Functional Analysis Of Copper-induced Rice Metallothionein OsMT2c

In recent years,heavy metal pollution has become more and more serious,contaminated soil and water has become one of the global environmental problems.During the course of evolution plants have developed unique mechanisms against heavy metal stress,not only maintaining the physiological concentration of nutrition elements,but also avoiding the toxicity of non-essential heavy metals.Once the metal ions entered into the cytoplasm,the most effective detoxification mechanism is chelation.The heavy metals can be binding with organic small molecular compounds in cytoplasm such as organic acid,amino acid,phytochelatins?PCs?and metallothioneins?MTs?,to reduce the toxicity.MT is a class of cysteine-rich low molecular weight polypeptides which play an important role in detoxicating heavy metal,maintaining homeostasis of intracellular metal ions,and regulating the balance of ROS in plant.In this study a rice MT gene,OsMT2c,was characterized and up-regulated by Cu and H₂O₂.We further analyzed the signal pathway in the process of Cu-induced the expression of OsMT2c,and then transformed OsMT2c into Arabidopsis and rice,obtained the transgenic plants materials to further study the function of OsMT2c under heavy metal stress.Searching the MSU Rice Genome Annotation Project and NCBI database,14 rice metallothionein genes were accessed,they distributed in 6 chromosomes,and belonged to four types of plant MTs.To analysis the phylogenetic relationship among this family protein,we made amino acid sequence alignment and found OsMTs contain two CRD domains,except OsMT4 with 3 CRD domains,were located on the N-and C-domain.Type-1 OsMTs has 7 members,mainly expressed in root,Type-2 and Type-3 MTs expressed higher during vegetative growth and seed development,and the expression of OsMT4 in different tissues was quite low.Further using the data from GEO public microarray database,we studied the expression characteristics of OsMTs in different tissues during growth and development,biotic and abiotic stress,and exogenous phytohormone treatments.The results were plotted as heat maps and showed that the expression of OsMTs genes were tissue-specificity and inducibility.We also analyzed the expression of some OsMTs genes in rice seedlings and seeds,in addition with Cu,H₂O₂ and salicylic acid treatments by semi-quantitative RT-PCR.Finally,we screened an OsMT gene,OsMT2c,simultaneously induced by heavy metal and H₂O₂.Excessive Cu elicited a rapid and sustained production and release of H₂O₂ in rice,and the expression of OsMT2c was also increased.Exogenous H₂O₂ scavengers DMTU and Asc decreased H₂O₂ production and OsMT2c expression in rice roots and leaves under copper stress.Furthermore,the expression of OsMT2c increased in the osapx2 mutant in which the H₂O₂ levels were higher than in wild-type?WT?plants.These results showed that Cu increased OsMT2c expression through the production and accumulation of Cu-induced H₂O₂ in O.sativa,OsMT2c may be a downstream protein of H₂O₂ signal pathway.Heterologous expression of OsMT2c in Arabidopsis thaliana significantly improved the Cu tolerance as they can grow in the medium containing 35?mol L-1 CuSO4 with little influence on root length and fresh weight.The transgenic Arabidopsis exhibited increased ROS scavenging ability which accumulated less H₂O₂ in leaves than that in WT and Ev.Under normal hydroponics no difference of Cu contents was observed in transgenic Arabidopsis and WT;but under 10?mol L-1 CuSO4 treatment,the Cu contents in shoots of transgenic plants were lower than in WT and Ev,and no differences in roots.In order to further analyze the function of OsMT2c,we constructed the plant expression vector pCAMBIA1304-OsMT2c for over expression of OsMT2c,and then transformed it into the callus induced from mature rice embryo,finally obtained rice transgenic suspension cells and seedlings.The Cu accumulation in rice suspension cells overexpressing OsMT2c did not change significantly,and other necessary metal elements?such as Mn,Zn and Fe?were significantly increased.Under Cu treatment,transgenic suspension cells accumulated more Cu,Mn and Fe than that in WT,but Zn content was not significantly increased.Overexpression of OsMT2c in rice obviously inhibited the development of roots system,observed.The root elongation and dry weight in OsMT2c transgenic rice and WT rice were obviously decreased and showed no differences between the two.However,the excessive OsMT2c still played a positive role in increasing roots activity,reducing the content of H₂O₂ in roots,Cu accumulation in shoots and increased the antioxidant enzyme activity.