A Study On Molecular Mechanism Of GIF1 In The Regulation Of Female Inflorescence Architecture And Floral Development In Maize

The development of inflorescences is highly associated with grain yield of maize(Zea mays L.),exploring the genetic basis on inflorescence development is important for improving maize grain yield.In this study,the grf-interacting factor 1(gif1)mutant previously studied were used to deeply analyze the botanical and cytological phenotypes on the female(ear)inflorescence and florets.The genes differentially expressed in the ear inflorescence of the gif1 and wild type were identified by RNA sequencing.The GIF1-interacting proteins were identified by the co-immunoprecipitation(Co-IP)experiments,the direct target genes of GIF1 were detected by chromatin immunoprecipitation sequencing(ChIP-seq).The main results are shown as follows: 1.Loss of function of Gif1 severely impairs the ear inflorescence architecture and floral organ development The ear inflorescence of the gif1 mutant was observed by stereomicroscope and scanning electron microscopy.The morphological and anatomical differences between wild type and gif1 mutant were compared.We found that The wild-type(WT)ears born paired spikelets in a highly ordered manner,and lacked any of long branches,while the gif1 ears displayed two extreme phenotypes: highly branched inflorescence(85%)and barren inflorescence(15%).On the barren inflorescence,spikelet meristems initiated were low active to differentiate consistently into fertile florets,while those branches on the highly branched inflorescence could produce variable number of meristems.In each of WT florets,three stamen primordia were arrested,carpel primordia were consistently developed to fused carpel.In the gif1 florets,stamen primordia were arrested,however,carpel primordia frequently failed to be initiated,or failed to develop into fused carpel.The ovule primordia in the gif1 florets were enlarged and were naked,showing expanded nucellus.In summary,the gif1 ear exhibited strong defective phenotypes with highly branched inflorescence,and florets on the gif1 was sterile.2.Overexpression of GIF1 compensates defective phenotypes of the gif1 mutant.Five transgenic lines were created by introducing GIF1-GFP fusion protein driven by Ubi promoter into maize ZZC01 line.The overexpressing GIF1-GFP transgenic lines showed normal plant status with higher plant height and ear height,and wider and larger leaves compared with the gif1 mutant.We crossed the OE2 to +/gif1 heterozygote,and then selected those compensated individuals they are homozygous gif1/gif1 genotype at gif1 locus and are overexpressing GIF1-GFP,referred to as gif1gif1/OE2.All of gif1gif1/OE2 individuals had normal plant status and inflorescence architectures,and both female and male florets were well developed and were fertile,showing overexpression of GIF1-GFP well compensates defective phenotypes of the gif1 mutant.3.GIF1-interacting proteins involve in a wide range of biological processesUsing OE2 overexpressing immature female inflorescences in transgenic families,using immunoprecipitation and mass spectrometry sequencing,56 GIF1 interaction proteins were detected in 3 biological replicates.Interacting proteins are involved in biological processes such as inflorescence development,hormone biosynthesis,chromatin remodeling,signal transduction,protein biosynthesis and degradation,metabolism,and DNA replication.Among them,a protein Nana Plant2(NA2)in brassinosteroid(BR)biosynthesis was verified to be interacted with GIF1 by luciferase complementary imaging(LCI).The NA2-GIF1 interaction demonstrated that GIF1 affects the hormone synthesis process to regulate female inflorescence development.4.GIF1 binds to inflorescence development-related genesUsing chromatin immunoprecipitation sequencing(ChIP-seq),a total of 11683 peaks and 1832 genes were identified in the immature ear inflorescence of the OE2 transgenic line.A total of 1982 peaks and 540 genes were identified in not less than 2 replicates.Combined with transcriptome sequencing(RNA-seq),a total of 177 genes were predicted as direct targets of GIF1.Among them,84 genes(47.5%)were down-regulated and 93 genes(52.5%)were up-regulated in the gif1 mutant compared to wild type.Importantly,GIF1 binds to genes such as RAMOSA2 and CLE4 a which are involved in the regulation of development of the maize inflorescences.Therefore,it is speculated that Gif1 is required for activity of inflorescence meristem,spikelet-paired meristem and carpel edge meristem.