Identification And Characterization Of Mac3a/3b(Suppressors Of Da3-1)in Arabidopsis Thaliana

As the continues increase of China's population,so does the demand for food.Although the output of China's grain has increased year by year in recent years,it is still unable to meet the growing needs of people,making China has to import large-scale food every year.Although China has 1.8 billion mu of cultivated land,the per capita cultivated area is very limited.Therefore,increasing the unit yield of grain is the key to solving the current food shortage.The organ size of the plant is closely related to the yield of the crop,so deciphering the underlying regulatory network of plant organ size is essential for further increasing the crop yields.The cell size of plants can be controlled mainly by regulating cell division and expansion,and ultimately regulate plant size.In Arabidopsis,although some genes involved in the regulation of organ size have been discovered,the knowledge is still far away from our comprehensive understanding of the regulatory networks of Arabidopsis organ size.At present,finding more genes involved in regulating plant organ size is the key to deciphering the regulatory network of organ size.Previous studies have identified a gene DA3(UBP14)that regulates plant organ size,da3-1 mutant shows large cotyledons,large floral organs,and leaf curled phenotypes.DA3 encodes the deubiquitinating enzyme UBP14,which binds to target proteins in plants and acts on downstream APC/C complexes,affecting cyclin activity,regulating nuclear replication,and ultimately affecting cell and organ size.To explore more substrates for DA3,we obtained a da3-1 repressor mutant library by EMS mutagenesis and successfully screened two key repressor mutants,5019b and 2223c.These two suppressor lines substantially rescued da3-1 to the wild-type phenotype.The regulatory genes of these two repressors were identified by map cloning and resequencing,were referred to as MAC3A(At1g04510)and MAC3B(At2g33340).It has been reported that MAC3A and MAC3B belong to the E3 ubiquitin ligase family members comprising the U-box domain,both of which have E3 ligase activity.The phylogenetic tree analysis of MAC3A and MAC3B shows that they have high homology and functional redundancy.To further determine the phenotype of the 5019b and 2223c repressor lines,we performed a statistical analysis of the cotyledon area and root length of them,and the results showed that both repressor lines inhibited the da3-1 phenotype.The results of cell ploidy analysis by flow cytometry further confirmed the phenotypes of 5019b and 2223c.Subcellular localization analysis indicated that both MAC3A and MAC3B were localized in the nucleus.Tissue qRT-PCR analysis showed that MAC3A and MAC3B were expressed in roots,stems,leaves and floral organs of plants,and the highest expression level was appeared in leaves.The tissue pattern analysis of genes by GUS staining indicated that MAC3A can be detected in roots,cotyledons and floral organs.The qRT-PCR analysis and GUS staining showed that both MAC3A and MAC3B showed constitutive expression in each tissue.Preliminary bimolecular fluorescence complementation experiments showed that DA3 interacted with both MAC3A and MAC3B,indicating that MAC3 A and M AC3B may severs as the direct substrates of DA3.These above results indicate that MAC3A and MAC3B are inhibitors of da3-1,and subcellular localization and tissue expression patterns demonstrated that MAC3A,MAC3B and DA3 have similar expression patterns.MAC3A and MAC3B may form a protein complex with DA3 to jointly regulate organ size of Arabidopsis.These studies provide new theoretical evidences to reveal the molecular mechanism by which DA3 regulates organ size of plants.