Molecular Regulatory Network Of Flower Color Formation In Hybrid Liriodendron ’Jinzhan’ Based On Transcriptomics

Liriodendron is a plant of the Magnoliaceae family of magnoliids,belonging to the tertiary relict species.At present,only two sister species,Liriodendron chinense(Hemsl.)Sargent.and Liriodendron tulipifera Linn.,exist in this genus.The former is naturally distributed in China and northern Vietnam,while the L.tulipifera is naturally distributed in the eastern United States to southern Canada,showing a typical east Asia-Eastern North America discontinuous distribution.Liriodendron can be hybridized to obtain hybrids with significant heterosis.While retaining the parental phenotype,it also shows stronger growth adaptability.The leaf shape has both the parental phenotype and flowers.Gorgeous and long flowering,it is an excellent tree species for garden viewing,road greening and fast-growing timber.In 2018,a hybrid Liriodendron with particularly bright flowers was found in the hybrid progeny forest of L.chinense and L.tulipifera in Longshan Forest Farm,Zhejiang Province.It was later named "Jinzhan",and its petals except the base were covered with Covered by orange-yellow pigments,it has higher ornamental and economic value than the common hybrid Liriodendron.In this study,the morphological observation was carried out using the petals of the hybrid Liriodendron ‘Jinzhan’,L.chinense,and L.tulipifera at different developmental stages as materials.Subjects were subjected to transcriptomic studies.The main findings obtained are as follows:1.At the morphological level,the petals of different species of Liriodendron exhibited different coloring patterns during development.The color of the upper part of the petals of L.chinense has always been dark green;the color of the upper part of the petals of L.tulipifera is green,while the middle part of the petals changes from light green to white and finally to orangeyellow during flower development;The petal coloring pattern of hybrid Liriodendron is divided into two types,one is similar to that of Liriodendron tulipifera,and the other is special,that is,in the process of flower development,the middle and upper parts of the petals change from green to orange-yellow.The sectioning results showed that the green petals of Liriodendron were mainly due to the accumulation of chloroplasts,while the accumulation of orange-yellow chromoplasts was the main reason for the orange-yellow petals.Among them,the process of chloroplast degradation and chromoplast accumulation existed in the petal mesophyll cells of hybrid Liriodendron in the process of flower development.2.The results of transcriptomic analysis showed that the expression of photosynthesis-related genes and carotenoid synthesis-related genes in the petals of hybrid Liriodendron was one of the important reasons for the different coloration patterns of the petals of L.tulipifera.In the photosynthesis-related pathways,it was found that the expression levels of 25 DAPs genes encoding photosystem I(PS I),photosystem II(PS II),ferredoxin,and chlorophyll ab-binding protein were found in the orange-yellow petals.The region was down-regulated,indicating that the photosynthetic capacity between different colored regions of petals changed,suggesting that chloroplast development may be affected in the process of petal coloring of hybrid tulipifera.In the carotenoid synthesis pathway,6 genes were found to be differentially expressed.The expression dynamics of Ls PSY,Ls PDS,Ls ZDS,Ls CRTISO,Ls LCY-ε and other genes all satisfied the trend of orange-yellow area > green area > white area.Expression leads to the entry of the carotenoid pathway into the α branch,and accumulation of ε-carotene and its derivatives in the orange-yellow region of the petals of hybrid Liriodendron.Aiming at the differential genes in the carotenoid synthesis pathway,we combined WGCNA,TGMI,SCCM and other algorithms to construct a regulatory network of transcription factors and target genes,and found that transcription factors such as Ls E2F3,Ls TIFY6,Ls HDZIP19,Ls PCF11,and Lsb HLH9 have significant effects on Ls PDS,Ls ZDS,Ls CRTISO,and Ls LCY-ε and others have potential regulatory functions,and there may be a mutual regulatory relationship between Ls E2F3 and Ls PCF11.3.Transcription factors that may be involved in flower color regulation were cloned and the gene families to which they belonged were identified and analyzed.Conserved motif analysis showed that Ls HDZIP19 had one more unique motif 9 compared to its paralogous genes,indicating that motif 9 may play a special role in the functional differentiation of Ls HDZIP19.Analysis of conserved motifs of other transcription factors showed that the composition of Ls PCF11 in the motif was very different from that of other members of the same subclade,suggesting that Ls PCF11 has a certain specialization in function.Fluorescence quantitative results showed that the expression patterns of the five transcription factors satisfy the trend of orangeyellow area>green area>white area.The Ls PDS,Ls CRTISO,Ls ZDS,Ls LCY-ε promoter sequences were cloned,and the corresponding sequences obtained were 2,460 bp,2,499 bp,2,502 bp and2,461 bp in length.The promoter sequences were analyzed,and the results showed that there were a variety of hormone response,stress response and growth and development-related elements on the promoters of these four genes,indicating that these genes may be involved in a variety of biological processes.At the same time,multiple possible binding sites for Ls E2F3,Ls PCF11,and Ls HDZIP11 were predicted on p Ls CRTSO,p Ls ZDS,and p Ls LCY-ε,and multiple transcriptional binding sites for Ls HDZIP11 were also predicted on p Ls PDS.This result further proves the accuracy of the constructed regulatory network.