Molecular Mechanism Of Transcription Factor OsWRKY37 Regulating Copper Uptake,translocation And Distribution In Rice

Copper is an essential element required for plant growth and development,which is important for both yield production and grain quality.Rice is one of the most important food crops in the world.More than 20% of rice grown area has been reported to be copper deficient in our country.Recent years,the highly intensive use of arable land and crop planting dramatically increasing the nutrient loss in soil,which may increase soil retirement and cause copper deficiency in arable land.Copper deficiency can easily lead to slow growth,delayed heading,decreased pollen activity,low fertility in rice.Therefore,it is of great significance for rice yield formation to explore the genes related to copper deficiency,analyze their biological functions and molecular regulation mechanisms to improve the absorption and utilization efficiency of copper in rice.However,the molecular mechanisms regulating copper uptake,transportation and distribution in rice under copper deficiency stress are still unclear.Previously,our research group obtained a function unknown WRKY transcription factor OsWRKY37 induced by copper deficiency through analyzing the transcriptome of rice plants treated with different copper concentrations.In this study,the expression patterns of OsWRKY37 in different growth stages and different tissues and organs of rice were systematically analyzed.The functions of OsWRKY37 in the process of copper absorption,transportation and distribution in rice and its effects on plant growth,pollen development and yield formation were studied by creating overexpression lines and knockout mutants.Furthermore,the downstream regulatory genes of OsWRKY37 were identified by RNA-seq,Yeast-one-hybridization,Transient expression assay in tobacco,EMSA and in situ RT-PCR,and its molecular regulatory module in copper absorption and transport was constructed.The research results can provide theoretical basis and genetic resources for further cultivating rice varieties with high copper nutrition,high efficiency,high yield and good quality.The main results are as follows:(1)Expression pattern of OsWRKY37 in rice.OsWRKY37 belongs to the Ib branch of WRKY transcription factor family and contains 4 exons.The results of subcellular localization showed that OsWRKY37 was located in the nucleus.Transcription activation experiment proved that OsWRKY37 can perform transcription function.The results of RT-q PCR analysis showed that OsWRKY37 was highly expressed in rice root at vegetative growth stage,and strongly expressed in root,basal node,old leaf,node,and immature panicles at reproductive growth stage.In Pr OsWRKY37:GUS transgenic lines,OsWRKY37 was highly expressed in the root,nodes,leaf vein,lamina joint,immature panicle,and anthers.At the same time,short-term copper deficiency can induce the expression of OsWRKY37 in the root and basal node of rice seedlings,while the recovery of copper supply can inhibit its expression level,but the external copper level has little effect on the expression level of OsWRKY37 in shoot.(2)Function of OsWRKY37 in absorption,transportation and distribution of copper in rice.During the vegetative growth period,compared with the wild type,the growth phenotype of OsWRKY37 over-expression lines and knockout mutants did not change significantly.However,under the condition of copper deficiency,the over-expression of OsWRKY37 significantly increased the copper concentration in all parts,improved the copper uptake capacity of root and increased the copper content in xylem sap,while the translocation of copper from root to shoot and the copper content in xylem sap in knockout mutants decreased significantly.In the reproductive growth period,the knockout mutants showed late heading,late flowering,and decreased yield under copper deficiency.Knocking out of OsWRKY37 reduced the transportation of copper from root to shoot and the distribution from source(leaf)to sink(grain),and decreased the accumulation of copper in anthers,resulting in cutin defect,decreased vitality,impaired germination in vitro and decreased fertility.This phenotype can be restored by spraying Cu SO4 solution on the top of rice.(3)OsWRKY37 regulates the expression levels of OsCOPT6 and OsYSL16.Through RNA-Seq analysis of OsWRKY37 knockout mutants and wild type,it was found that the expression levels of 11 copper transport-related genes in OsWRKY37 knockout mutants changed significantly.Furthermore,yeast-one-hybridization,transient expression assay in tobacco and EMSA confirmed that OsWRKY37 could bind to the promoters of copper transporters OsCOPT6 and OsYSL16.The results of RT-q PCR and in situ RT-PCR showed that OsWRKY37 could positively regulate the expression levels of OsCOPT6 and OsYSL16.(4)Function of OsCOPT6 in copper uptake of rice.The results of subcellular localization showed that OsCOPT6 was located in the cell membrane.The analysis of OsCOPT6 knockout mutants showed that the biomass,copper concentration,accumulation and uptake capacity of root in rice were reduced,and the mutants also showed the phenomenon of late flowering under the condition of copper deficiency.In general,copper deficiency can induce the transcription of OsWRKY37.OsWRKY37 can bind with the promoters of OsCOPT6 and OsYSL16 to positively regulate their expression levels,which can promote the uptake of copper by rice plants and the transportation of copper from root to the shoot and reproductive organs for the development of floral organs and yield formation.