Prospects for Knockout of MYB60, a Transcriptional Repressor of Anthocyanin Biosynthesis, in Brassicaceae Plants by Genome Editing


The Brassicaceae plant family contains many economically important crops such as Brassica napus L., Brassica rapa L., Brassica oleracea L., Brassica juncea L., Eruca sativa Mill., Camelina sativa L. and Raphanus sativus L. Insufficient data on the genetic regulation of agronomic traits in these species complicates the editing of their genomes. In recent years, the attention of the academic community has been drawn to anthocyanin hyperaccumulation. This trait is not only beneficial for human health, but can also increase plant resistance to stress. MYB transcription factors are the main regulators of flavonoid biosynthesis in plants. Some of them are well studied in Arabidopsis thaliana. The AtMYB60 gene is a transcriptional repressor of anthocyanin biosynthesis, and it also negatively impacts plant responses to drought stress. Myb60 is one of the least studied transcription factors with similar functions in Brassicaceae. There is a high degree of homology between predicted MYB60 genes of A. thaliana and related plant species. However, functions of these homologous genes have never been studied. Gene knockout by CRISPR/Cas technology remains the easiest way to perform genome editing in order to discover the role of individual plant genes. Disruption of genes acting as negative regulators of anthocyanin biosynthesis could result in color staining of plant tissues and an increase in stress tolerance. In the present study, we investigated the AtMYB60 gene and its homologs in Brassicaceae plants and suggested universal gRNAs to knockout these genes.

Keywords: CRISPR, Brassicaceae, MYB60, knockout, anthocyanin

[1] Warwick SI. (2011). Genetics and genomics of the Brassicaceae. New York: Springer; 2011. Brassicaceae in Agriculture.

[2] Kuluev, Bulat & Gumerova, Gulnar & Mikhaylova, Elena et al. Delivery of CRISPR/Cas components into higher plant cells for genome editing. Russian Journal of Plant Physiology. 2019;66(5):694-706.

[3] Shoeva OY, Khlestkina EK. Anthocyanins participate in the protection of wheat seedlings against cadmium stress. Cereal Research Communications. 2018;46(2):242-252.

[4] Mitsunami T, Nishihara M, Galis I, et al. Overexpression of the PAP1 transcription factor reveals a complex regulation of flavonoid and phenylpropanoid metabolism in Nicotiana tabacum plants attacked by Spodoptera litura. PLoS One. 2014;9(9). 108849-108849

[5] Gandikota, Madhuri & De Kochko, Alexandre & Chen, Lili & Ithal, Nagabhushana & Fauquet, Claude & Reddy, Arjula. Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance. Molecular Breeding. 2001;7(1):73-83.

[6] Kangatharalingam, N. & Pierce, Margaret & Bayles, Melanie & Essenberg, Margaret Epidermal anthocyanin production as an indicator of bacterial blight resistance in cotton. Physiological and Molecular Plant Pathology. 2002;61(3):189-195.

[7] Li S. Transcriptional control of flavonoid biosynthesis: Fine-tuning of the MYB-bHLHWD40 (MBW) complex. Plant Signaling & Behavior. 2014;9(1) 27522- 27522

[8] Park JS, Kim JB, Cho KJ, et al. (2008). Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa). Plant Cell Reports. 2008;27(6):985-994.

[9] Zhu HF, Fitzsimmons K, Khandelwal A, Kranz RG CPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis. Molecular Plant. 2009;2(4):790-802,

[10] Matsui K, Umemura Y, OhmeTakagi M. AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. The Plant Journal. 2008;55(6):954-967.

[11] Millard PS, Kragelund BB, Burow M. R2R3 MYB transcription factors–functions outside the DNA-binding domain. Trends in Plant Science. 2019;24(10):934-946.

[12] Wang B, Luo Q, Li Y, et al. Structural insights into target DNA recognition by R2R3- MYB transcription factors. Nucleic Acids Research. 2020;48(1):460-471.

[13] Oh JE, Kwon Y, Kim JH et al. A dual role for MYB60 in stomatal regulation and root growth of Arabidopsis thaliana under drought stress. Plant Molecular Biology. 2011;77(1-2):91-103.

[14] Lu S, Wang J, Chitsaz F, et al. CDD/SPARCLE: The conserved domain database in 2020. Nucleic Acids Research. 2020;48(D1):265-268.

[15] Cominelli E, Galbiati M, Vavasseur A et al. A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance. Current Biology. 2005;15(13):1196-1200

[16] Cominelli E, Galbiati M, Albertini A et al. DOF-binding sites additively contribute to guard cell-specificity of AtMYB60promoter. BMC Plant Biology. 2011;11(1):162-167.

[17] Falginella L, Di Gaspero G, Castellarin SD. Expression of flavonoid genes in the red grape berry of ‘Alicante Bouschet’ varies with the histological distribution of anthocyanins and their chemical composition. Planta. 2012;236(4):1037-1051.

[18] Batista, V G L, Pedro Dantas Fernandes, R.C. dos Santos, Péricles A. Melo Filho and Liziane Maria de Lima. (2019) Expression profile of MYB60 and GUSP1 genes during early growth of cotton genotypes submitted to water stress. Genetics and Molecular Research. 2019;18(4). 1-18

[19] Galbiati M, Matus JT, Francia P, et al. The grapevine guard cell-related VvMYB60 transcription factor is involved in the regulation of stomatal activity and is differentially expressed in response to ABA and osmotic stress. BMC Plant Biology. 2011;11(1):142- 142.

[20] Waterhouse A, Bertoni M, Bienert S, et al. SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Research. 2018;46(W1):296-303.

[21] Kuznetsov, Dmitry & Tegenfeldt, Fredrik & Manni, Mosè & Dias, Renata & Simão, Felipe & Zdobnov, Evgeny. OrthoDB v10: Sampling the diversity of animal, plant, fungal, protist, bacterial and viral genomes for evolutionary and functional annotations of orthologs. Nucleic Acids Research. 2019;47(D1):807-D811.

[22] Haeussler M, Schönig K, Eckert H, et al. (2016). Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biology. 2016;17(1):148-148.