Stress

In recent years,the rapid development of industry and agriculture has made the heavy metal pollution in the environment increasingly serious.Excessive heavy metals in soil and water not only affect the normal growth of plants,but also endanger human health through the food chain.Heavy metals can impair cell membrane structural integrity,inhibit normal cell division and the synthesis of chlorophyll,sugar and starch.In the course of long-term evolution,plants have developed various physiological mechanisms that resistance to heavy metals toxicity.Plants can limit the absorption of heavy metals in the environment through root systems,and can also chelate heavy metal ions through organic compounds in plants such as organic acids,amino acids,phytochelations(PCs)and metallothioneins(MTs).In addition,plants produce excessive amounts of reactive oxygen species under heavy metal stress,but the antioxidant system can remove excess active oxygen to maintain the plant's homeostasis.In this paper,copper-resistant rice varieties(No.B1139)and sensitive copper rice varieties(No.B1195)selected in the laboratory were selected as experimental materials to analyze the differences in gene expression levels of two rice varieties under copper stress.The gene functions of rice glutathione s-transferases(GSTs)and germin like proteins(GLPs)under heavy metal stress were analyzed.Copper is an essential trace element in plants,but high concentrations of copper can have a significant effect on the expression characteristics of genes in plants.Bioinformatics techniques were used to analyze the transcriptome sequencing data of two rice varieties(B1139 and B1195)under copper stress.The results showed that Cu stress induced the expression of 7331 and 9670 genes in B1139 and B1195 roots,respectively.GO(Gene ontology)function annotation results show that commonly up-regulated genes in two rice varieties under Cu stress are mainly associated with stimuli response,stress response,biotic stress,catabolic pathways,abiotic stress responses,endogenous stimuli responses,and macromolecule modification,secondary metabolic pathways,catalytic activity,transcriptional regulation activity,transcription factor activity,and binding function.Meanwhile the commonly down-regulated genes mainly associated with gene expression,carbohydrate metabolism pathways,biosynthetic pathways,cell macromolecular metabolic pathways,DNA metabolism pathways,cell composition pathway,cell cycle,cell biosynthetic pathway,structural molecular activity,and RNA binding.Cu stress induces the differentially expression level of ROS scavenger genes,heat shock proteins,pathogenesis-related proteins,secondary metabolic biosynthesis-related genes,transcription factors,Cu metabolic pathway related proteins,alternative splicing events and long non-coding RNAs in two rice varieties.Splicing factors have the function of regulating intracellular alternative splicing events,and splicing factors can direct spliceosomes to specific splice sites in genes.The results show that the expression level of 4 and 9 splicing factors were differentially expressed under Cu stress in rice B1139 and B1195,respectively.The results of GO enrichment analysis showed that the genes whose expression levels were significantly higher in B1195 than B1139 were mainly related to the stress response.Glutathione S-transferases(GSTs)are enzyme proteins commonly found in animals and plants.Their role is to catalyze the coupling of certain endogenous or exotic electrophilic groups with reduced glutathione.In conjunction,increasing its hydrophobicity makes it easy to cross the cell membrane and is eliminated from the body after being decomposed,thereby achieving the purpose of detoxification.Transcriptome sequencing showed that there were 20 differentially expressed GST genes between rice B1139 and B1195 under 8 ?M Cu stress.The activities of GST in two rice varieties were significantly reduced under copper stress,and the GST activity of B1195 decreased more than that of B1139.By analyzing the GST genes expression profile in rice RGAP(Rice Genome Annotation Project)database and the FPKM(Fragments Per Kilobase Million)values of the genes in the transcriptome sequencing,the results showed that the rice OsGSTF2 had the highest expression level in the GST genes family.The expression of OsGSTF2 in B1195 was significantly decreased under copper stress.In normal LB culture medium,the growth of wild-type and OsGSTF2-overexpressed E.coli tends to be consistent,while OsGSTF2-overexpressed E.coli growth better than wild-type E.coli under copper stress.As a Cu binding protein,OsGSTF2 protein has two predicted Cu binding motifs,H-(X)4-H and C-(X)3-H.The His-OsGSTF2 protein was purified using a Ni-IDA column,and its GST activity was significantly inhibited by copper ions in vitro.Furthermore,it is predicted that 14 amino acids in the OsGSTF2 protein are related to the GST activity by constructing a three-dimensional model of the OsGSTF2 protein.The histidine 41 in the OsGSTF2 protein is predicted to be the Cu binding site and the enzyme activity site.The histidine(H)in the 41th amino acid sequence of-the OsGSTF2 protein was replaced by glycine(G).The results showed that the GST activity of the OsGSTF2H41G protein was significantly lower than that of the native OsGSTF2 protein.The two osgstf2 mutants(osgstf2-1 and osgstf2-2)were more inhibited than wild type rice under copper stress,however,the GSH content and copper ion content between the two mutants and wild type rice were not significant under copper stress.Germin like proteins(GLPs)are widely distributed in plants and regulate plant growth and development.Studies have shown that GLPs may have activities such as superoxide dismutase(SOD),oxalate oxidase(OXO),or polyphenol oxidase(PPO).Therefore,GLPs play an important role in plant resistance to abiotic and abiotic stresses.By recognizing the Cupin motif of the germin-like proteins,43 and 32 GLPs were identified in rice and Arabidopsis,respectively.Transcriptome sequencing showed that there were 21 differentially expressed GLP genes between rice B1139 and B1195 under 8 ?M Cu stress.The data in the NCBI database was used to reveal gene expression profiles of GLPs at different developmental stages and under different biological and abiotic stress conditions.The promoter region of the GLP genes contains many stress-related cis-acting elements,such as ABA response elements(ABREs),anaerobic response elements(AREs),low-temperature response elements(LTRs),myb binding sites(MBSs),and heat shock elements(HSEs),endosperm expression elements(GCN4),TC-rich repeat elements(TC-RICHs),and wounding and pathogen response elements(W-BOXs).Chromosome localization results showed that GLPs existed mainly in the form of tandem duplicated genes in rice,and the expression level of tandem duplicated genes tended to be consistent.The phylogenetic tree of GLPs gene was constructed using Bayesian algorithm.The results showed that GLPs gene can be divided into 6 branches.The gene structure of GLPs in different branches is highly similar.The expression levels of 3 GLPs(OsGLP4-1 OsGLP8-7 and OsGLP8-11)were significantly induced by heavy metal stress.Agrobacterium-mediated contained FLG-or GFP-tagged GLP proteins(OsGLP4-1,OsGLP8-7,and OsGLP8-11)were transiently expressed in N.benthamiana.Using immunoblot,SOD enzyme activity assay and subcellular localization analysis,the results showed that the three GLPs proteins had the activity of superoxide dismutase(SOD),and were located in the cell wall.These results provide theoretical and experimental evidence for the further study of the function of rice GLP genes.