Cloning Of Genes Related To Anthocyanin Biosynthesis And Relationship Between Their Differential Expression And Flower Color Mutation In Tulip

Tulip(Tulipa L.) is a perennial, bulbous plant in the genus Tulipa, which belongs to the family Liliaceae. It is one of the most important commercial bulbous flowers in the floricultural industry. Flower color has been regarded as an important character in deciding the horticultural value of tulip plants. Anthocyanins have been implicated as major color pigments in tulip, hence, getting information of genes related to anthocyanin biosynthesis in tulip is important for carrying out molecular breeding programs towards flower colors in the future. Through selective breeding, two bud sports Tulipa fosteriana ‘Shangnong Zaoxia’ with red flower and ‘Shangnong 09’ with orange-red flower were bred through spontaneous mutants from a pink flower tulip cultivar T. fosteriana ‘Albert heijn’. Most of the biological features of ‘Shangnong Zaoxia’ and ‘Shangnong 09’, except for flower color, are identical with those of ‘Albert heijn’. However, the biochemical and molecular basis underlying the color change is still unclear. In this study, full-length c DNA sequences of nine structural genes and six transcription factors were firstly cloned from T. fosteriana. Furthermore, ‘Shangnong Zaoxia’ and ‘Shangnong 09’ were compared with ‘Albert heijn’ in terms of total anthocyanins and flavonols contents, anthocyanin components, expression and transcriptional regulation of genes related to anthocyanin biosynthesis to analyze the relationship between the expression profiles of these genes and the flower color change in ‘Shangnong Zaoxia’ and ‘Shangnong 09’. Main results were as follows:1. Flower color, total anthocyanins and flavonols contents, and anthocyanin components in petals were significantly different among ‘Albert heijn’, ‘Shangnong Zaoxia’, and ‘Shangnong 09’. Flower color were described by RHSCC(Royal Horticultural Society Color Card), result showed that the flower color of ‘Albert heijn’ belongs to ‘Red-purple’ group, while the flower color of ‘Shangnong Zaoxia’ and ‘Shangnong 09’ belongs to ‘Red’ and ‘Orange-red’ group, respectively. Total anthocyanins and flavonols contents in petals were analyzed by ULPC-PDA(Ultra performance liquid chromatography with a photodiode array detector), the result showed that the total anthocyanins content in petals of ‘Shangnong Zaoxia’ were significantly higher than that in ‘Albert heijn’, but the total anthocyanins content in petals of ‘Shangnong 09’ showed no significant difference with that in ‘Albert heijn’. The total flavonols content in petals of ‘Shangnong Zaoxia’ and ‘Shangnong 09’ were significantly higher than that in ‘Albert heijn’. Anthocyanin components in petals of ‘Albert heijn’, ‘Shangnong Zaoxia’, and ‘Shangnong 09’ were analyzed by UPLC-Q-TOF-MS(Ultra Performance Liquid Chromatography & Quadrupole-Time-of- Flight Mass Spectrometer), the result demonstrated that major cyanidin 3-O-rutinoside(79.33% of total anthocyanins content) and minor pelargonidin 3-O-rutinoside(20.67% of total anthocyanins content) were accumulated in the petals of ‘Albert heijn’, whereas major pelargonidin anthocyanins(more than 90% of total anthocyanins content, pelargonidin 3-O-acetylrutinoside and pelargonidin 3-O-rutinoside) and minor cyanidin 3-O-glucoside were detected in petals of ‘Shangnong Zaoxia’ and ‘Shangnong 09’.2. Full-length c DNA sequences of nine anthocyanin biosynthetic structural genes were firstly isolated from T. fosteriana with homologous cloning and RACE technique. They were named as Tf CHS1(1470 bp), Tf CHI1(998 bp), Tf CHI2(823 bp), Tf F3H1(1241 bp), Tf F3’H1(1766 bp), Tf DFR1(1394 bp), Tf ANS1(1371 bp), Tf3GT1(1673 bp), and Tf FLS1(1193 bp). The deduced amino acids of these genes share high identity with each corresponding protein reported in other species previously and have motifs, amino residues, binding sites and activity sites that conversed in each corresponding protein.3. The expression levels of anthocyanin biosynthetic structural genes during flower development in ‘Shangnong Zaoxia’ and ‘Shangnong 09’ were significantly different from that in ‘Albert heijn’. The changes in expression levels of Tf CHS1, Tf CHI2, Tf F3H1, Tf DFR1, Tf ANS1, Tf FLS1, and Tf F3’H1 in ‘Shangnong Zaoxia’ and ‘Shangnong 09’ were positively correlated with the changes in total anthocyanins and flavonols contents and cyanidin anthocyanin content in petals, compared with that in ‘Albert heijn’. During flower development, the expression levels of Tf CHS1, Tf CHI2, Tf F3H1, Tf DFR1, Tf ANS1, and Tf3GT1 were continuously increased, which were correlated with the accumulation pattern of total anthocyanins in petals. Tf FLS1 transcript level was slowly decreased during flower development, corresponding to the accumulation pattern of total flavonols. The expression levels of Tf CHS1, Tf CHI2, Tf F3H1, Tf DFR1, Tf ANS1, and Tf FLS1 in ‘Shangnong Zaoxia’ were significantly higher than those in ‘Albert heijn’, corresponding to the significantly higher total anthocyanins and flavonols contents in ‘Shangnong 09’ compared with that in ‘Albert heijn’. The expression levels of Tf CHS1, Tf CHI2, and Tf F3H1 in ‘Shangong 09’ were significantly higher than that in ‘Albert heijn’, but Tf DFR1 and Tf ANS1 transcript levels in ‘Shangong 09’ were not significantly different from that in ‘Albert hejin’, corresponding to the the significantly higher total flavonols content in ‘Shangnong 09’ compared with that in ‘Albert heijn’. The transcription of Tf F3’H1 in ‘Shangnong Zaoxia’ and ‘Shangnong 09’ were significantly lower than that in ‘Albert heijn’ throughout flower development, corresponding to the significantly lower content of cyanidin anthocyanin in petals of ‘Shangnong Zaoxia’and ‘Shangnong 09’ compared with that in ‘Albert heijn’.4. Reason for the significant reduction of Tf F3’H1 transcription in ‘Shangnong Zaoxia’ and ‘Shangnong 09’ was investigated. The promoter sequences of Tf F3’H1 in ‘Albert heijn’, ‘Shangnong Zaoxia’ and ‘Shangnong 09’ were compared, the result showed that an extra fragment(255 bp in length) was found in the 5’flanking region of Tf F3’H1 at 326 bp upstream of the ATG translation start codon in ‘Shangnong Zaoxia’ and ‘Shangnong 09’. Consequently, the promoters of Tf F3’H1 in ‘Albert heijn’, ‘Shangnong Zaoxia’ and ‘Shangnong 09’were isolated, fused with the β-glucuronidase(GUS), and then transformed to Arabidopsis thaliana. Results demonstrated that the GUS activity in Arabidopsis thaliana seedlings with Tf F3’H1 promoter from ‘Shangnong Zaoxia’ and ‘Shangnong 09’ was only 4.5% and 5.4% of that from ‘Albert heijn’, respectively, suggesting that the insert mutation in the Tf F3’H1 promoter resulted in the significant reduction of Tf F3’H1 transcription in ‘Shangnong Zaoxia’ and ‘Shangnong 09’.5. By homologous cloning in combination with RACE technique, and based on the tulip petal transcriptome database, full-length c DNA sequences of four R2R3 MYB and two b HLH genes were firstly isolated from petal of T. fosteriana, they were named as Tf MYB1(856 bp), Tf MYB2(840 bp), Tf MYB3(906 bp), Tf MYB4(851 bp), Tfb HLH1(2527 bp), and Tfb HLH2(2321 bp). Phylogenetic analysis indicated that Tf MYBs have high homology with Arabidopsis PAP1, PAP2, and petunia AN2, while Tfb HLH1 and Tfb HLH2 have high homology with Arabidopsis At EGL3, At TT8, and At GL3, and petunia AN1. The expression profiles of these transcription factors in petals of ‘Albert heijn’, ‘Shangnong Zaoxia’ and ‘Shangnong 09’ during flower development were analyzed by real-time PCR, results demonstrated that the temporal expression patterns of Tf MYB2, Tf MYB3, Tf MYB4, and Tfb HLH1 were correlated with the temporal expression patterns of Tf CHS1, Tf CHI2, Tf F3H1, Tf DFR1, Tf ANS1 and Tf3GT1, and the accumulation pattern of total anthocyanins in petals. In addition, the differential transcriptional profiles of Tf MYB2, Tf MYB3, and Tfb HLH1 among ‘Albert heijn’, ‘Shangnong Zaoxia’ and ‘Shangnong 09’ during flower development were consistent with the differential expression profiles of Tf DFR1, Tf ANS1 and Tf3GT1 among ‘Albert heijn’, ‘Shangnong Zaoxia’ and ‘Shangnong 09’, while the expression profile of Tf MYB4 in ‘Albert heijn’, ‘Shangnong Zaoxia’ and ‘Shangnong 09’ were consistent with the expression profiles of Tf CHS1, Tf CHI2 and Tf F3H1 in ‘Albert heijn’, ‘Shangnong Zaoxia’ and ‘Shangnong 09’. These results suggested that Tf MYB2, Tf MYB3 and Tfb HLH1 may be involved in regulation of Tf CHS1, Tf CHI2 and Tf F3H1 in petals while Tf MYB4 may be involved in regulation of Tf DFR1, Tf ANS1 and Tf3GT1 in petals in tulip.6. CHS and ANS is the key gene in early and later steps in anthocyanin biosynthetic pathway, respectively. To further analyze the regulation of transcription factors on anthocyanin structural genes, the interaction of Tf MYB2, Tf MYB3, Tf MYB4, and Tfb HLH1 with the promoters of Tf CHS1 and Tf ANS1 were investigated. The coding sequence of Tf MYB2, Tf MYB3, Tf MYB4, and Tfb HLH1 was constructed under 35 S promoter, respectively, and the promoter sequence for Tf CHS1 and Tf ANS1 was fused with LUC, respectively. These two kinds of vectors were co-transformed to tobacco leaves to test the interaction of transcription factors with promoters by testing the relative LUC activity in transgenic tobacco leaves. The results showed that Tf MYB2 and Tf MYB3 with or without Tfb HLH1 could significantly enhance the promoter activity of Tf ANS1, while Tf MYB4 with or without Tfb HLH1 could significantly enhance the promoter activity of Tf CHS1, suggesting the differential expression levels of Tf CHS1 and Tf ANS1 in Shangnong Zaoxia’ and ‘Shangnong 09’ compared with that in ‘Albert heijn’ may related to the differential expression levels of Tf MYB2, Tf MYB3, Tf MYB4, and Tfb HLH1, in other words, the differential expression levels of Tf MYB2, Tf MYB3, Tf MYB4, and Tfb HLH1 in ‘Shangnong Zaoxia’ and ‘Shangnong 09’ compared with that in ‘Albert heijn’ may contribute to the flower color mutation.