A Study On Map-Based Cloning And Biological Function Of A GRF-Interacting Factor1(gif1)Mutant In Maize

Plant morphogenesis results from a balance of indeterminate and determinate cell fates.Cells with indeterminate fates are located in meristems.Groups of pluripotent cells that produce lateral organs.We identified a mutant that has a defect in balancing determinacy and indeterminacy.The mutant with narrow blades and shortened internodes indicated that there were fewer determinate cells in the leaves and stem cells.On the contrary,mutants fail to control indeterminacy in shoot meristems.Inflorescence meristems was apical fasciated which indicated that the lateral meristems had lost the determinacy.In this study,a GROWTH REGULATING FACTOR-INTERACTING FACTOR1(GIF1)was identified as the candidate gene responsible for mutant phenotype through positional cloning.These results provide new insight into the biological functions of GIF1.The major achievement of this study is as following:1.Morphological and cytological phenotypes of gif1 plants Most important agronomic traits were different significantly between wild type(WT)and gif1.The mutant plants were almost semi-dwarf with narrow and smaller leaves.The branch number of tassel was reduced,while the ear developped extra branches at the base and were fasciated.The mutants showed male and female abortion,and they could not produce offspring through reciprocal cross with other inbred lines.Scanning Electron Microscopy(SEM)was used to observe the developing tassels and ears.The result indicated that the female and male inflorescence meristems of gif1 mutant were dysplastic.The apical inflorescence meristems were fasciated in both ear and tassel and lost determinacy.The branches number of gif1 tassel reduced and the development of branch meristems delayed.But increased short branches in the ear were observed.Glume primordia produced from the SM of male florets were arrested and degeneration of the lower floret and stamen primordia of the female inflorescence was delayed compared to wild type.2.ZmGIF1 was identified as the mutated gene through map-based cloning.Genetic analysis showed that the gif1 mutant was controlled by a pair of recessive single genes.The gene was mapped to the long arm of chromosome 1 and encoded a GIF1 transcription factor(GRMZM2G180246).And it was homologous to At GIF1/ANGUSTIFOLIA3(AN3)and OsGIF1.Through resequencing analysis ofGRMZM2G180246,significant difference was found in the gene between WT and gif1 mutant.A 51 bp sequence insertion in exon3 was the major variation site.According to the further analysis results,a 51 bp insertion composed of a 43 bp Popin transposon and an 8bp positive repetitive sequence.The transposon inserted into the protein domain,causing the termination of transcription.This sequence was also a direct cause of the reduction of transcription efficiency of the gene.A Mu7 transposon inserted in the 5'-UTR of gif1-mum1.The Mu insertion of gif1-mum1 led to a significant reduction in GIF1 expression in immature tassels and developed semi-dwarf phenotype relative to that in W22.All gif1-mum1 plants had narrower and shorter leaves,and the development of female and male inflorescences were inhibited.The result indicated GIF1 was the cloned gene.The full and CDS length of GIF1 was 3656 bp and 686 bp respectively,which coded 227 amino acids.GIF1 belongs to GIF family.These tissue specific GRF/GIF complexes were formed through specific identification between the highly conserved SNH structure domain of GIF and the GROWTH-FACTOR(GRF)N-end conservative structure domain QLQ.It was a kind of co-transcriptional activator.Different GRF/GIF complexes participated in the regulation of different development processes of plant growth.3.Specific expression of GIF1 Spatiotemporal expression analysis showed that GIF1 was highly expressed in dividing tissues including shoot apical meristems(SAMs),immature tassels and ears.The expression pattern analysis of GIFs and GRFs indicated that ZmGIF1 had obvious specificity in inflorescencethe development and was higher in the early stage than in the late stage.The expression of GRFs varies from different organizations.The expression of GIF1 in IM and SPM were higher than in SM and FM of ears.mRNA in situ hybridization showed that GIF1 transcription were enriched in the SAM but were excluded from the tip.Strong expression was found in young leaf primordia.Expression at the base of the SAM,adjacent to the recently initiated leaf,was internode in the future.The SAM and leaf expression was absent in gif1.High levels of expression were seen in the IM except at the tip,and in or surrounding SPM and SM.Expression at slightly later stages includes a ring around the floral meristem(FM)and in the lemma and palea primordia.Expression was not detected in the mutant.The expression pattern overlapped with that of ramosa2(ra2)in the SPM and SM meristems.Expression of ra2 was greatlyreduced in the mutant.That indicated that the two genes may overlap in function.4.Regulation and interaction pathway of GIF1 We identified 350(24%)DEGs were up-regulated,while 1,118(76%)DEGs were down-regulated in the gif1 developing tassel relative to wild type.Gene function classification analysis showed that down-regulated genes were highly enriched in biological processes associated with biological processes and molecular function.Several important genes such as ub3,ra2,te1,zfl1/2 and vt2 involved in inflorescence development were down-regulated genes in the gif1 mutant.They were key regulators of axillary meristem identity and determinacy in maize.Many genes that were significantly down-regulated in gif1 were involved in cell cycle and auxin synthesis and response.Of the GRF family members,GRF3 was up-regulated,while both GRF7 and GRF17 were significantly down-regulated.Expression of GIF2 and GIF3 were not affected.Chromatin Immunoprecipitation sequencing(ChIP-seq)analysis showed that GIF1 mainly bound in gene region with a high proportion(32.4%)of all the sites.And12.0% of binding within 1.0 kb promoter regions upstream of the transcriptional start site(TSS).Within 10 kb of high-confidence peaks,we identified 1275 genes as putative targets of GIF1.Putative GIF1 targets included a number of known transcription regulators.We found 50 genes differentially expressed in gif1 mutant that were directly regulated by GIF1 through comparison analysis between ChIP-seq and RNA-seq,including 37 down-regulated and 13 up-regulated genes.GIF1 interacted in vitro with 13 of 16 GRFs excluding GRF7,GRF11 and GRF20.GIF1 could self-regulate confidence at the transcription level and regulated the branch development of tassel through directly binding UB3.5.Association sites of candidate gene By association analysis of candidate genes with rich source of natural variation and different maize inbred lines for sequencing,there were 22 polymorphic sites,5 of which were significantly correlated with plant and inflorescence development as the smallest allele frequency(MAF)was greater than 0.05.The polymorphic sites were mainly focused on the position of the transcription start site upstream about 1.0 kb and5 'UTR region.The genetic variation was mainly distributed in the transcription of the gene regulatory regions.GIF1 may be in the expression levels of regulation.Thephenotypic data and the genetic variation of GIF1 were analyzed in the candidate gene,and the two SNP sites of A/G and intron2 C/G in exon1 were closely related to the branch number of the male.