Molecular Mechanism Of XsMYB113 Regulating Yellowhorn Flower Color Change

Xanthoceras sorbifolium(yellowhorn),an important woody shrub or small tree that originated in China,bears oil-rich seeds.It has strong physiological characteristics of cold resistance,drought resistance,and salinity resistance,and can usually be used as a pioneer tree species for soil and water conservation and greening of barren hills.The kernel of yellowhorn has high oil content and can be used as high-quality edible oil,it is called "Northern Camellia Oil ".In addition,yellowhorn is a precious landscaping tree species,with multiple varieties and bright colors,which has high ornamental value.Therefore,yellowhorn has a wide range of ecological,agricultural,horticultural and commercial significance.Among them,the color of inner base of petals gradually changes in white-flowered trees,the color of flowers has graduality.However,in nature,there are few reports on the spontaneous change of flower color in different developmental stages.The graduality of flower color makes the plants more ornamental.Compared with the plants with a single flower color,the plants with gradient colors are more competitive in terms of ornamental value.In the process of yellowhorn flower color change,the DNA sequence did not change,but the gene expression did undergo heritable changes.Several studies have shown that epigenetic regulation is related to fruit development and color formation processes in apple,sweet orange,strawberry,tomato and other species,and plays an important role in these processes.Therefore,a similar regulatory mechanism may also be related to the process of yellowhorn flower color change.In order to study the regulatory mechanism of flower color change,this project adopts the methods of metabolome,transcriptome and epigenome.Using metabolome to analyze the external causes of the yellowhorn flower color change.Using the methods of epigenome and transcriptome to analyze the intrinsic molecular mechanism of epigenetic regulation in the process of yellowhorn flower color change.The main findings are as follows:1.Targeted metabolism and gene expression analysis during yellowhorn flower color changeAccording to the color and developmental state,the process of yellowhorn flower color change was divided into four different developmental stages(S1–S4),and the whole flowers of the four stages were used as materials for targeted metabolism and transcriptome sequencing.The results of targeted metabolism showed that the reason for the yellowhorn flower color change was mainly due to the gradual accumulation of cyanidin and peonidin at the inner base of the petals.Differentially expressed genes(DEGs)in each stage were analyzed by transcriptome data.Commonly up-regulated genes in S2 vs S1,S3 vs S1,S4 vs S1 were analyzed by GO and KEGG enrichment.These DEGs include biological processes mainly involved in glucose metabolism,fatty acid metabolism,pathogen resistance,plant hormone signal transduction,and flavonoid metabolism.Through the analysis of gene expression in flavonoid metabolic pathway,it was found that except for the decrease of F3’5’H gene expression,the gene expression in most anthocyanin biosynthesis pathways(ABP)increased significantly.Among them,F3’H and OMT-1 are the key genes for the synthesis of cyanidin and peonidin.The two genes were overexpressed in tobacco,respectively,and the transgenic results showed that both genes could increase the total anthocyanin content in transgenic tobacco flowers.This proves the biological function of the two genes in the synthesis of anthocyanins in yellowhorn flowers.2.Epigenome analysis in the process of yellowhorn flower color changeBy mapping the single-base resolution DNA methylation maps of flowers in four developmental stages of yellowhorn,the results showed that DNA methylation changes dynamically with the gradual change of flower color.The genome-wide total DNA methylation increased from S1 through S4,which was mainly due to the increase of CHH methylation on transposons in yellowhorn genome.Through the study of DNA methylase and demethylase,it was found that the increase of genome-wide DNA methylation was related to the decrease of ROS1 gene expression.Further research showed that Rd DM regulated the expression of ROS1 gene.Furthermore,in this process we specifically analyzed the levels of 21-nt,22-nt and 24-nt si RNAs.It was found that the abundance of genome-wide 21-nt and 22-nt si RNAs showed an upward trend,while the abundance of 24-nt si RNAs showed a downward trend.Further analysis found that changes in 24-nt si RNA levels correlated with gene expression levels of RDR2 and NRPE in the Rd DM pathway.3.Epigenetic regulation mechanism of anthocyanin biosynthesis in the process of flower color changeFour MYB genes(Xs MYB113-1,Xs MYB113-2,Xs MYB113-3 and Xs MYB113-4)related to anthocyanins were obtained by analyzing the R2R3-MYB transcription factor in yellowhorn genome.Transcriptome and RT-PCR results showed that their expression levels were upregulated in the process of flower color change and correlated with anthocyanin accumulation.Through gene structure analysis,it was found that four Xs MYB113 genes were located on the same chromosome and formed a tandem structure gene cluster with the transposons.By further analysis of DNA methylation and small RNAs on transposons near genes.It was found that CHH methylation levels on these transposons decreased with decreasing abundance of 24-nt si RNA.It indicated that the expression of Xs MYB113 genes were regulated by Rd DM.By transforming four Xs MYB113 genes into tobacco for gene function verification,it was found that all four genes can positively regulate the synthesis of anthocyanin,among which the Xs MYB113-1 gene has the strongest ability to regulate the synthesis of anthocyanin in tobacco.In conclusion,the analysis of the regulation mechanism of the yellowhorn flower color change is helpful to study the similarities and differences of the regulation modes of anthocyanins in plants with different color rendering modes.This study provides a theoretical basis for the creation of new varieties of ornamental yellowhorn,and has great guiding significance and practical application value.