Identification And Gene Cloning Of Topp4-1 Suppressor Mutants In Arabidopsis

In previous study,our lab has identified a dwarfed Arabidopsis mutant named topp4-1,in order to further understand the function of TOPP4 in Arabidopsis,we identified thirteen suppressors of topp4-1 by screening an EMS-mutagenized topp4-1 population.In this study,we characterized the phenotypes of 2731 and 2141 mutants,and identified the genes by map-based cloning and whole genome sequencing.Studies on genetic and function analysis of these genes were done and the results provide deep insights into the important function for PP1 to regulate plant growth and development.2731 mutant inhibits the phenotype of topp4-1 mutant and exhibits normal apical dominance,roundish and increasing rosette leaves and abnormal inflorescence.Genetic evidence showed that the phenotype of 2731 is controlled by a single recessive nuclear locus.The locus was finally located in a 730 kb DNA region between markers F1N18 and T19E23 on Chromosome 1 by map-based cloning.Whole genome sequencing analysis of AFO allele in the mutant revealed that the 1030th base is substituted from C to T in the first exon,which results in an amino acid change from proline to serine.AFO encodes an F-box protein,which is required for the proper identity of the floral meristem.To study the physiological function of AFO,we examined afo-11 and afo-11 topp4-]phenotypes in the presence of hormones.In response to GA3 treatment,the hypocotyls of afo-11 and afo-11 topp4-1 lines were dramatically shorter than WT.The decreased sensitivity of afo-11 and afo-11 topp4-1 to GA3 suggests that mutants are partially defective in GA responses.The root-length assays showed that afo-11 topp4-1 was not sensitive to exogenous IAA comparing with WT.The decreased sensitivity of afo-11 and afo-11 topp4-1 to IAA suggests that AFO may be involved in auxin biosynthesis or responses.2141 mutant exhibits normal plant height and rosette leaves,abnormal carpel post-fertilization,short and small siliques,abnormal seeds.Map-based cloning and sequencing analysis showed that the phenotype of the mutant is controlled by FUL,which belongs to MADS box gene.The identity of ful-2 was subsequently confirmed by genetic complementation experiments.These results showed that the ful-2 mutation at this position was not responsible for the phenotypes of the suppressor mutant.To map the locus,large F2 mapping population showing topp4-1 phenotype were used for map-based cloning.We mapped the RPT gene on Chromosome 5,which has strong linkage relationship with FUL.Comparison of the sequences of RPT allele revealed that the 1108th base is substituted from C to T in the first exon,which results in an amino acid change from arginine to cysteine.RPT,which belongs to CC-NBS-LRR family,encodes a disease resistance protein and involves in plant defense response.