Transcriptional Regulation Mechanism Of Anthocyanin Biosynthesis In Oriental Hybrid Lily

Flower color is one of the most important factors in ornamental plants.Oriental hybrid lily has large and colorful flowers.Anthocyanin is one of the main pigments that determines flower color in lily.However,the main factors and mechanisms involved in the transcriptional regulation of anthocyanin biosynthesis in oriental hybrid lily have not been carefully analyzed.In this study,the pink cultivar ’Sorbonne’and the white cultivar ’Siberia’ of oriental hybrid lily were used as research materials.On the basis of the determination of flower color phenotype,anatomical structure and physiological indicators,the transcriptome and s RNA sequencing were performed to screen the genes related to anthocyanin biosynthesis and analyze the main factors and mechanisms involved in transcriptional regulation of in oriental hybrid lily.The main findings are as follows:(1)Differences in flower color traits between ’Sorbonne’ and ’Siberia’ depended on the anthocyanin accumulation.In ’Siberia’,there was no obvious pigment accumulation in petals.In ’Sorbonne’,pigments mainly accumulated in adaxial epidermal cells of the petals.Anthocyanin content increased with development of flower buds: anthocyanin started to accumulate significantly at the initial color developmental stage of flower buds,and reached the peak value when tepals were fully colored.UPLC-MS/MS method was performed to analyze the anthocyanin contents of ’Sorbonne’ and ’Siberia’ tepals.The results showed that the main anthocyanins in ’Sorbonne’ were cyanidin and delphinium,while no anthocyanin was detected in ’Siberia’.(2)Lh MYBA1-Lh TT8 were the key regulatory modules of anthocyanin biosynthesis in ’Sorbonne’.A total of 213 structural gene and 843 transcription factor transcripts related to anthocyanin biosynthesis were screened from the transcriptome data.The cloning,sequence analysis,bioinformatics analysis and expression pattern analysis of differentially expressed structural genes and transcription factors were performed respectively.The results of q RT-PCR analysis showed that the expression patterns of structural genes(Lh CHS,Lh CHI,Lh F3 H,Lh F3’H,Lh FLS,Lh DFR,Lh ANS)were consistent with the trend of anthocyanin accumulation in ’Sorbonne’ during the bud developmental process.In ’Sorbonne’,up-regulated R2R3-MYB type transcription factor Lh MYBA1 could be clustered with the reported anthocyanin activators into the same branch of the phylogenetic tree.Meanwhile,there was no difference in the coding sequence and genome sequence of Lh MYBA1 between ’Sorbonne’ and ’Siberia’.Lh MYBD1,annotated as the R3-MYB repressor,was down-regulated expressed in ’Sorbonne’ and had higher expression in ’Siberia’.Lh TT8 was up-regulated in both two cultivars,and its expression pattern was consistent with Lh MYBA1 in ’Sorbonne’.In addition,287,169,039 high-quality tags were obtained by s RNA sequencing.According to combined transcriptome-s RNA analysis and q RT-PCR analysis,the results showed that mi RNA and si RNA might target on anthocyanin biosynthesis-related structural genes and transcription factors to participate in the transcriptional regulation of anthocyanin biosynthesis.(3)Lh MYBA1 promotes anthocyanin accumulation.The subcellular localization analysis of 9 structural genes and 6 transcription factors related to anthocyanin biosynthesis showed that Lh MYBA1,Lh MYBC1,Lh MYBC2,Lh MYBC3,Lh MYBD1 and Lh TT8 proteins were localized in the nucleus,most structural gene proteins(Lh CHSA,Lh CHSB,Lh CHI,Lh F3 H,Lh DFR,Lh ANS,Lh ANR)were localized in the cytoplasm,and Lh F3’H protein was localized in the endoplasmic reticulum.Stable overexpression of Lh MYBA1 could significantly promote the accumulation of anthocyanins in wild-type Arabidopsis and various tissue parts of tobacco plants.The expression of most anthocyanin biosynthesis genes in transgenic plants were activated to varying degrees,which proved that Lh MYBA1 was a positive regulator of anthocyanin biosynthesis.(4)Sequence differences in the promoter region of Lh MYBA1 lead to differential interaction patterns with transcription factors in ’Sorbonne’ and ’Siberia’.The promoter sequences of 5 structural genes and 2transcription factors related to anthocyanin biosynthesis were cloned respectively.The analysis of cisacting elements in the promoters showed that the sequences contained core promoter elements,enhancer promoter elements and multiple transcription factor binding sites respectively.Notably,comparing with’Sorbonne’,two insertions were found in the promoter sequence of Lh MYBA1 in ’Siberia’,where these sequence differences were conserved among wild parents,hybrid progeny of oriental hybrid lily,and oriental hybrid lily cultivars with different flower colors.The results of GUS staining to identify the transcriptional activity of promoters showed that these gene promoters could activate the expression of reporter genes in the petals of the white cultivar ‘Siberia’.The transcription factor proteins Lh TT8,Lh MYBA1,Lh MYBC2,Lh MYBC3 and Lh MYBD1 could bind to the promoter of the structural gene Lh CHSA.Lh MYBA1 promoter of ’Sorbonne’ could specifically bind to its own protein,but the Lh MYBA1 promoter of ’Siberia’ had no such function.(5)The positive regulation transcription factor Lh MYBA1 and the negative regulation transcription factor Lh MYBD1 competed for the interaction site with Lh TT8,and jointly regulate the accumulation of anthocyanin in oriental hybrid lily.Lh TT8,Lh MYBA1,Lh MYBA2 and Lh MYBC1 could interact with their own proteins.The positive regulators Lh MYBA1 and negative regulators Lh MYBC2,Lh MYBD1 could interact with Lh TT8 protein.What’s more,in the amino acid sequence of Lh TT8,the b HLH domain was the key domain for Lh TT8 to interact with itself,and the MIR domain was the key domain for the interaction with Lh MYBA1 protein.In Lh MYBA1,Lh MYBA1 could interact with Lh TT8 only when the R3 domain of the amino acid sequence of Lh MYBA1 was intact.Based on the above results,the transcription factor Lh TT8,the positive regulator Lh MYBA1 and the negative regulator Lh MYBD1 are involved in the transcriptional regulation of anthocyanin biosynthesis in oriental hybrid lily.It can be confirmed that the interaction between the promoter of Lh MYBA1 and transcription factors is changed due to the insertion of the fragments,resulting in the inhibition of anthocyanin biosynthesis in ’Siberia’.Therefore,Lh MYBA1 promoter region may be one of the key sites of natural and artificial selection in oriental hybrid lilies which has important breeding value.