Cloning And Functional Analysis Of Osmotic Stress Resistant Mutant Osr1 In Maize

Water is one of the most important factors that affect the yield and quality of crops.More than 1/3 of the total land in the world is arid or semi-arid.In China,the arid and semi-arid areas reach 50% of the total land.Drought has adverse effects on the growth of plants,which has become one of the serious threats to food security both in China and in the world.Therefore,the study on molecular mechanism of drought stress response in plants is crucial for improving water use efficiency,and enhancing drought resistance of crops in order to increase the quality and the yield of crops in dryland.To study the molecular mechanism of plant response to drought stress,our Lab generated several maize mutant libraries for screening of mutants with altered drought stress resistance.Here,a mutant with high osmosis resistance was identified.This mutant showed no differences with the wild type under drought conditions,but higher sensitivity to mannitol induced osmotic stress than wild type plant.Therefore,it is defined as an osmotic stress resistant mutant(osr1).The phenotypic analysis reveals that osr1 mutant germinates normally on the sixth day after sowing,but shows higher leaf surface temperature than wild type one week after mannitol treatment.osr1 grows normally in two weeks after treated with mannitol(i.e.at the third week after sowing)while wild type wilted.The survival rate of osr1 is significantly higher than that of WT,indicating that osr1 is insensitive to osmotic stress compared to WT under mannitol treatment.The leaf water loss of osr1 is significantly lower than that of WT,while the relative water content of leaves is significantly higher than that of WT under mannitol treatment,which indicates that the water retention ability of osr1 is higher than that of the WT.Moreover,statistical analysis shows that there is no significant differences in stomatal morphological characteristics and stomatal density between osr1 and WT.Under mannitol treatment,however,both the stomatal conductance and transpiration rate of osr1 are significantly lower than those of WT,indicating that the phenotype of osr1 is not related to the stomatal development,but to the stomatal conductance,suggesting that the water retention capacity of leaves in osr1 is higher than that in WT.In addition,the number of lateral roots,the root surface area and the relative underground biomass content of osr1 and WT were compared under different treatments.The results show that under normal condition or mannitol treatment,the number of lateral roots,the root surface area and the relative underground biomass content of osr1 were significantly higher than that of WT.These findings indicate that osmotic stress insensitity of osr1 may be attributed to its strong root system.Both soluble sugar and proline are small organic compounds induced by abiotic stress in plants.The results show that the soluble sugar and proline content,as well as leaf osmotic potential of osr1 is higher than that of WT under mannitol treatment,indicating that osr1 has stronger water absorption than WT most likely by accumulating more small molecules such as soluble sugar and proline.At the same time,the MDA content of leaves in osr1 is much higher than that in WT,while SOD and POD activity of leaves in osr1 are significantly higher than that in WT under mannitol treatment.These results demonstrate that osr1 is insensitive to osmotic stress.To study the molecular mechanism of OSR1 action,genetic analysis was carried out.It shows that the phenotype of osr1 is controlled by a single recessive gene.The result of map-based cloning shows that the OSR1 was located in the Bin 6.06 of chromosome 6,AC209257.4?FG006(Zm00001d038446).The protein encoded by OSR1 is a member of the AP2/ERF transcription factor superfamily.OSR1 contained 205 amino acids,including one AP2/ERF domain that could bind to the specific DNA sequence(DRE core motif).In addition,subcellular localization analysis reveals that OSR1 is localized to the nucleus,and EMSA experiment shows that OSR1 is specifically associated with DRE box.Therefore,OSR1 may act as a transcription factor to regulate the expression of its target genes.Tissue expression analysis shows that OSR1 has the highest expression in leaves and the lowest expression in kernels.Compared with the WT,osr1 has five mutations: four nonsynonymous substitutions and one 12 nucleotide delection.Based on the protein structure prediction,both secondary and steric structures of osr1 mutant protein is altered in comparison with the WT.In addition,both quantitative PCR and digital PCR results show that the OSR1 expression level in osr1 is lower than that in WT.The yeast one hybrid assay shows that the transcription activity of osr1 is lower than that of wild type.For the identification of downstream targets of OSR1,RNA-seq analysis was performed to identify differentially expressed genes(DEGs)in WT and osr1 under control and osmotic stress conditions.Results show that the number of DEGs induced by osmotic stress are far less in osr1 than in WT.In addition,most genes are down-regulated in osr1 under osmotic stress conditions.GO enrichment analysis indicates that the DEGs between the control group and osmotic stress treatment group are involved more diversified biological processes in osr1 than WT.q RT-PCR were also conducted and verify the results of transcriptome analysis.The analysis of DEGs between WT and osr1 under osmotic stress reveals that auxin related factors,such as PPR and TPR family members are up-regulated,and might be involved in osmotic stress response by promoting root development.In summary,map-based cloning of osr1 mutant demonstrates that OSR1 is a member of the AP2/ERF transcription factor superfamily.osr1 contains four nonsynonymous substitutions and one 12 nucleotide deletion,and has reduced expression level of OSR1.Gene functional analysis reveals that OSR1 is localized to the nucleus and can bind to DRE element.It is assumued that OSR1 acts as a negative regulator of osmotic stress response.Loss of or reduced function of OSR1 confers osmotic resistance by regulating root development.The results of this study would provide useful information for elucidating the molecular mechanism underlying plant osmotic sress resistance.