Abstract
Lignin biosynthesis occurs via the phenylpropanoid pathway and is regulated by transcription factors (TFs) including R2R3-MYB family members. In this study, we functionally characterized the R2R3-MYB TF VcMYB4a from blueberry (Vaccinium corymbosum) in lignin biosynthetic pathway. Phylogenetic analysis indicated that VcMYB4a clusters in a subclade with other TFs that act as transcriptional repressors of lignin and phenolic acid biosynthesis. Furthermore, lignin accumulation appeared to be negatively correlated with VcMYB4a expression during fruit development. Heterologous expression of VcMYB4a repressed lignin accumulation in Arabidopsis. Overexpression of VcMYB4a decreased lignin content in blueberry calli, whereas inhibition of VcMYB4a expression increased lignin accumulation in blueberry leaves. Finally, the transcriptome sequencing showed that overexpressing VcMYB4a in blueberry calli downregulated the expression of Vc4CL (Vc4CL5 and Vc4CL7), VcCOMT (VcCOMT1 and VcCOMT2), and VcCAD (VcCAD1 and VcCAD9) genes involved in lignin biosynthetic pathway. The heterologously expressing VcMYB4a in Arabidopsis downregulated the expression of genes, including AtC4H, At4CL (At4CL1 and At4CL5), AtCAD (AtCAD5 and AtCAD9), and AtCOMT1. The promoter sequences of these genes all contain MYB binding sites, and VcCAD9 and AtCAD9 genes have the most MYB binding sites. At the same time, VcCAD9 is more closely related to AtCAD9 than other CAD homologs from blueberry and Arabidopsis according to phylogenetic analysis. These findings suggested that VcMYB4a functions as a repressor of lignin biosynthesis by downregulating expression of 4CL, COMT, and CAD family members, especially CAD9 homologs. Our studies provide prospects for breeding new blueberry varieties with high lignin contents.
This is a preview of subscription content, access via your institution.







Data availability
All data generated or analyzed during this study in this published article (and its supplementary information files) and are available from the corresponding author on reasonable request.
Change history
22 July 2022
Handling editor name correction.
References
Aharoni A, De Vos C, Wein M, Sun Z, Greco R, Kroon A (2001) The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J 28(3):319–332. https://doi.org/10.1046/j.1365-313X.2001.01154.x
An XH, Tian Y, Chen K, Wang X, Hao Y (2012) The apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation. J Plant Physiol 169(7):710–717. https://doi.org/10.1016/j.jplph.2012.01.015
An X, Tian Y, Chen K, Liu X, Liu D, Xie X, Cheng C, Cong P, Hao Y (2015) MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant Cell Physiol 56(4):650–662. https://doi.org/10.1093/pcp/pcu205
Cavallini E, Matus JT, Finezzo L, Zenoni S, Loyola R, Guzzo F, Schlechter R, Ageorges A, Arce-Johnson P, Tornielli GB (2015) The phenylpropanoid pathway is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine. Plant Physiol 167(4):1448–1470. https://doi.org/10.1104/pp.114.256172
Clough SJ, Bent AF (1998) Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743. https://doi.org/10.1046/j.1365-313x.1998.00343.x
Dauwe R, Morreel K, Goeminne G, Gielen B, Rohde A, Van Beeumen J, Ralph J, Boudet A, Kopka J, Rochange SF, Halpin C, Messens E, Boerjan W (2007) Molecular phenotyping of lignin-modified tobacco reveals associated changes in cell-wall metabolism, primary metabolism, stress metabolism and photorespiration. Plant J 52(2):263–285. https://doi.org/10.1111/j.1365-313X.2007.03233.x
Dubos C, Gourrierec JL, Baudry A, Huep G, Lanet E, Debeaujon I, Routaboul J, Alboresi A, Weisshaar B, Lepiniec L (2008) MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. Plant J 55(6):940–953. https://doi.org/10.1111/j.1365-313X.2008.03564.x
Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L (2010) MYB transcription factors in Arabidopsis. Trends Plant Sci 15(10):573–581. https://doi.org/10.1016/j.tplants.2010.06.005
Fornalé S, Shi X, Chai C, Encina A, Irar S, Capellades M, Fuguet E, Torres JL, Rovira P, Puigdoménech P (2010) ZmMYB31directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux. Plant J 64(4):633–644. https://doi.org/10.1111/j.1365-313X.2010.04363.x
Fornalé S, Lopez E, Salazar-Henao JE, Fernández-Nohales RJ, Caparros-Ruiz D (2014) AtMYB7, a new player in the regulation of UV-Sunscreens in Arabidopsis thaliana. Plant Cell Physiol 55(3):507–516. https://doi.org/10.1093/pcp/pct187
Giongo L, Vrhovsek U, Gasperi F, Endrizzi L, Palmieri L, Saviane A, Camin F, Perini M, Bontempo L, Danek I, Krol K, Mladin P, Mattivi F (2009) A three-year highbush blueberry survey in different European locations for the fresh and the processing markets. Acta Horti 810:887–894. https://doi.org/10.17660/ActaHortic.2009.810.118
Goujon T, Sibout R, Eudes A, Mackay J, Jouanin L (2003) Genes involved in the biosynthesis of lignin precursors in Arabidopsis thaliana. Plant Physiol Bioch 41(8):677–687. https://doi.org/10.1016/S0981-9428(03)00095-0
Häkkinen SH, Törrönen AR (2000) Content of flavonols and selected phenolic acids in strawberries and Vaccinium species: influence of cultivar, cultivation site and technique. Food Res Int 33(6):517–524. https://doi.org/10.1016/S0963-9969(00)00086-7
Jaakola L, Määttä K, Pirttilä AM, Törrönen R, Kärenlampi S, Hohtola A (2002) Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin, proanthocyanidin, and flavonol levels during bilberry fruit development. Plant Physiol 130(2):729–739. https://doi.org/10.1104/pp.006957
Jia N, Liu J, Sun Y, Tan P, Cao H, Xie Y, Wen B, Gu T, Liu J, Li M, Huang Y, Lu J, Jin N, Sun L, Xin F, Fan B (2018) Citurs sinensis MYB transcription factors CsMYB330 and CsMYB308 regulate fruit juice sac lignification through fine-tuning expression of the Cs4CL1. Plant Sci 277:334–343. https://doi.org/10.1016/j.plantsci.2018.10.006
Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C, Weisshaar B, Martin C (2000) Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J 19(22):6150–6161. https://doi.org/10.1093/emboj/19.22.6150
Kagale S, Pozwadowski K (2011) EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression. Epigenetics 6(2):141–146. https://doi.org/10.4161/epi.6.2.13627
Kim S, Kim K, Cho M, Franceschi VR, Davin LB, Lewis NG (2007) Expression of cinnamyl alcohol dehydrogenases and their putative homologues during Arabidopsis thaliana growth and development: lessons for database annotations? Phytochemistry 68(14):1957–1974. https://doi.org/10.1016/j.phytochem.2007.02.032
Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12(4):357–360. https://doi.org/10.1038/nmeth.3317
Kranz HD, Denekamp M, Greco R, Jin H, Leyva A, Meissner RC, Petroni K, Urzainqui A, Bevan M, Martin C, Smeekens S, Tonelli C, Paz-Ares J, Weisshaar B (1998) Towards functional characterization of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant J 16(2):263–276. https://doi.org/10.1046/j.1365-313x.1998.00278.x
Legay S, Sivadon P, Blervacq A, Pavy N, Baghdady A, Tremblay L, Levasseur C, Ladouce N, Lapierre C, Séguin A, Hawkins S, Mackay J, Grima-Pettenati J (2010) EgMYB1, an R2R3 MYB transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar. New Phytol 188(3):774–786. https://doi.org/10.1111/j.1469-8137.2010.03432.x
Liu Q, Luo L, Zheng L (2018) Lignins: biosynthesis and biological functions in plants. Int J Mol Sci 19:335. https://doi.org/10.3390/ijms19020335
Matsui K, Umemura Y, Ohme-Takagi M (2008) AtMYBL2, a protein with a sing MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. Plant J 55(6):954–967. https://doi.org/10.1111/j.1365-313X.2008.03565.x
Matus JT, Loyola R, Vega A, Peña-Neira A, Bordeu E, Arce-Johnson P, Alcalde JA (2009) Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera. J Exp Bot 60(3):853–867. https://doi.org/10.1093/jxb/ern336
Mellerowicz EJ, Baucher M, Sundberg B, Boerjan W (2001) Unravelling cell wall formation in the woody dicot stem. Plant Mol Biol 47(1-2):239–274. https://doi.org/10.1023/A:1010699919325
Meng D, Li C, Park H, González J, Wang J, Dandekar AM, Turgeon BG, Cheng L (2018) Sorbitol modulates resistance to Alternaria alternata by regulating the expression of an NLR resistance Gene in Apple. Plant Cell 30(7):1562–1581. https://doi.org/10.1105/tpc.18.00231
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Omer S, Kumar S, Khan BM (2013) Over-expression of a subgroup 4 R2R3 type MYB transcription factor gene from Leucaena leucocephala reduces lignin content in transgenic tobacco. Plant Cell Rep 32(1):161–171. https://doi.org/10.1007/s00299-012-1350-9
Pfaffl MW (2001) A new mathematical model for relative qunatification in realtime RT-PCR. Nucl Acids Res. 29(9):e45. https://doi.org/10.1093/nar/29.9.e45
Plunkett BJ, Espley RV, Dare AP, Warren BAW, Grierson ERP, Cordiner S, Turner JL, Allan AC, Albert NW, Davies KM, Schwinn KE (2018) MYBA from blueberry (Vaccinium section cyanococcus) is a subgroup 6 type R2R3MYB transcription factor that activates anthocyanin production. Front Plant Sci 9:1300. https://doi.org/10.3389/fpls.2018.01300
Poupin MJ, Federici F, Medina C, Matus JT, Timmermann T, Arce-Johnson P (2007) Isolation of the three grape sub-lineages of B-class MADS-box TM6, PISTILLATA and APETALA3 genes which are differentially expressed during flower and fruit development. Gene 404:10–24. https://doi.org/10.1016/j.gene.2007.08.005
Preston J, Wheeler J, Heazlewood J, Li SF, Parish RW (2004) AtMYB32 is required for mormal pollen development in Arabidopsis thaliana. The Plant J 40(6):979–995. https://doi.org/10.1111/j.1365-313X.2004.02280.x
Rabino I, Mancinelli AL (1986) Light, temperature, and anthocyanin production. Plant Physiol 81(3):922–924. https://doi.org/10.1104/pp.81.3.922
Shen H, He X, Poovaiah CR, Wuddineh WA, Ma J, Mann DGJ, Wang H, Jackson L, Tang Y, Neal Stewart Jr C, Chen F, Dixon RA (2012) Functional characterization of the switchgrass (Panicum virgatum) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks. New Phytologist 193(1):121–136. https://doi.org/10.1111/j.1469-8137.2011.03922.x
Sonbol FM, Fornalé S, Capellades M, Encina A, Touriño S, Torres J, Rovira P, Ruel K, Puigdomènech P, Rigau J, Caparrós-Ruiz D (2009) The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana. Plant Mol Biol 70(3):283–296. https://doi.org/10.1007/s11103-009-9473-2
Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1(4):2019–2025. https://doi.org/10.1038/nprot.2006.286
Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4(5):447–456. https://doi.org/10.1016/s1369-5266(00)00199-0
Tak H, Negi S, Ganapathi TR (2017) Overexpression of MusaMYB31, a R2R3 type MYB transcription factor gene indicate tis role as a negative regulator of lignin biosynthesis in banana. PLoS ONE 12(2):e0172695. https://doi.org/10.1371/journal.pone.0172695
Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, Roberts K, Martin C (1998) The AmMYB308 and AmMYB330 transcription factors from Antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell 10(2):135–154. https://doi.org/10.2307/3870694
Tamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. Mol Bio Evol 38(7):3022–3027. https://doi.org/10.1093/molbev/msab120
Thévenin J, Pollet B, Letarnec B, Saulnier L, Gissot L, Maia-Grondard A, Lapierre C, Jouanin L (2011) The simultaneous repression of CCR and CAD, two enzymes of the lignin biosynthetic pathway, results in sterility and dwarfism in Arabidopsis thaliana. Mol Plant 4(1):70–82. https://doi.org/10.1093/mp/ssq045
Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) Suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956. https://doi.org/10.1046/j.1365-313X.2003.01676.x
Wang A, Liang K, Yang S, Cao Y, Wang L, Zhang M, Zhou J, Zhang L (2021) Genome-wide analysis of MYB transcription factors of Vaccinium corymbosum and their positive responses to drought stress. BMC Genomics 22:565. https://doi.org/10.1186/s12864-021-07850-5
Zhang X, Henriques R, Lin S, Niu Q, Chua N (2006) Agrobacterimu-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2):641–646. https://doi.org/10.1038/nprot.2006.97
Zhang C, Liu H, Jia C, Liu Y, Wang F, Wang J (2016) Cloning, characterization and functional analysis of a flavonol synthase from Vaccinium corymbosum. Trees 30(5):1595–1605. https://doi.org/10.1007/s00468-016-1393-6
Zhang C, Guo Q, Liu Y, Liu H, Wang F, Jia C (2017) Molecular cloning and functional analysis of a flavanone 3-hydroxylase gene from blueberry. J Hortic Sci Biotech 92(1):57–64. https://doi.org/10.1080/14620316.2016.1224604
Zhang C, Liu H, Zhang X, Guo Q, Bian S, Wang J, Zhai L (2020) VcMYB4a, an R2R3-MYB transcription factor from Vaccinium corymbosum, negatively regulates salt, drought, and temperature stress. Gene 757:144935. https://doi.org/10.1016/j.gene.2020.144935
Zhang S, Yang J, Li H, Chiang V, Fu Y (2021) Cooperative regulation of flavonoid and lignin biosynthesis in plants. Crit Rev Plant Sci 40(2):109–126. https://doi.org/10.1080/07352689.2021.1898083
Zhong R, Lee C, Ye Z (2010) Evolutionary conservation of the transcriptional network regulating secondary cell wall biosynthesis. Trends Plant Sci 15(11):625–632. https://doi.org/10.1016/j.tplants.2010.08.007
Zhou J, Lee C, Zhang R, Ye Z (2009) MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell 21(1):248–266. https://doi.org/10.1105/tpc.108.063321
Zhu L, Shan H, Chen S, Jiang J, Gu C, Zhou G, Chen Y, Song A, Chen F (2013) The heterologous expression of the Chrysanthemum R2R3-MYB transcription factor CmMYB1 alters lignin composition and represses flavonoid synthesis in Arabidopsis thaliana. PLoS ONE 8(6):e65680. https://doi.org/10.1371/journal.pone.0065680
Zifkin M, Jin A, Ozga JA, Zaharia I, Schernthaner JP, Gesell A, Abrams SR, Kennedy JA, Constabel CP (2012) Gene expression and metabolite profiling of developing highbush blueberry fruit indicates transcriptional regulation of flavonoid metabolism and activation of abscisic acid metabolism. Plant Physiol. 158(1):200–224. https://doi.org/10.1104/pp.111.180950
Acknowledgements
We thank the Beijing BioMarker Bioinformatics Technology Co., Ltd. (Beijing, China) for the high throughput sequencing in this study.
Data archiving statement
We uploaded the RNA-Seq data generated in this study to BioProject in the NCBI repository (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA831018) with the accession number of PRJNA831018. This Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GJYR00000000. The version described in this paper is the first version, GJYR01000000.
Funding
This study was supported by the National Natural Science Foundation of China (grant number 31700260).
Author information
Authors and Affiliations
Contributions
All the authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Bofei Yang, Yuening Li, Yan Song, Xianglong Wang, Qingxun Guo, Lianxia Zhou, Xue Xue, and Chunyu Zhang. The first draft of the manuscript was written by Bofei Yang, and all the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Additional information
Communicated by A.M. Dandekar
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Yang, B., Li, Y., Song, Y. et al. The R2R3-MYB transcription factor VcMYB4a inhibits lignin biosynthesis in blueberry (Vaccinium corymbosum). Tree Genetics & Genomes 18, 27 (2022). https://doi.org/10.1007/s11295-022-01560-z
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11295-022-01560-z
Keywords
- VcMYB4a
- Lignin
- Blueberry
- Transcription factor
- Transcriptome