A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes
Abstract
:1. Introduction
2. Results
2.1. Molecular Cloning and Sequence Analysis of CsWD40
2.2. CsWD40 Interacts with MYB and bHLH TFs
2.3. CsWD40 Complements the Arabidopsis ttg1 Deficient Phenotype
2.4. Anthocyanin and PA Accumulation in the Flowers of CsWD40-Overexpressing Tobacco Plants
2.5. Analysis of Expression of Genes Involved in Flavonoid Biosynthesis in the Flowers of CsWD40-Overexpressing Tobacco Plants
2.6. Anthocyanin and PA Content in CsWD40- and CsMYB5e-Overexpressing Tobacco Plants
2.7. Expression Analysis of Genes Involved in Flavonoid Biosynthesis in CsWD40 and CsMYB5e Transgenic Tobacco
2.8. Expression Patterns of CsWD40 in Tea Leaves under Different Abiotic Stresses
3. Discussion
4. Materials and Methods
4.1. Plant Materials and Growth Conditions
4.2. Cloning CsWD40 and CsMYB5e
4.3. Extraction and Analysis of Anthocyanins and PAs
4.4. Agrobacterium-Mediated Transformation of Arabidopsis and Tobacco
4.5. Hybridization of Different Transgenic Tobacco Lines
4.6. Abiotic Stress Treatment of Tea Shoots
4.7. Yeast Two-Hybrid Assay
4.8. Quantitative Real-Time PCR
Supplementary Materials
Author Contributions
Acknowledgments
Conflicts of Interest
Abbreviations
ANR | Anthocyanidinreductase |
ANS | Anthocyanidin synthase |
CHS | Chalcone synthase |
DFR | Dihydroflavonolreductase |
DMACA | Dimethylaminocinnamaldehyde |
F3H | Flavanone 3-hydroxylase |
F3′H | Flavonoid 3-hydroxylase |
FLS | Flavonol synthase |
MBW | MYB-bHLH-WD40 |
Pas | Proanthocyanidins |
ttg1 | Transparent testa glabra 1 |
References
- Winkel-Shirley, B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 2001, 126, 485–493. [Google Scholar] [CrossRef] [PubMed]
- Ferreyra, M.L.F.; Rius, S.P.; Casati, P. Flavonoids: Biosynthesis, biological functions, and biotechnological applications. Front. Plant Sci. 2012, 3, 222. [Google Scholar]
- Hernández, I.; Alegre, L.; Van Breusegem, F.; Munné-Bosch, S. How relevant are flavonoids as antioxidants in plants? Trends Plant Sci. 2009, 14, 125–132. [Google Scholar] [CrossRef] [PubMed]
- Nakabayashi, R.; Yonekurasakakibara, K.; Urano, K.; Suzuki, M.; Yamada, Y.; Nishizawa, T.; Matsuda, F.; Kojima, M.; Sakakibara, H.; Shinozaki, K.; et al. Enhancement of oxidative and drought tolerance in Arabidopsis by over accumulation of antioxidant flavonoids. Plant J. 2014, 77, 367–379. [Google Scholar] [CrossRef] [PubMed]
- Tohge, T.; Fernie, A.R. An overview of compounds derived from the shikimate and phenylpropanoid pathways and their medicinal importance. Mini-Rev. Med. Chem. 2016, 17, 1013–1027. [Google Scholar] [CrossRef] [PubMed]
- Khan, N.; Adhami, V.M.; Mukhtar, H. Apoptosis by dietary agents for prevention and treatment of prostate cancer. Endocr-Relat. Cancer 2010, 17, R39–R52. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ross, J.A.; Kasum, C.M. Dietary flavonoids: Bioavailability, metabolic effects, and safety. Annu. Rev. Nutr. 2002, 22, 19–34. [Google Scholar] [CrossRef] [PubMed]
- Holton, T.A.; Cornish, E.C. Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 1995, 7, 1071–1083. [Google Scholar] [CrossRef] [PubMed]
- Pang, Y.; Peel, G.J.; Wright, E.; Wang, Z.; Dixon, R.A. Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula. Plant Physiol. 2007, 145, 601–615. [Google Scholar] [CrossRef] [PubMed]
- Gonzalez, A.; Zhao, M.; Leavitt, J.M.; Lloyd, A.M. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 2008, 53, 814–827. [Google Scholar] [CrossRef] [PubMed]
- Zhou, L.L.; Shi, M.Z.; Xie, D.Y. Regulation of anthocyanin biosynthesis by nitrogen in TTG1-GL3/TT8-PAP1-programmed red cells of Arabidopsis thaliana. Planta 2012, 236, 825–837. [Google Scholar] [CrossRef] [PubMed]
- Shi, M.Z.; Xie, D.Y. Engineering of red cells of Arabidopsis thaliana and comparative genome-wide gene expression analysis of red cells versus wild-type cells. Planta 2011, 233, 787–805. [Google Scholar] [CrossRef] [PubMed]
- Tohge, T.; Nishiyama, Y.; Hirai, M.Y.; Yano, M.; Nakajima, J.; Awazuhara, M.; Inoue, E.; Takahashi, H.; Goodenowe, D.B.; Kitayama, M.; et al. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. Plant J. 2005, 42, 218–235. [Google Scholar] [CrossRef] [PubMed]
- Wilkins, O.; Nahal, H.; Foong, J.; Provart, N.; Campbell, M. Expansion and diversification of the populus R2R3-MYB family of transcription factors. Plant Physiol. 2009, 149, 981–993. [Google Scholar] [CrossRef] [PubMed]
- Shi, M.Z.; Xie, D.Y. Biosynthesis and metabolic engineering of anthocyanins in Arabidopsis thaliana. Recent Pat. Biotechnol. 2014, 8, 47–60. [Google Scholar] [CrossRef] [PubMed]
- Deng, Y.; Lu, S. Biosynthesis and regulation of phenylpropanoids in plants. Crit. Rev. Plant Sci. 2017, 36, 1–34. [Google Scholar] [CrossRef]
- Xu, W.; Grain, D.; Bobet, S.; Le, G.J.; Thévenin, J.; Kelemen, Z.; Lepiniec, L.; Dubos, C. Complexity and robustness of the flavonoid transcriptional regulatory network revealed by comprehensive analyses of MYB–bHLH–WDR complexes and their targets in Arabidopsis seed. New Phytol. 2014, 202, 132–144. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ramsay, N.A.; Glover, B.J. MYB–bHLH–WD40 protein complex and the evolution of cellular diversity. Trends Plant Sci. 2005, 10, 63–70. [Google Scholar] [CrossRef] [PubMed]
- Lloyd, A.M. Progress in the molecular genetic analysis of trichome initiation and morphogenesis in Arabidopsis. Trends Plant Sci. 2000, 5, 214–219. [Google Scholar]
- Baudry, A.; Heim, M.A.; Dubreucq, B.; Caboche, M.; Weisshaar, B.; Lepiniec, L. TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J. 2010, 39, 366–380. [Google Scholar] [CrossRef] [PubMed]
- Sompornpailin, K.; Makita, Y.; Yamazaki, M.; Saito, K. A WD-repeat-containing putative regulatory protein in anthocyanin biosynthesis in Perilla frutescens. Plant Mol. Biol. 2002, 50, 485–495. [Google Scholar] [CrossRef] [PubMed]
- De, N.V.; Quattrocchio, F.; Mol, J.; Koes, R. The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast, plants, and animals. Genes Dev. 1997, 11, 1422–1434. [Google Scholar]
- Humphries, J.A.; Walker, A.R.; Timmis, J.N.; Orford, S.J. Two WD-repeat genes from cotton are functional homologues of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1 (TTG1) gene. Plant Mol. Biol. 2005, 57, 67–81. [Google Scholar] [CrossRef] [PubMed]
- An, X.H.; Tian, Y.; Chen, K.Q.; Wang, X.F.; Hao, Y.J. The apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation. J. Plant Physiol. 2012, 169, 710–717. [Google Scholar] [CrossRef] [PubMed]
- Ben-Simhon, Z.; Judeinstein, S.; Nadler-Hassar, T.; Trainin, T.; Bar-Ya’akov, I.; Borochov-Neori, H.; Holland, D. A pomegranate (Punica granatum L.) WD40-repeat gene is a functional homologue of Arabidopsis TTG1 and is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development. Planta 2011, 234, 865–881. [Google Scholar] [CrossRef] [PubMed]
- Pang, Y.; Wenger, J.P.; Saathoff, K.; Peel, G.J.; Wen, J.; Huhman, D.; Allen, S.N.; Tang, Y.; Cheng, X.; Tadege, M. A WD40 repeat protein from Medicago truncatula is necessary for tissue-specific anthocyanin and proanthocyanidin biosynthesis but not for trichome development. Plant Physiol. 2009, 151, 1114–1129. [Google Scholar] [CrossRef] [PubMed]
- Xie, X.; Zhao, J.; Hao, Y.J.; Fang, C.; Wang, Y. The ectopic expression of apple MYB1 and bHLH3 differentially activates anthocyanin biosynthesis in tobacco. Plant Cell Tissue Organ Cult. 2017, 131, 183–194. [Google Scholar] [CrossRef]
- Kong, D.; Li, M.; Dong, Z.; Ji, H.; Li, X. Identification of TaWD40D, a wheat WD40 repeat-containing protein that is associated with plant tolerance to abiotic stresses. Plant Cell Rep. 2015, 34, 395–410. [Google Scholar] [CrossRef] [PubMed]
- Lepiniec, L.; Debeaujon, I.; Routaboul, J.M.; Baudry, A.; Pourcel, L.; Nesi, N.; Caboche, M. Genetics and biochemistry of seed flavonoids. Annu. Rev. Plant Biol. 2006, 57, 405–430. [Google Scholar] [CrossRef] [PubMed]
- Nocker, S.V.; Ludwig, P. The WD-repeat protein superfamily in Arabidopsis: Conservation and divergence in structure and function. BMC Genom. 2003, 4, 50. [Google Scholar]
- Yamazaki, M.; Makita, Y.; Springob, K.; Saito, K. Regulatory mechanisms for anthocyanin biosynthesis in chemotypes of Perilla frutescens var. crispa. Biochem. Eng. J. 2003, 14, 191–197. [Google Scholar] [CrossRef]
- Koornneef, M. The complex syndrome of ttg mutanis. Arab. Inf. Serv. 1981, 18, 45–51. [Google Scholar]
- Pang, Y.; Peel, G.J.; Sharma, S.B.; Tang, Y.; Dixon, R.A. A transcript profiling approach reveals an epicatechin-specific glucosyltransferase expressed in the seed coat of Medicago truncatula. Proc. Natl. Acad. Sci. USA 2008, 105, 14210–14215. [Google Scholar] [CrossRef] [PubMed]
- Zhao, L.; Gao, L.; Wang, H.; Chen, X.; Wang, Y.; Yang, H.; Wei, C.; Wan, X.; Xia, T. The R2R3-MYB, bHLH, WD40, and related transcription factors in flavonoid biosynthesis. Funct. Integr. Genom. 2013, 13, 75–98. [Google Scholar] [CrossRef] [PubMed]
- Xie, D.Y.; Dixon, R.A. Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science 2003, 299, 396–399. [Google Scholar] [CrossRef] [PubMed]
- Baudry, A.; Caboche, M.; Lepiniec, L. TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and bHLH factors, allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana. Plant J. 2006, 46, 768–779. [Google Scholar] [CrossRef] [PubMed]
- Xu, W.; Grain, D.; Gourrierec, J.L.; Harscoët, E.; Berger, A.; Jauvion, V.; Scagnelli, A.; Berger, N.; Bidzinski, P.; Kelemen, Z.; et al. Regulation of flavonoid biosynthesis involves an unexpected complex transcriptional regulation of TT8 expression, in Arabidopsis. New Phytol. 2013, 198, 59–70. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Xie, D.Y.; Shi, M.Z. Differentiation of programmed Arabidopsis cells. Bioengineered 2012, 3, 54–59. [Google Scholar] [CrossRef] [PubMed]
- He, X.; Zhao, X.; Gao, L.; Shi, X.; Dai, X.; Liu, Y.; Xia, T.; Wang, Y. Isolation and characterization of key genes that promote flavonoid accumulation in purple-leaf tea (Camellia sinensis L.). Sci. Rep. 2018, 8, 130. [Google Scholar] [CrossRef] [PubMed]
- Jiang, X.; Huang, K.; Zheng, G.; Hou, H.; Wang, P.; Jiang, H.; Zhao, X.; Li, M.; Zhang, S.; Liu, Y. CsMYB5a and CsMYB5e from Camellia sinensis differentially regulate anthocyanin and proanthocyanidin biosynthesis. Plant Sci. 2018, 270, 209–220. [Google Scholar] [CrossRef] [PubMed]
- Zhao, L.; Jiang, X.L.; Qian, Y.M.; Wang, P.Q.; Xie, D.Y.; Gao, L.P.; Xia, T. Metabolic characterization of the anthocyanidin reductase pathway involved in the biosynthesis of flavan-3-ols in Elite Shuchazao tea (Camellia sinensis) cultivar in the field. Molecules 2017, 22, 2241. [Google Scholar] [CrossRef] [PubMed]
- Bogs, J.; Jaffé, F.W.; Takos, A.M.; Walker, A.R.; Robinson, S.P. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol. 2007, 143, 1347–1361. [Google Scholar] [CrossRef] [PubMed]
- Qian, Y.M.; Zhang, S.X.; Yao, S.B.; Xia, J.X.; Liu, Y.J.; Gao, L.P.; Xia, T. The effects of vitro sucrose on quality components of tea plants (Camellia sinensis) based on transcriptomic and metabolic analysis. BMC Plant Biol. 2018, in press. [Google Scholar]
- Li, M.; Li, Y.; Guo, L.; Gong, N.; Pang, Y.; Jiang, W.; Liu, Y.; Jiang, X.; Zhao, L.; Wang, Y. Functional characterization of tea (Camellia sinensis) MYB4a transcription factor using an integrative approach. Front. Plant Sci. 2017, 8, 943. [Google Scholar] [CrossRef] [PubMed]
- Jiang, X.; Liu, Y.; Wu, Y.; Tan, H.; Meng, F.; Wang, Y.S.; Li, M.; Zhao, L.; Liu, L.; Qian, Y.; et al. Analysis of accumulation patterns and preliminary study on the condensation mechanism of proanthocyanidins in the tea plant (Camellia sinensis). Sci. Rep. 2015, 5, 8742. [Google Scholar] [CrossRef] [PubMed]
- Wang, Y.S.; Xu, Y.J.; Gao, L.P.; Yu, O.; Wang, X.Z.; He, X.J.; Jiang, X.L.; Liu, Y.J.; Xia, T. Functional analysis of flavonoid 3′,5′-hydroxylase from Tea plant (Camellia sinensis): Critical role in the accumulation of catechins. BMC Plant Biol. 2014, 14, 347. [Google Scholar] [CrossRef] [PubMed]
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Liu, Y.; Hou, H.; Jiang, X.; Wang, P.; Dai, X.; Chen, W.; Gao, L.; Xia, T. A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes. Int. J. Mol. Sci. 2018, 19, 1686. https://doi.org/10.3390/ijms19061686
Liu Y, Hou H, Jiang X, Wang P, Dai X, Chen W, Gao L, Xia T. A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes. International Journal of Molecular Sciences. 2018; 19(6):1686. https://doi.org/10.3390/ijms19061686
Chicago/Turabian StyleLiu, Yajun, Hua Hou, Xiaolan Jiang, Peiqiang Wang, Xinlong Dai, Wei Chen, Liping Gao, and Tao Xia. 2018. "A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes" International Journal of Molecular Sciences 19, no. 6: 1686. https://doi.org/10.3390/ijms19061686