The Molecular Mechanism Of Flower Color Variation Of Iris Laevigata Based On Multi-omics Analysis

Iris laevigata is an extremely cold-resistant species in Iris,and is a very important aquatic flower in landscaping in Northeast China.The flower color of I.laevigata in nature is mostly blue,occasionally light blue.Because its flower color is not rich enough,it is hoped to make a breakthrough in the breeding of I.laevigata flower color in garden application to meet people’s demand for flower color diversity.With the application of genetic engineering in flower color breeding,it provides a new idea for flower color breeding of I.laevigata.The study of flower color and variation mechanism is very necessary for the development of genetic engineering breeding.The white flower variant plant of I.laevigata var.alba was found in the field by the research group,however,the mechanism of flower color variation is still unclear.The discovery of I.laevigata var.alba provides a very good material for analyzing the mechanism of flower color variation of I.laevigata.In this study,the perianth slices of I.laevigata（IB）and I.laevigata var.alba（IW）were used as materials.Based on metabolomics analysis,starting from the material basis of flower color variation,combined with transcriptome and proteome analysis,the key genes of flower color regulation were obtained and the regulatory function was verified,and the mechanism of flower color variation of swallow flower was deeply analyzed.The main results were as follows:（1）Widely targeted metabolomics analysis showed that anthocyanins is the material basis of IB coloration.IW also contains anthocyanins,but the type and content of anthocyanins in IB are significantly higher than those in IW,especially delphinidin.The content of proanthocyanidins in IW increased significantly,and the accumulation of anthocyanin synthesis was blocked,which was an important reason for the formation of IW flower color.（2）Two Pac Bio Iso Seq libraries were constructed using third-generation transcriptome sequencing,and a total of 835,813 transcripts were obtained after redundancy removal.The second-generation transcriptome sequencing was performed with the third-generation full-length transcript as a reference,and a total of 15,930 differentially expressed genes（DEGs）were identified in IB and IW samples.GO and KEGG enrichment analysis were performed on DEGs,and 23 structural genes and 9 transcription factors related to anthocyanin synthesis were screened.Combined transcriptome and metabolome analysis identified 11 DEGs involved in flower color variation,including Il CHS,Il CHI-1,Il CHI-2,Il F3H,Il ANS,Il ANR,Il MYB3,Il MYB4,Il MYB5,Ilb HLH2 and Ilb HLH3.（3）A total of 1723 differentially expressed proteins（DEPs）were identified in IB and IW by i TRAQ technology.GO and KEGG enrichment analysis was performed on DEPs,and 7structural genes and 3 transcription factors involved in anthocyanin synthesis were screened.Six DEGs involved in flower color variation were identified by transcriptome and proteome analysis,including Il CHI-1,Il CHI-2,Il ANR,Il MYB4,Il MYB5 and Ilb HLH3.Multi-omics analysis showed that Il ANR was the key structural gene for IW to produce white flower variation,and Il MYB4,Il MYB5 and Ilb HLH3 were the key transcription factors to regulate the flower color variation of I.laevigata.（4）Four key genes involved in flower color variation,Il MYB4（OQ243217）,Il MYB5（OQ243218）,Ilb HLH3（OQ243219）and ANR（OQ433948）,were cloned from the I.laevigata.The plant overexpression vectors were constructed and transformed into tobacco by leaf disc method.T₃homozygous transgenic lines（OE-Il MYB4,OE-Il MYB5,OE-Ilb HLH3,OE-Il ANR）were obtained.Compared with the wild type（WT）,OE-Il MYB4 and OE-Il ANR made the corolla color of tobacco lighter,and Il MYB4 changes the flower color by up-regulating the expression of ANR gene.OE-Il MYB5 and OE-Ilb HLH3 advanced the coloration period and deepened the color of transgenic tobacco corolla by up-regulating the expression of HY5 and UFGT genes.In addition,Il MYB4 was also found to be involved in the regulation of leaf shape,leaf color,pistil development,and branching in leaf axils.（5）RT-q PCR was used to detect the expression patterns of Il MYB4,Il MYB5 and Ilb HLH3in four different stages and different tissues of IB and IW,including uncolored perianth（S1）,beginning coloring（S2）,completing coloring（S3）and fully blooming（S4）.It was found that Il MYB4,Il MYB5 and Ilb HLH3 were all preferentially expressed in flowers.In the S1-S3 period of IW,the expression of Ilb HLH3 was the highest,followed by Il MYB4.In the S4 period,the relative expression of Ilb HLH3 and Il MYB5 decreased,and both were lower than Il MYB4.Il MYB4,Il MYB5 and Ilb HLH3 proteins all acted on the nucleus.Yeast two-hybrid and bimolecular fluorescence complementation experiments showed that Il MYB4 and Il MYB5interacted with Ilb HLH3 protein.（6）The 1520 bp promoter sequence of Il ANR gene was cloned by FPNI-PCR,which not only contained the promoter elements of higher plants,but also contained some other functional elements and MYB binding sites related to plant growth and development.Yeast one-hybrid assay showed that Il MYB4 could bind to the P1 site of Il ANR promoter to regulate its expression.Dual luciferase experiments showed that the interaction between Il MYB4 and Ilb HLH3 could promote the transcriptional expression of Il ANR,resulting in the synthesis of proanthocyanidins such as epicatechin in the flavonoid biosynthesis pathway in IW,and the anthocyanin synthesis was blocked.