Functional and transcriptional regulation of the anthocyanidin acyl modifier gene Gs5AT of Gentiana sino-ornata
Han Meng A # , Siqi Chen A # , Yanmei Wu A and Xuehua Jin A *A
Abstract
The Chinese gentian, Gentiana sino-ornata produces brilliant blue flowers. To investigate the biological function and transcriptional regulation mechanism of the anthocyanin 5-O-acyltransferase gene (Gs5AT) in the corolla, it is beneficial to analyse the mechanism of blue flower colour presentation. In this investigation, we obtained the CDS and promoter sequences of the gene Gs5AT. Yeast one-hybrid experiments were used to identify the transcription factor GsbHLH7 that activates the gene Gs5AT. According to quantitive reverse transcription polymerase chain reaction analysis, the expression of the gene Gs5AT was significantly and positively correlated with the gene GsbHLH7. The colour phenotype of the flowers was significantly altered by the virus-induced gene silencing transduction of Gs5AT and GsbHLH7, with GsbHLH7 silencing producing more pronounced changes in the corolla colour than Gs5AT. The expression of GsF3′5′H, GsDFR, GsANS, Gs3GT, and Gs5GT all fell to varying degrees after GsbHLH7 silencing, indicating that GsbHLH7 may regulate transcription of these genes as well as Gs5AT. The results of this study indicate that Gs5AT was positively regulated by the GsbHLH7, and thus affects the colour presentation of the blue corolla.
Keywords: anthocyanin, blue-flowering, gene function validation, Gentiana sino-ornata, Gs5AT, promoter, transcription factor, VIGS.
References
Brugliera F, Tao G-Q, Tems U, Kalc G, Mouradova E, Price K, Stevenson K, Nakamura N, Stacey I, Katsumoto Y, Tanaka Y, Mason JG (2013) Violet/blue chrysanthemums – metabolic engineering of the anthocyanin biosynthetic pathway results in novel petal colors. Plant and Cell Physiology 54(10), 1696-1710.
| Crossref | Google Scholar | PubMed |
Chen K, Liu H, Lou Q, Liu Y (2017) Ectopic expression of the grape hyacinth (Muscari armeniacum) R2R3-MYB transcription factor gene, MaAN2, induces anthocyanin accumulation in tobacco. Frontiers in Plant Science 8, 965.
| Crossref | Google Scholar |
Cominelli E, Gusmaroli G, Allegra D, Galbiati M, Wade HK, Jenkins GI, Tonelli C (2008) Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana. Journal of Plant Physiology 165(8), 886-894.
| Crossref | Google Scholar | PubMed |
D’Auria JC (2006) Acyltransferases in plants: a good time to be BAHD. Current Opinion in Plant Biology 9(3), 331-340.
| Crossref | Google Scholar | PubMed |
Feller A, Machemer K, Braun EL, Grotewold E (2011) Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. The Plant Journal 66(1), 94-116.
| Crossref | Google Scholar | PubMed |
Fujiwara H, Tanaka Y, Fukui Y, Nakao M, Ashikari T, Kusumi T (1997) Anthocyanin 5-aromatic acyltransferase from Gentiana triflora: purification, characterization and its role in anthocyanin biosynthesis. European Journal of Biochemistry 249(1), 45-51.
| Crossref | Google Scholar | PubMed |
Fujiwara H, Tanaka Y, Fukui Y, Ashikari T, Yamaguchi M, Kusumi T (1998) Purification and characterization of anthocyanin 3-aromatic acyltransferase from Perilla frutescens. Plant Science 137(1), 87-94.
| Crossref | Google Scholar |
Gonzalez A, Zhao M, Leavitt JM, Lloyd AM (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. The Plant Journal 53(5), 814-827.
| Crossref | Google Scholar | PubMed |
Heim MA, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey PC (2003) The basic helix–loop–helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Molecular Biology and Evolution 20(5), 735-747.
| Crossref | Google Scholar | PubMed |
Hong Y, Tang X, Huang H, Zhang Y, Dai S (2015) Transcriptomic analyses reveal species-specific light-induced anthocyanin biosynthesis in chrysanthemum. BMC Genomics 16(1), 202.
| Crossref | Google Scholar | PubMed |
Hosokawa K, Fukushi E, Kawabata J, Fujii C, Ito T, Yamamura S (1997) Seven acylated anthocyanins in blue flowers of Gentiana. Phytochemistry 45(1), 167-171.
| Crossref | Google Scholar |
Hsu C-C, Chen Y-Y, Tsai W-C, Chen W-H, Chen H-H (2015) Three R2R3-MYB transcription factors regulate distinct foral pigmentation patterning in Phalaenopsis spp. Plant Physiology 168(1), 175-191.
| Crossref | Google Scholar | PubMed |
Hu K, Han KS, Dai S (2010) Mechanisms of environmental factors regulating the synthesis and coloration of anthocyanins in plants. Chinese Bulletin of Botany 45(03), 307-317.
| Crossref | Google Scholar |
Hu YW, Li SS, Liu Y, Ge HY (2022) Functional characterization of SmCDF2 in anthocyanin biosynthesis and flowering in Solanu melongena L. Journal of Nanjing Agricultural University 45, 1117-1125.
| Google Scholar |
Koes R, Verweij W, Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends in Plant Science 10(5), 236-242.
| Crossref | Google Scholar | PubMed |
Lai Y, Li H, Yamagishi M (2013) A review of target gene specificity of flavonoid R2R3-MYB transcription factors and a discussion of factors contributing to the target gene selectivity. Frontiers in Biology 8(6), 577-598.
| Crossref | Google Scholar |
Lai B, Li X-J, Hu B, Qin Y-H, Huang X-M, Wang H-C, Hu G-B (2014) LcMYB1 is a key determinant of differential anthocyanin accumulation among genotypes, tissues, developmental phases and ABA and light stimuli in Litchi chinensis. PLoS ONE 9(1), e86293.
| Crossref | Google Scholar | PubMed |
Lai B, Du L-N, Liu R, Hu B, Su W-B, Qin Y-H, Zhao J-T, Wang H-C, Hu G-B (2016) Two LcbHLH transcription factors interacting with LcMYB1 in regulating late structural genes of anthocyanin biosynthesis in Nicotiana and Litchi chinensis during anthocyanin accumulation. Frontiers in Plant Science 7, 166.
| Crossref | Google Scholar | PubMed |
Li C, Qiu J, Yang G, Huang S, Yin J (2016a) Isolation and characterization of a R2R3-MYB transcription factor gene related to anthocyanin biosynthesis in the spathes of Anthurium andraeanum (Hort.). Plant Cell Reports 35, 2151-2165.
| Crossref | Google Scholar | PubMed |
Li Y, Shan X, Gao R, Yang S, Wang S, Gao X, Wang L (2016b) Two IIIf Clade-bHLHs from Freesia hybrida play divergent roles in flavonoid biosynthesis and trichome formation when ectopically expressed in Arabidopsis. Scientific Reports 6, 30514.
| Crossref | Google Scholar | PubMed |
Li C, Qiu J, Huang S, Yin J, Yang G (2019) AaMYB3 interacts with AabHLH1 to regulate proanthocyanidin accumulation in Anthurium andraeanum (Hort.) – another strategy to modulate pigmentation. Horticulture Research 6, 14.
| Crossref | Google Scholar | PubMed |
Li H, Yang Z, Zeng Q, Wang S, Luo Y, Huang Y, Xin Y, He N (2020) Abnormal expression of bHLH3 disrupts a flavonoid homeostasis network, causing differences in pigment composition among mulberry fruits. Horticulture Research 7, 83.
| Crossref | Google Scholar |
Liang L, Yang Y, Wang E (2018) Advances in research on the biosynthesis and regulation of plant anthocyanins. Anhui Agricultural Sciences 46, 18-24.
| Google Scholar |
Lu C, Li Y, Cui Y, Ren J, Qi F, Qu J, Huang H, Dai S (2021) Isolation and functional analysis of genes involved in polyacylated anthocyanin biosynthesis in blue Senecio cruentus. Frontiers in Plant Science 12, 640746.
| Crossref | Google Scholar | PubMed |
Luo J, Nishiyama Y, Fuell C, Taguchi G, Elliott K, Hill L, Tanaka Y, Kitayama M, Yamazaki M, Bailey P, Parr A, Michael AJ, Saito K, Martin C (2007) Convergent evolution in the BAHD family of acyl transferases: identification and characterization of anthocyanin acyl transferases from Arabidopsis thaliana. The Plant Journal 50(4), 678-695.
| Crossref | Google Scholar | PubMed |
Morita Y, Saitoh M, Hoshino A, Nitasaka E, Iida S (2006) Isolation of cDNAs for R2R3-MYB, bHLH and WDR transcriptional regulators and identification of c and ca mutations conferring white flowers in the Japanese morning glory. Plant and Cell Physiology 47(4), 457-470.
| Crossref | Google Scholar | PubMed |
Nakatsuka T, Haruta KS, Pitaksutheepong C, Abe Y, Kakizaki Y, Yamamoto K, Shimada N, Yamamura S, Nishihara M (2008) Identification and characterization of R2R3-MYB and bHLH transcription factors regulating anthocyanin biosynthesis in gentian flowers. Plant and Cell Physiology 49(12), 1818-1829.
| Crossref | Google Scholar | PubMed |
Ni M, Tepperman JM, Quail PH (1998) PIF3, a phytochrome-interacting factor necessary for normal photoinduced signal transduction, is a novel basic helix-loop-helix protein. Cell 95, 657-667.
| Crossref | Google Scholar | PubMed |
Otsuki T, Matsufuji H, Takeda M, Toyoda M, Goda Y (2002) Acylated anthocyanins from red radish (Raphanus sativus L.). Phytochemistry 60(1), 79-87.
| Crossref | Google Scholar | PubMed |
Pires N, Dolan L (2010) Origin and diversification of basic-helix-loop-helix proteins in plants. Molecular Biology and Evolution 27, 862-874.
| Crossref | Google Scholar | PubMed |
Premathilake AT, Ni J, Bai S, Tao R, Ahmad M, Teng Y (2020) R2R3-MYB transcription factor PpMYB17 positively regulates flavonoid biosynthesis in pear fruit. Planta 252, 59.
| Crossref | Google Scholar | PubMed |
Prior RL, Wu X (2006) Anthocyanins: structural characteristics that result in unique metabolic patterns and biological activities. Free Radical Research 40(10), 1014-1028.
| Crossref | Google Scholar | PubMed |
Rinaldo AR, Cavallini E, Jia Y, Moss SMA, McDavid DAJ, Hooper LC, Robinson SP, Tornielli GB, Sara Z, Ford CM, Boss PK, Walker AR (2015) A grapevine anthocyanin acyltransferase, transcriptionally regulated by VvMYBA, can produce most acylated anthocyanins present in grape skins. Plant Physiology 169, 1897-1916.
| Crossref | Google Scholar | PubMed |
Tanaka Y, Tsuda S, Kusumi T (1998) Metabolic engineering to modify flower color. Plant and Cell Physiology 39, 1119-1126.
| Crossref | Google Scholar |
Tanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. The Plant Journal 54, 733-749.
| Crossref | Google Scholar | PubMed |
Tao R, Yu W, Gao Y, Ni J, Yin L, Zhang X, Li H, Wang D, Bai S, Teng Y (2020) Light-induced basic/helix-loop-helix64 enhances anthocyanin biosynthesis and undergoes CONSTITUTIVELY PHOTOMORPHOGENIC1-mediated degradation in pear. Plant Physiology 184, 1684-1701.
| Crossref | Google Scholar | PubMed |
Tasaki K, Higuchi A, Watanabe A, Sasaki N, Nishihara M (2019) Effects of knocking out three anthocyanin modification genes on the blue pigmentation of gentian flowers. Scientific Reports 9, 15831.
| Crossref | Google Scholar |
Tuominen LK, Johnson VE, Tsai C-J (2011) Differential phylogenetic expansions in BAHD acyltransferases across five angiosperm taxa and evidence of divergent expression among Populus paralogues. BMC Genomics 12, 236.
| Crossref | Google Scholar |
Unno H, Ichimaida F, Suzuki H, Takahashi S, Tanaka Y, Saito A, Nishino T, Kusunoki M, Nakayama T (2007) Structural and mutational studies of anthocyanin malonyltransferases establish the features of BAHD enzyme catalysis. Journal of Biological Chemistry 282, 15812-15822.
| Crossref | Google Scholar | PubMed |
Vidana Gamage GC, Lim YY, Choo WS (2022) Sources and relative stabilities of acylated and nonacylated anthocyanins in beverage systems. Journal of Food Science and Technology 59(3), 831-845.
| Crossref | Google Scholar | PubMed |
Xiang L-L, Liu X-F, Li X, Yin X-R, Grierson D, Li F, Chen K-S (2015) A novel bHLH transcription factor involved in regulating anthocyanin biosynthesis in Chrysanthemums (Chrysanthemum morifolium Ramat.). PLoS ONE 10(11), e0143892.
| Crossref | Google Scholar | PubMed |
Xie X-B, Li S, Zhang R-F, Zhao J, Chen Y-C, Zhao Q, Yao Y-X, You C-X, Zhang X-S, Hao Y-J (2012) The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples. Plant, Cell & Environment 35, 1884-1897.
| Crossref | Google Scholar | PubMed |
Xie Y, Sun Y, Huang J (2013) Anthocyanin modification in Arabidopsis. Plant Physiology Journal 49, 101-110.
| Google Scholar |
Xu ZR, Li CL, Cui G, Sun Y (2008) MYB protein of anthocyanin biosynthesis in plant. Plant Physiology 44, 597-604.
| Google Scholar |
Xu W, Dubos C, Lepiniec L (2015) Transcriptional control of flavonoid biosynthesis by MYB–bHLH–WDR complexes. Trends in Plant Science 20, 176-185.
| Crossref | Google Scholar | PubMed |
Yonekura-Sakakibara K, Tanaka Y, Fukuchi-Mizutani M, Fujiwara H, Fukui Y, Ashikari T, Murakami Y, Yamaguchi M, Kusumi T (2000) Molecular and biochemical characterization of a novel hydroxycinnamoyl-CoA: anthocyanin 3-O-Glucoside-6″ -O-Acyltransferase from Perilla frutescens. Plant and Cell Physiology 41, 495-502.
| Crossref | Google Scholar | PubMed |
Yu B, Zhang D, Huang C, Qian M, Zheng X, Teng Y, Su J, Shu Q (2012) Isolation of anthocyanin biosynthetic genes in red Chinese sand pear (Pyrus pyrifolia Nakai) and their expression as affected by organ/tissue, cultivar, bagging and fruit side. Scientia Horticulturae 136, 29-37.
| Crossref | Google Scholar |
Yuan Y, Shi Y, Tang D (2020) Isolation and characterization of R2R3-MYB and basic helix–loop–helix (bHLH) transcription factors involved in anthocyanin biosynthesis in tulip tepals. Acta Physiologiae Plantarum 42, 32.
| Crossref | Google Scholar |