Identification And Functional Analysis Of The Arabidopsis Thaliana ANGUSTIFOLIA Enhancer IPGA1 That Regulates Petal Growth

In order to survive and adapt to the environment,plants have evolved diverse organs and cells.For example,in order to reproduce,the petals of angiosperms evolved a variety of shapes and colors,and the epidermis of petals evolved into unique conical cells in order to attract pollinators.How plant organs and cells develop into specific forms is an important scientific question in plant biology.Plant organs,such as petals and leaves,evolve in various forms,which depends on sophisticated genetic regulation.The morphogenesis of organs mainly depends on anisotropic growth,that is,nonuniform growth in space.Many genes have been identified to regulate the size and morphology of organs,but the molecular and genetic regulation mechanisms of organ growth and morphology remain to be further studied.More studies are needed in terms of the contribution of changes at the cell level to organ growth.Therefore,genetic screening for mutants and the identification of novel genes will advance our understanding of organ growth and morphogenesis.Arabidopsis thaliana ANGUSTIFOLIA?AN?is a homologous protein of animal CtBP/BARs?C-terminal binding protein / BFA-induced adenosine diphosphate glycosylation substrate?.The function of CtBP / BARs has been widely studied in animals.However,the functional study of AN in plants is verylimited.AN is involved in the morphogenesis,microtubule skeleton arrangement and stress defense of Arabidopsis thaliana.The organs?leaves,petals,etc.?with deletion of AN function have narrower,more anisotropic phenotypes than the wild type.But how AN cooperates with other genes to regulate organ growth and the molecular and genetic mechanism regulating morphology is largely unclear.In this study,based on petal phenotype,we screened the enhancer mutants of an-2,with loss of AN function,by EMS chemical mutagenesis.We obtained an enhancer mutant ipga1-1,which could enhance the petal anisotropic growth of an-2?increased petal growth anisotropy?.Compared with the wild type,the petals of ipga1-1 showed longer and narrower phenotype.A single base mutation at base 1401 of the At4g18570 gene was found in the ipga1-1 mutant,which changed from TGG?tryptophan?to TGA?termination codon?,by map-based cloning and high-throughput genomic sequencing.We named this gene as IPGA1.Furthermore,analysis of two T-DNA insertion mutants ipga1-2?SALK₁37332C?and ipga1-3?SAIL₅23_C10?showed that they displayed a similar petal phenotype as the ipga1-1.In addition,the petal phenotypes of F1 plants crossed by ipga1-1 and ipga1-2,ipga1-3,respectively,were similar to those observed in the ipga1-1 mutant which further demonstrated that IPGA1 mutation was the cause of ipga1 mutant phenotypes.In addition,the expression vector of IPGA1 gene driven byIPGA1 promoter was constructed,and the ipga1-1 mutant phenotype was restored by complementary analysis.Therefore,we demonstrated that the mutation of At4g18570 is the reason for the phenotype of ipga1-1 mutant.After obtaining the information of IPGA1 gene?At4g18570?,we found that it was a previously uncharacterized gene with unknown function.Subcellular localization analysis showed that IPGA1 was localized to cortical microtubules.Amino acid sequence analysis revealed that the N-terminal of IPGA1 protein had a coiled-coil domain.In Arabidopsis thaliana,there is no highly homologous protein with the full length of IPGA1,but three genes share homology with the C-terminal of IPGA1.The purified His-IPGA1 protein was obtained by prokaryotic expression and was shown to bind to microtubule in vitro by microtubule coprecipitation analysis.Analysis of IPGA1 by FRAP?fluorescence bleaching recovery?technique,the recovery time of fluorescent bleaching was similar to that of previously known microtubule-binding protein.The transient expression of IPGA1 truncated protein in Arabidopsis thaliana cotyledons was analyzed.We found that IPGA1 1-200 aa protein was localized to cortical microtubules.Therefore,we conclude that IPGA1 is a newly discovered microtubule-binding protein.In order to study the gene expression pattern of IPGA1,the fusion GUS expression vector of IPGA1 promoter was constructed and transformed into the wild type Arabidopsis thaliana plants.GUS activityassay showed that GUS was expressed in cotyledons,roots,petals,sepals and other organs and tissues of Arabidopsis thaliana.At the same time,qRT-PCR results showed that the expression of IPGA1 in petals was higher than that in other organs,especially in the late stage of petal development.In addition,the expression of GFP-IPGA1 protein was analyzed in a stably transgenic line expressing pACT2-GFP-IPGA1/ipga1-2.In order to study the physiological function of IPGA1,the phenotypes of organs and cells of ipga1 mutants and IPGA1 overexpression lines were analyzed in detail.The results showed that in ipga1 mutants,both organs and cells had enhanced anisotropic phenotypes?longer and narrower?: mutant cotyledons,true leaves,petals and sepals had significantly increased anisotropy compared with those of the wild type.At the same time,the base of the petal adaxial?close to the stylus side?cells was stretched.And the abaxial cells also had the elongated phenotype,and the number of cell lobes decreased significantly at the mature stage,and the cell edges were smooth.In IPGA1 overexpression lines,the organ anisotropy was decreased: the cotyledons became round.IPGA1 mutation had no effect on cell division process,but had an effect on cell expansion and growth anisotropy,resulting in morphological changes at the organ level.In conclusion,IPGA1 is a negative regulator of organ growth anisotropy.IPGA1 is a new microtubule-binding protein.In order to further study the effect of IPGA1 on microtubule cytoskeleton arrangement,ipga1-1 was crossed to GFP-Tubulin6?GFP-TUA6?,a microtubule marker line.It was found that the microtubule arrangement of the ipga1-1mutant petal abaxial epidermis cells was more parallel and orderly than that of the control GFP-TUA6 cells.And during petal development from stage8 to stage14,the arrangement of microtubule in mutant cells gradually became orderly.And the microtubule arrangement in IPGA1 overexpressed lines became disordered.This indicates that IPGA1 is a negative regulatory factor for the ordering of microtubule in the process of cell growth.In order to investigate the genetic relationship between IPGA1 and AN,we analyzed the organ phenotypes of ipga1 and an-2 single mutants and ipga1 an-2 double mutant in detail.The results showed that ipga1 enhanced the organ anisotropy of an-2,and the phenotype of ipga1 an-2double mutant was not only a simple superposition effect of single mutation phenotype,but also a synergistic effect on genetic relationship.This indicates that IPGA1 genetically interacts with AN in the pathway of petal organ growth.Taken together,we found that IPGA1 is an enhancer of AN by screening genetic mutants.IPGA1 was found to be a new microtubule-binding protein.And the physiological function of IPGA1 was further analyzed.IPGA1 played an important role in the microtubule organization in petal epidermal cells.It played an important role in cell growth and organ morphogenesis of Arabidopsis thaliana.Genetic analysis showed that there was genetic synergy between IPGA1 and AN.Therefore,it is of great scientific significance to understand the molecular and genetic regulation network of organ and cell morphogenesis.