Screening And Identification Of Esf,an Enhancer Of Fk-J3158 Mutant In Arabidopsis

In previous study,our lab identified a stomatal development mutant named fk-J3158.Through a series of studies,it demonstrated that early sterol synthesis pathway is essential for cell fate commitment and maintenance of stomatal precursor cells after first asymmetric division.To further study the mechanism of early sterol synthesis in stomatal development and find out the relevant factor or downstream gene of FK,we carried out a secondary EMS mutagenesis of fk-J3158,and screened the enhancer of stomatal development mutants or revertant mutants.In this study,we identified 303#(fk-J3158 background)from 1570 plants.303# mutant exhibited rounded leaves and reduced trichomes,and the number of small cell clusters and stomata clusters significantly increased in the rosette leaf epidermal.Therefore,303# is an enhancer of fk-J3158 which has enhanced the stomatal development defects in fk-J3158 mutant.To find out the enhancer gene,we backcrossed 303# with Col-0,and isolated a single mutant esf.Compared to wild type,esf had no obvious stomatal defects,but there were large pavement cells uniformly distributed throughout the epidermis.Also,esf mutant exhibited some other phenotypes: reduced trichomes,increased lateral branches and partially siliques abortion.In addition,the roots and hypocotyls were slightly shorter than Col-0.In order to determine whether the enhanced stomatal development defects in 303# mutant was caused by esf,we made genetic cross with esf and fk-J3158,and identified the phenotype of F2 generation plants.In the same F2 segregation population,four phenotypes including wild type,fk-J3158,esf and esf fk-J3158 plant were identified,and the separation ratio was close to 9:3:3:1.Thus,esf mutant could exacerbate the stomatal development defects phenotype of fk-J3158 mutant.Through map-based cloning,the gene of esf was finally positioned between makers MTG13 and F7K24 on chromosome 5,approximately 1042 kb interval.Using whole genome sequencing analysis,we found there was only a G to A substitution at the 992 base of At5g19220 which caused premature termination of translation.The results indicated that At5g19220 is the possible candidate gene of esf.