Molecular Mechanism Of Carpel Fusion In The Process Of Ovarv Formation In Maize

The normal development of maize kernel has an important effect on realizing the economic value of maize.Maize kernel is composed of pericarp,embryo and endosperm.Pericarp,the outer most layer of the kernel,protects the development of embryo and endosperm.The incomplete pericarp or a gap on the pericarp of maize kernel will affect the normal development of maize kernels.Maize kernel is much more vulnerable to pathogens without the protection of a complete pericarp.Maize kernels infested with pathogens will affect their own edibility,feed and industrial value.Pericarp plays an important role in the protection of seed,and incomplete pericarp affects the seed vigor.Previous studies found that pericarp is derived from the development of ovary wall,and the ovary wall results from the carpels which compose the maize pistil in the floret development.Currently,the variation phenotype reported in pistil development is limited,and there are few studies on the development of carpel fusion during maize pistil formation.Thus few studies have been done on the genes involved in development of carpel fusion and on the regulation of these genes.In this study,the experiment was conducted on the maize inbred line with incomplete kernel pericarp.The aim of this study was to analyze the morphological changes of the pericarp-deficient kernels during pistil formation,and to find the genes which take part in the regulation of the variation of this morphological structure,and finally to build the molecular network that the main genes are subjected.The preliminary results of this study are as follows:1.Results of morphological and histological observations during the IFC kernels development:In this study,after continuous observation of the development of floret and ovary,it was found that the pericarp defect resulted from the incompletely fused carpels when the pistil formed.2.Gene expression differences between IFC and CFC ovaries:The maize ovary derives from fusion of three carpels;however,the molecular networks regulating maize carpel fusion remain largely unclear.In this study,RNA sequencing(RNA-seq)was performed on completely fused carpels(CFC)and IFC ovaries that were collected after carpel fusion defects could be morphologically distinguished.In total,877 differentially expressed genes were identified.Functional analysis revealed overexpression of genes related to "DNA binding","transcription regulation""hormones",and "stress responses".Among the 88 differentially expressed transcription factor(TF)genes,five showed a high degree of conservation(77.7-88.0%amino acid identity)of their conserved domains with genes associated with carpel fusion deficiency in Arabidopsis thaliana,suggesting that these five genes might control carpel fusion in maize.In addition,30 genes encoding components of hormone synthesis and signaling pathways were differentially expressed between IFC and CFC ovaries,indicating complex hormonal regulation during carpel fusion.These results help elucidate the underlying mechanisms that regulate carpel fusion,supporting the functional analysis of genes involved in producing this phenotype.3.The miRNA regulation and phytohormone changes in icompletely fused carpels ovary:Although the molecular basis of carpel fusion in maize ovary development remains largely unknown,increasing evidence suggests a critical role of microRNAs(miRNAs).Here,a combination of miRNA sequencing,degradome and physiological analysis was used to characterize carpel fusion development in incompletely fused carpels(IFC)ovary and completely fused carpels(CFC)ovary in maize.A total of 162 known miRNAs distributed across 33 families were identified,of which 20 were differentially expressed.In addition,53 miRNA candidates were identified,and 10 of them were found to be differentially expressed in IFC and CFC ovaries.In degradome analysis,a total of 113 and 11 target genes were predicted for known and novel miRNAs,respectively.Thereinto,24(60%)target genes of the differentially expressed known miRNAs were found to code transcription factors,including auxin response factors(ARF),TB1-CYC-PCFs(TCP),APETALA2(AP2),growth regulating factor(GRF),MYB,NAC,NF-YA and so on,which have been proved to have roles on regulating carpel fusion development.Correlation analysis of differentially expressed known miRNAs and their targets to phytohormone signals revealed that either of the miRNA or of its target had a significant correlation to at least one examined phytohormone signal,the main regulator of carpel fusion development.These results suggested that incomplete carpel fusion is partly the result of differential expression of certain miRNAs and their targets.Taking together,the findings improve our knowledge about the effect of miRNA regulation on target expression,providing a useful resource for further analysis of the interactions between miRNAs,target genes and phytohormones during carpel fusion development in maize.4.The regulation DNA methylation and IncRNAs in icompletely fused carpels ovary:Both DNA methylation level and the expression of lncRNA were changed between IFC and CFC ovaries.The number of genes with hypomethylation is 4.8 times that of hypermethylation genes.The number of miRNA genes with hypomethylation is 9.2 times that of hypermethylation miRNA genes.The number of repeats with hypomethylation is 2.4 times that of hypermethylation repeats.The number of down-regulated IncRNA is 6.7 times that of up-regulated genes.These results indicate that both hypomethylation of DNA methylation and down-regulated expression of lncRNA may be partly responsible for incompletely fused carpels.5.Improvement and characterization of carpel fusion deficient of maize kernel:As we all know,the periparp plays a role in the protection of seed,and incomplete pericarp affects the seed vigor.In this study,the results showed that the number of incomplete pericarp kernel on an ear decreased gradually,and the defect kernel was about 1%on an ear after the complete of two cycles of improvement.At maturity,compared to the normal kernels the one hundred kernel volum,fresh weight,dry weight and filling rate of the IFC kernels decreased.