Studying The Molecular Mechanisms Of Two WRKY Transcription Factors Regulating Leaf Senescence And Ethylene Signaling,respectively In Arabidopsis Thaliana

Arabidopsis thaliana,as an important model organism,is an ideal material for studying the response of plants to biotic or abiotic stresses.Development and senescence are two important stages for plant growth.Therefore,exploring the AtWRKY genes involved in leaf senescence and plant development can provide some important theoretical basis and ideas for crop plant improvement.WRKY transcription factors constitute an important gene family in plants.There are 72 members in Arabidopsis,which are mainly involved in plants against various biotic and abiotic stresses.So far,the studying on WRKY transcription factors' functions has made great progress,however the function of many members of WRKY family remains to be further explored.Reactive oxygen species(ROS)is an important signal molecule produced in plants,but too much accumulation leads to toxic effect and cell death.Ethylene(ET)is an important plant hormone regulating different developmental processes and a weak change may have a great impact on plant development.Therefore,it is necessary to explore how the WRKY transcription factors affect the ethylene content in Arabidopsis.In this study,the constitutive and inducible overexpression lines of AtWRKYa gene were obtained by flower diping.In the natural senescence test,we found that senescence of overexpression lines was promoted while mutant lines were delayed.Then we analyzed the expression changes of marker genes associated with production and scavenging of ROS as well as leaf senescence by q RT-PCR.We found three putative downstream target genes of At SAG13,At Rboh D and At Rboh F.The results were further confirmed by dual luciferase assay and electrophoretic mobility shift assay(EMSA),and we found that AtWRKYa could bind to the promoters of downstream genes of At Rboh D and At Rboh F and regulate ROS accumulation in Arabidopsis leaves.At the same time,we obtained constitutive and inducible overexpression of AtWRKYb and its homologous gene AtWRKYc.We found that the constitutive overexpression lines were very small and had more branches than wild type.Through triple response assay,we observed that the length of hypocotyls of the overexpression lines was significantly shorter than the control,whereas the mutants were much shorter.These phenotypes imply a relationship between the AtWRKYb and the ethylene content in the Arabidopsis.In order to explore the link between them,we screened some ACS genes by q RT-PCR and found some ACS genes can be regulated by AtWRKYb.Then we conducted a dual luciferase assay,and confirmed that AtWRKYb can actually activate the expression of ACS genes to affect the content of ethylene in Arabidopsis.To summarize,AtWRKb participates in regulation of plant growth and development,possibly through ethylene signaling pathway.