LvbHLH13 Regulates Anthocyanin Biosynthesis by Activating the LvMYB5 Promoter in Lily (Lilium ‘Viviana’)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. RNA Extraction, cDNA Synthesis, and qRT-PCR
2.3. Anthocyanin Concentration Analysis
2.4. Virus-Induced Gene Silencing (VIGS) of LvMYB5 and LvbHLH13 Genes in Lily Petals
2.5. Overexpression of LvMYB5 and LvbHLH13 Genes in Lily Petals
2.6. Subcellular Localization of LvMYB5 and LvbHLH13
2.7. Yeast One-Hybrid Assay of LvbHLH13 and LvMYB5 Promoter
2.8. Electrophoretic Mobility Shift Assay (EMSA) of LvbHLH13 and LvMYB5 Promoter
2.9. Dual-Luciferase Transient Assay
2.10. Yeast Two-Hybrid Assay of LvbHLH13 and LvMYB5 Promoter
2.11. Statistical Measurement
3. Results
3.1. Spatiotemporal Differences in Gene Expression during Anthocyanin Accumulation in Lily Petals
3.2. Functional Analysis of the Transcription Factors LvMYB5 and LvbHLH13
3.3. LvbHLH13 Gene Silencing Inhibits Anthocyanin Synthesis in Lily Petals
3.4. Overexpression of LvbHLH13 Promotes Anthocyanin Accumulation in Lily Petals
4. Discussion
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Alappat, B.; Alappat, J. Anthocyanin Pigments: Beyond Aesthetics. Molecules 2020, 25, 5500. [Google Scholar] [CrossRef]
- 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. 2004, 39, 366–380. [Google Scholar] [CrossRef] [PubMed]
- Carretero-Paulet, L.; Galstyan, A.; Roig-Villanova, I.; Martínez-García, J.F.; Bilbao-Castro, J.R.; Robertson, D.L. Genome-Wide Classification and Evolutionary Analysis of the bHLH Family of Transcription Factors in Arabidopsis, Poplar, Rice, Moss, and Algae. Plant Physiol. 2010, 153, 1398–1412. [Google Scholar] [CrossRef] [PubMed]
- Deng, G.-M.; Zhang, S.; Yang, Q.-S.; Gao, H.-J.; Sheng, O.; Bi, F.-C.; Li, C.-Y.; Dong, T.; Yi, G.-J.; He, W.-D.; et al. MaMYB4, an R2R3-MYB Repressor Transcription Factor, Negatively Regulates the Biosynthesis of Anthocyanin in Banana. Front. Plant Sci. 2021, 11, 600704. [Google Scholar] [CrossRef] [PubMed]
- Deng, J.; Li, J.; Su, M.; Lin, Z.; Chen, L.; Yang, P. A bHLH gene NnTT8 of Nelumbo nucifera regulates anthocyanin biosynthesis. Plant Physiol. Biochem. 2021, 158, 518–523. [Google Scholar] [CrossRef]
- Deng, J.; Wu, D.; Shi, J.; Balfour, K.; Wang, H.; Zhu, G.; Liu, Y.; Wang, J.; Zhu, Z. Multiple MYB Activators and Repressors Collaboratively Regulate the Juvenile Red Fading in Leaves of Sweetpotato. Front. Plant Sci. 2020, 11, 941. [Google Scholar] [CrossRef]
- Espley, R.V.; Hellens, R.P.; Putterill, J.; Stevenson, D.E.; Kutty-Amma, S.; Allan, A.C. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J. 2007, 49, 414–427. [Google Scholar] [CrossRef]
- Fernández-Calvo, P.; Chini, A.; Fernández-Barbero, G.; Chico, J.-M.; Gimenez-Ibanez, S.; Geerinck, J.; Eeckhout, D.; Schweizer, F.; Godoy, M.; Franco-Zorrilla, J.M.; et al. The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses. Plant Cell 2011, 23, 701–715. [Google Scholar] [CrossRef]
- Hellens, R.P.; Allan, A.C.; Friel, E.N.; Bolitho, K.; Grafton, K.; Templeton, M.D.; Karunairetnam, S.; Gleave, A.P.; Laing, W.A. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 2005, 1, 13. [Google Scholar] [CrossRef]
- Hichri, I.; Barrieu, F.; Bogs, J.; Kappel, C.; Delrot, S.; Lauvergeat, V. Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J. Exp. Bot. 2011, 62, 2465–2483. [Google Scholar] [CrossRef]
- Huang, H.; Gao, H.; Liu, B.; Fan, M.; Wang, J.; Wang, C.; Tian, H.; Wang, L.; Xie, C.; Wu, D.; et al. bHLH13 Regulates Jasmonate-Mediated Defense Responses and Growth. Evol. Bioinform. 2018, 14, 1176934318790265. [Google Scholar] [CrossRef] [PubMed]
- Jiang, L.; Yue, M.; Liu, Y.; Zhang, N.; Lin, Y.; Zhang, Y.; Wang, Y.; Li, M.; Luo, Y.; Zhang, Y.; et al. A novel R2R3-MYB transcription factor FaMYB5 positively regulates anthocyanin and proanthocyanidin biosynthesis in cultivated strawberries (Fragaria× ananassa). Plant Biotechnol. J. 2023, 21, 1140–1158. [Google Scholar] [CrossRef] [PubMed]
- Kim, J.; Kim, D.-H.; Lee, J.-Y.; Lim, S.-H. The R3-Type MYB Transcription Factor BrMYBL2.1 Negatively Regulates Anthocyanin Biosynthesis in Chinese Cabbage (Brassica rapa L.) by Repressing MYB–bHLH–WD40 Complex Activity. Int. J. Mol. Sci. 2022, 23, 3382. [Google Scholar] [CrossRef]
- Lai, B.; Du, L.-N.; Liu, R.; Hu, B.; Su, W.-B.; Qin, Y.-H.; Zhao, J.-T.; Wang, H.-C.; Hu, G.-B. Two LcbHLH Transcription Factors Interacting with LcMYB1 in Regulating Late Structural Genes of Anthocyanin Biosynthesis in Nicotiana and Litchi chinensis During Anthocyanin Accumulation. Front. Plant Sci. 2016, 7, 166. [Google Scholar] [CrossRef] [PubMed]
- Li, H.; Yang, Z.; Zeng, Q.; Wang, S.; Luo, Y.; Huang, Y.; Xin, Y.; He, N. Abnormal expression of bHLH3 disrupts a flavonoid homeostasis network, causing differences in pigment composition among mulberry fruits. Hortic. Res. 2020, 7, 83. [Google Scholar] [CrossRef] [PubMed]
- Li, H.; Yao, Y.; An, L.; Li, X.; Cui, Y.; Bai, Y.; Yao, X.; Wu, K. Isolation and expression analysis of the HvnAnt2 gene in qingke barley (Hordeum vulgare L. var. nudum Hook. f.) varieties with different grain colours. Czech J. Genet. Plant Breed. 2024, 60, 107–118. [Google Scholar] [CrossRef]
- Liu, X.; Gu, J.; Wang, J.; Lu, Y. Lily breeding by using molecular tools and transformation systems. Mol. Biol. Rep. 2014, 41, 6899–6908. [Google Scholar] [CrossRef]
- Liu, Y.; Zhang, J.; Yang, X.; Wang, J.; Li, Y.; Zhang, P.; Mao, J.; Huang, Q.; Tang, H. Diversity in flower colorations of Ranunculus asiaticus L. revealed by anthocyanin biosynthesis pathway in view of gene composition, gene expression patterns, and color phenotype. Environ. Sci. Pollut. Res. 2018, 26, 13785–13794. [Google Scholar] [CrossRef]
- Lloyd, A.; Brockman, A.; Aguirre, L.; Campbell, A.; Bean, A.; Cantero, A.; Gonzalez, A. Advances in the MYB–bHLH–WD Repeat (MBW) Pigment Regulatory Model: Addition of a WRKY Factor and Co-option of an Anthocyanin MYB for Betalain Regulation. Plant Cell Physiol. 2017, 58, 1431–1441. [Google Scholar] [CrossRef]
- Lorenzo, O.; Chico, J.M.; Saénchez-Serrano, J.J.; Solano, R. JASMONATE-INSENSITIVE1Encodes a MYC Transcription Factor Essential to Discriminate between Different Jasmonate-Regulated Defense Responses in Arabidopsis[W]. Plant Cell 2004, 16, 1938–1950. [Google Scholar] [CrossRef]
- Lotkowska, M.E.; Tohge, T.; Fernie, A.R.; Xue, G.-P.; Balazadeh, S.; Mueller-Roeber, B. The Arabidopsis transcription factor MYB112 promotes anthocyanin formation during salinity and under high light stress. Plant Physiol. 2015, 169, 1862–1880. [Google Scholar] [CrossRef]
- Lu, R.; Li, Y.; Zhang, J.; Wang, Y.; Zhang, J.; Li, Y.; Zheng, Y.; Li, X.-B. The bHLH/HLH transcription factors GhFP2 and GhACE1 antagonistically regulate fiber elongation in cotton. Plant Physiol. 2022, 189, 628–643. [Google Scholar] [CrossRef] [PubMed]
- Ma, D.; Constabel, C.P. MYB Repressors as Regulators of Phenylpropanoid Metabolism in Plants. Trends Plant Sci. 2019, 24, 275–289. [Google Scholar] [CrossRef]
- Marasek-Ciolakowska, A.; Nishikawa, T.; Shea, D.J.; Okazaki, K. Breeding of lilies and tulips—Interspecific hybridization and genetic background. Breed. Sci. 2018, 68, 35–52. [Google Scholar] [CrossRef] [PubMed]
- Mekapogu, M.; Vasamsetti, B.M.K.; Kwon, O.-K.; Ahn, M.-S.; Lim, S.-H.; Jung, J.-A. Anthocyanins in Floral Colors: Biosynthesis and Regulation in Chrysanthemum Flowers. Int. J. Mol. Sci. 2020, 21, 6537. [Google Scholar] [CrossRef]
- Mrkvicová, E.; Pavlata, L.; Karásek, F.; Šťastník, O.; Doležalová, E.; Trojan, V.; Kizek, R.; Holeksová, V.; Mareš, J.; Brabec, T.; et al. The influence of feeding purple wheat with higher content of anthocyanins on antioxidant status and selected enzyme activity of animals. Acta Vet. Brno 2017, 85, 371–376. [Google Scholar] [CrossRef]
- Muhammad, N.; Uddin, N.; Khan, M.K.U.; Ali, N.; Ali, K.; Jones, D.A. Diverse role of basic Helix-Loop-Helix (bHLH) transcription factor superfamily genes in the fleshy fruit-bearing plant species. Czech J. Genet. Plant Breed 2023, 59, 1–13. [Google Scholar] [CrossRef]
- Nakata, M.; Ohme-Takagi, M. Two bHLH-type transcription factors, JA-ASSOCIATED MYC2-LIKE2 and JAM3, are transcriptional repressors and affect male fertility. Plant Signal. Behav. 2014, 8, e26473. [Google Scholar] [CrossRef]
- Nesi, N.; Debeaujon, I.; Jond, C.; Pelletier, G.; Caboche, M.; Lepiniec, L. The TT8 Gene Encodes a Basic Helix-Loop-Helix Domain Protein Required for Expression of DFR and BAN Genes in Arabidopsis Siliques. Plant Cell 2000, 12, 1863–1878. [Google Scholar] [CrossRef]
- Nguyen, C.T.; Tran, G.B.; Nguyen, N.H. The MYB–bHLH–WDR interferers (MBWi) epigenetically suppress the MBW’s targets. Biol. Cell 2019, 111, 284–291. [Google Scholar] [CrossRef]
- Qi, T.; Song, S.; Ren, Q.; Wu, D.; Huang, H.; Chen, Y.; Fan, M.; Peng, W.; Ren, C.; Xie, D. The Jasmonate-ZIM-Domain Proteins Interact with the WD-Repeat/bHLH/MYB Complexes to Regulate Jasmonate-Mediated Anthocyanin Accumulation and Trichome Initiation in Arabidopsis thaliana. Plant Cell 2011, 23, 1795–1814. [Google Scholar] [CrossRef] [PubMed]
- Fonseca, S.; Fernández-Calvo, P.; Fernández, G.M.; Díez-Díaz, M.; Gimenez-Ibanez, S.; López-Vidriero, I.; Godoy, M.; Fernández-Barbero, G.; Van Leene, J.; De Jaeger, G.; et al. bHLH003, bHLH013 and bHLH017 Are New Targets of JAZ Repressors Negatively Regulating JA Responses. PLoS ONE 2014, 9, e86182. [Google Scholar] [CrossRef] [PubMed]
- Shin, J.; Park, E.; Choi, G. PIF3 regulates anthocyanin biosynthesis in an HY5-dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis. Plant J. 2007, 49, 981–994. [Google Scholar] [CrossRef]
- Silva, S.; Costa, E.M.; Calhau, C.; Morais, R.M.; Pintado, M.E. Anthocyanin extraction from plant tissues: A review. Crit. Rev. Food Sci. Nutr. 2015, 57, 3072–3083. [Google Scholar] [CrossRef]
- Sun, Y.; Zhang, Y.; Ni, N.; Wang, B.; Chen, J.; Sun, S.; Han, W.; Zhang, X.; Muhammad, I.; Chen, L.; et al. Interplay between LvCOP1 and LvMYB1 governs light-induced anthocyanin biosynthesis in lily (Lilium ‘Viviana’). Sci. Hortic. 2024, 332, 113197. [Google Scholar] [CrossRef]
- Tao, R.; Yu, W.; Gao, Y.; Ni, J.; Yin, L.; Zhang, X.; Li, H.; Wang, D.; Bai, S.; Teng, Y. Light-Induced Basic/Helix-Loop-Helix64 Enhances Anthocyanin Biosynthesis and Undergoes CONSTITUTIVELY PHOTOMORPHOGENIC1-Mediated Degradation in Pear. Plant Physiol. 2020, 184, 1684–1701. [Google Scholar] [CrossRef]
- Tominaga-Wada, R.; Iwata, M.; Nukumizu, Y.; Wada, T. Analysis of IIId, IIIe and IVa group basic-helix-loop-helix proteins expressed in Arabidopsis root epidermis. Plant Sci. 2011, 181, 471–478. [Google Scholar] [CrossRef]
- Wang, J.; Cao, K.; Wang, L.; Dong, W.; Zhang, X.; Liu, W. Two MYB and Three bHLH Family Genes Participate in Anthocyanin Accumulation in the Flesh of Peach Fruit Treated with Glucose, Sucrose, Sorbitol, and Fructose In Vitro. Plants 2022, 11, 507. [Google Scholar] [CrossRef] [PubMed]
- Wang, L.; Tang, W.; Hu, Y.; Zhang, Y.; Sun, J.; Guo, X.; Lu, H.; Yang, Y.; Fang, C.; Niu, X.; et al. A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang. Plant J. 2019, 99, 359–378. [Google Scholar] [CrossRef]
- Wang, Y.; Wu, J.; Li, J.; Liu, B.; Wang, D.; Gao, C. The R2R3-MYB transcription factor ThRAX2 recognized a new element MYB-T (CTTCCA) to enhance cadmium tolerance in Tamarix hispida. Plant Sci. 2023, 329, 111574. [Google Scholar] [CrossRef]
- Xu, F.; Tang, J.; Wang, S.; Cheng, X.; Wang, H.; Ou, S.; Gao, S.; Li, B.; Qian, Y.; Gao, C.; et al. Antagonistic control of seed dormancy in rice by two bHLH transcription factors. Nat. Genet. 2022, 54, 1972–1982. [Google Scholar] [CrossRef] [PubMed]
- Yamagishi, M. How genes paint Lily flowers: Regulation of colouration and pigmentation patterning. Sci. Hortic. 2013, 163, 27–36. [Google Scholar] [CrossRef]
- Yamagishi, M.; Shimoyamada, Y.; Nakatsuka, T.; Masuda, K. Two R2R3-MYB Genes, Homologs of Petunia AN2, Regulate Anthocyanin Biosyntheses in Flower Tepals, Tepal Spots and Leaves of Asiatic Hybrid Lily. Plant Cell Physiol. 2010, 51, 463–474. [Google Scholar] [CrossRef]
- Yan, H.-X.; Fu, D.-Q.; Zhu, B.-Z.; Liu, H.-P.; Shen, X.-Y.; Luo, Y.-B. Sprout vacuum-infiltration: A simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species. Plant Cell Rep. 2012, 31, 1713–1722. [Google Scholar] [CrossRef] [PubMed]
- Yin, X.; Lin, X.; Liu, Y.; Irfan, M.; Chen, L.; Zhang, L. Integrated metabolic profiling and transcriptome analysis of pigment accumulation in diverse petal tissues in the Lily cultivar ‘Vivian’. BMC Plant Biol. 2020, 20, 446. [Google Scholar] [CrossRef] [PubMed]
- Yin, X.; Zhang, Y.; Zhang, L.; Wang, B.; Zhao, Y.; Irfan, M.; Chen, L.; Feng, Y. Regulation of MYB Transcription Factors of Anthocyanin Synthesis in Lily Flowers. Front. Plant Sci. 2021, 12, 761668. [Google Scholar] [CrossRef]
- Yu, C.; Huang, J.; Wu, Q.; Zhang, C.; Li, X.-L.; Xu, X.; Feng, S.; Zhan, X.; Chen, Z.; Wang, H.; et al. Role of female-predominant MYB39-bHLH13 complex in sexually dimorphic accumulation of taxol in Taxus media. Hortic. Res. 2022, 9, uhac062. [Google Scholar] [CrossRef]
- Yu, H.; Song, S.; Qi, T.; Fan, M.; Zhang, X.; Gao, H.; Huang, H.; Wu, D.; Guo, H.; Xie, D. The bHLH Subgroup IIId Factors Negatively Regulate Jasmonate-Mediated Plant Defense and Development. PLoS Genet. 2013, 9, e1003653. [Google Scholar]
- Yu, J.Q.; Gu, K.D.; Sun, C.H.; Zhang, Q.Y.; Wang, J.H.; Ma, F.F.; You, C.X.; Hu, D.G.; Hao, Y.J. The apple bHLH transcription factor MdbHLH3 functions in determining the fruit carbohydrates and malate. Plant Biotechnol. J. 2020, 19, 285–299. [Google Scholar] [CrossRef]
- Zhang, F.; Gonzalez, A.; Zhao, M.; Payne, C.T.; Lloyd, A. A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis. Development 2003, 130, 4859–4869. [Google Scholar] [CrossRef]
- Zhang, L.; Duan, Z.; Ma, S.; Sun, S.; Sun, M.; Xiao, Y.; Ni, N.; Muhammad, I.; Chen, L.; Sun, Y. SlMYB7, an AtMYB4-Like R2R3-MYB Transcription Factor, Inhibits Anthocyanin Accumulation in Solanum lycopersicum Fruits. J. Agric. Food Chem. 2023, 71, 18758–18768. [Google Scholar] [CrossRef] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
An, W.; Sun, Y.; Gao, Z.; Liu, X.; Guo, Q.; Sun, S.; Zhang, M.; Han, Y.; Irfan, M.; Chen, L.; et al. LvbHLH13 Regulates Anthocyanin Biosynthesis by Activating the LvMYB5 Promoter in Lily (Lilium ‘Viviana’). Horticulturae 2024, 10, 926. https://doi.org/10.3390/horticulturae10090926
An W, Sun Y, Gao Z, Liu X, Guo Q, Sun S, Zhang M, Han Y, Irfan M, Chen L, et al. LvbHLH13 Regulates Anthocyanin Biosynthesis by Activating the LvMYB5 Promoter in Lily (Lilium ‘Viviana’). Horticulturae. 2024; 10(9):926. https://doi.org/10.3390/horticulturae10090926
Chicago/Turabian StyleAn, Wenzhong, Yibo Sun, Zhenhua Gao, Xiaoye Liu, Qi Guo, Shaokun Sun, Minghui Zhang, Yutong Han, Muhammad Irfan, Lijing Chen, and et al. 2024. "LvbHLH13 Regulates Anthocyanin Biosynthesis by Activating the LvMYB5 Promoter in Lily (Lilium ‘Viviana’)" Horticulturae 10, no. 9: 926. https://doi.org/10.3390/horticulturae10090926