Register      Login
Functional Plant Biology Functional Plant Biology Society
Plant function and evolutionary biology
RESEARCH ARTICLE

MdMYBL2 helps regulate cytokinin-induced anthocyanin biosynthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana) callus

Yicheng Wang https://orcid.org/0000-0003-1319-6957 A , Jingjing Sun B , Nan Wang A , Haifeng Xu A , Changzhi Qu A , Shenghui Jiang A , Hongcheng Fang A , Mengyu Su A , Zongying Zhang A and Xuesen Chen A C
+ Author Affiliations
- Author Affiliations

A State Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An, Shandong, China.

B College of Forestry, Shandong Agricultural University, Tai-An, Shandong, China.

C Corresponding author. Email: chanyetixi@163.com

Functional Plant Biology 46(2) 187-196 https://doi.org/10.1071/FP17216
Submitted: 2 August 2017  Accepted: 20 September 2018   Published: 18 October 2018

Abstract

Anthocyanin biosynthesis is induced by cytokinins, and is regulated by MYB transcription factors. However, the underlying molecular mechanisms have not been fully characterised. In the present study, red-fleshed apple callus were induced from the leaves of an R6/R6 homozygous line, which was the hybrid offspring of Malus sieversii f. niedzwetzkyana and ‘Fuji’. We analysed the callus anthocyanin contents in response to different cytokinin concentrations. We observed that cytokinin treatments upregulated the expression of anthocyanin structural genes MdDFR and MdUFGT and transcription factor genes MdMYB10 and MdbHLH3. Additionally, the expression of MdMYBL2, which encodes the bHLH and EAR motifs, was inhibited by cytokinin treatments. The MdMYBL2-overexpressing callus had lower anthocyanin contents than the wild-type controls. We noted that the expression levels of anthocyanin biosynthesis structural genes MdDFR and MdUFGT and transcription factor genes MdMYB10 and MdbHLH3 were strongly suppressed in the transgenic callus. Subsequent yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MdMYBL2 interacts with MdbHLH3, which may influence the expression of anthocyanin biosynthesis-related genes. Our findings may provide new insights into how MYB transcription factors influence the cytokinin-regulated anthocyanin biosynthesis in red-fleshed apples.

Additional keywords: anthocyanin, cytokinins, MdbHLH3, MdMYBL2.


References

Aharoni A, De Vos CH, Wein M, Sun Z, Greco R, Kroon A, Mol JN, O’Connell AP (2001) The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. The Plant Journal 28, 319–332.
The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco.Crossref | GoogleScholarGoogle Scholar |

Albert NW, Lewis DH, Zhang H, Schwinn KE, Jameson PE, Davies KM (2011) Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning. The Plant Journal 65, 771–784.
Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning.Crossref | GoogleScholarGoogle Scholar |

Albert NW, Davies KM, Lewis DH, Zhang H, Montefiori M, Brendolis C, Boase MR, Ngo H, Jameson PE, Schwinn KE (2014) A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. The Plant Cell 26, 962–980.
A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots.Crossref | GoogleScholarGoogle Scholar |

Bac-Molenaar JA, Fradin EF, Rienstra JA, Vreugdenhil D, Keurentjes JJB (2015) GWA mapping of anthocyanin accumulation reveals balancing selection of MYB90 in Arabidopsis thaliana. PLoS One 10, e0143212–e0143231.
GWA mapping of anthocyanin accumulation reveals balancing selection of MYB90 in Arabidopsis thaliana.Crossref | GoogleScholarGoogle Scholar |

Bai YC, Li CL, Zhang JW, Li SJ, Luo XP, Yao HP, Chen H, Zhao HX, Park SU, Wu Q (2014) Characterization of two tartary buckwheat R2R3-MYB transcription factors and their regulation of proanthocyanidin biosynthesis. Physiologia Plantarum 152, 431–440.
Characterization of two tartary buckwheat R2R3-MYB transcription factors and their regulation of proanthocyanidin biosynthesis.Crossref | GoogleScholarGoogle Scholar |

Ballare C (2003) Stress under the sun: spotlight on ultraviolet-B responses. Plant Physiology 132, 1725–1727.
Stress under the sun: spotlight on ultraviolet-B responses.Crossref | GoogleScholarGoogle Scholar |

Ban Y, Honda C, Hatsuyama Y, Igarashi M, Bessho H, Moriguchi T (2007) Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant & Cell Physiology 48, 958–970.
Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin.Crossref | GoogleScholarGoogle Scholar |

Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. The Plant Cell 12, 2383–2393.
Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis.Crossref | GoogleScholarGoogle Scholar |

Chen DQ, Li ZY, Pan RC, Wang XJ (2006) Anthocyanin accumulation mediated by blue light and cytokinin in Arabidopsis seedlings. Journal of Integrative Plant Biology 48, 420–425.

Colquhoun TA, Kim JY, Wedde AE, Levin LA, Schmitt KC, Schuurink RC, Clark DG (2011) PhMYB4 fine-tunes the floral volatile signature of Petunia × hybrida through PhC4H. Journal of Experimental Botany 62, 1133–1143.
PhMYB4 fine-tunes the floral volatile signature of Petunia × hybrida through PhC4H.Crossref | GoogleScholarGoogle Scholar |

Das PK, Dong HS, Choi SB, Yoo SD, Choi G, Park YI (2012) Cytokinins enhance sugar-induced anthocyanin biosynthesis in Arabidopsis. Molecules and Cells 34, 93–101.
Cytokinins enhance sugar-induced anthocyanin biosynthesis in Arabidopsis.Crossref | GoogleScholarGoogle Scholar |

Dedio W, Clark KW (1971) Influence of cytokinins on isoflavone and anthocyanin synthesis in red clover seedlings. Pesticide Science 2, 65–68.
Influence of cytokinins on isoflavone and anthocyanin synthesis in red clover seedlings.Crossref | GoogleScholarGoogle Scholar |

Deikman J, Hammer PE (1995) Induction of anthocyanin accumulation by cytokinins in Arabidopsis thaliana. Plant Physiology 108, 47–57.
Induction of anthocyanin accumulation by cytokinins in Arabidopsis thaliana.Crossref | GoogleScholarGoogle Scholar |

Dubos C, Le GJ, Baudry A, Huep G, Lanet E, Debeaujon I, Routaboul JM, Alboresi A, Weisshaar B, Lepiniec L (2008) MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. The Plant Journal 55, 940–953.
MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana.Crossref | GoogleScholarGoogle Scholar |

Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC (2007) Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. The Plant Journal 49, 414–427.
Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10.Crossref | GoogleScholarGoogle Scholar |

Espley RV, Brendolise C, Chagné D, Kutty-Amma S, Green S, Volz R, Putterill J, Schouten HJ, Gardiner SE, Hellens RP, Allan AC (2009) Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. The Plant Cell 21, 168–183.
Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples.Crossref | GoogleScholarGoogle Scholar |

Feng S, Sun S, Chen X, Wu S, Wang D, Chen X (2015) PyMYB10 and PyMYB10.1 interact with bHLH to enhance anthocyanin accumulation in pears. PLoS One 10, e0142112–e0142128.
PyMYB10 and PyMYB10.1 interact with bHLH to enhance anthocyanin accumulation in pears.Crossref | GoogleScholarGoogle Scholar |

Gao JJ, Shen XF, Zhang Z, Peng RH, Xiong AS, Xu J, Zhu B, Zheng JL, Yao QH (2011) The MYB transcription factor MdMYB6 suppresses anthocyanin biosynthesis in transgenic Arabidopsis. Plant Cell, Tissue and Organ Culture 106, 235–242.
The MYB transcription factor MdMYB6 suppresses anthocyanin biosynthesis in transgenic Arabidopsis.Crossref | GoogleScholarGoogle Scholar |

Gatica-Arias A, Farag MA, Häntzschel KR, Matoušek J, Weber G (2012) The transcription factor ATMTB75/PAP1 regulates the expression of flavonoid biosynthesis genes in transgenic hop (Humulus Lupulus L.). Brewingscience 65, 103–111.

Hemm MR, Herrmann KM, Chapple C (2001) AtMYB4: A transcription factor general in the battle against UV. Trends in Plant Science 6, 135–136.
AtMYB4: A transcription factor general in the battle against UV.Crossref | GoogleScholarGoogle Scholar |

Ibrahim RK, Thakur ML, Permanand B (1971) Formation of anthocyanins in callus tissue cultures. Lloydia 34, 175–182.

Ji XH, Wang YT, Zhang R, Wu SJ, An MM, Li M, Wang CZ, Chen XL, Zhang YM, Chen XS (2015) Effect of auxin, cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of redfleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell, Tissue and Organ Culture 120, 325–337.
Effect of auxin, cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of redfleshed apple (Malus sieversii f. niedzwetzkyana).Crossref | GoogleScholarGoogle Scholar |

Jin HL, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C (2000) Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO Journal 19, 6150–6161.
Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis.Crossref | GoogleScholarGoogle Scholar |

Kim B, Tenessen D, Last R (1998) UVB induced photomorphogenesis in Arabidopsis thaliana. The Plant Journal 15, 667–674.
UVB induced photomorphogenesis in Arabidopsis thaliana.Crossref | GoogleScholarGoogle Scholar |

Kim JH, Hyun WY, Nguyen HN, Jeong CY, Xiong L, Hong SW, Lee H (2015) AtMYB7, a subgroup 4 R2R3 MYB, negatively regulates ABA-induced inhibition of seed germination by blocking the expression of the bZIP transcription factor ABI5. Plant, Cell & Environment 38, 559–571.
AtMYB7, a subgroup 4 R2R3 MYB, negatively regulates ABA-induced inhibition of seed germination by blocking the expression of the bZIP transcription factor ABI5.Crossref | GoogleScholarGoogle Scholar |

Klein AO, Hagen CW (1961) Anthocyanin production in detached petals of lmpatiens balsamina L. Plant Physiology 36, 1–9.
Anthocyanin production in detached petals of lmpatiens balsamina L.Crossref | GoogleScholarGoogle Scholar |

Li YY, Mao K, Zhao C, Zhao XY, Zhang HL, Shu HR, Hao YJ (2012) MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple. Plant Physiology 160, 1011–1022.
MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple.Crossref | GoogleScholarGoogle Scholar |

Lotkowska ME, Tohge T, Fernie AR, Xue GP, Balazadeh S, Muellerroeber B (2015) The Arabidopsis transcription factor MYB112 promotes anthocyanin formation during salinity and under high light stress. Plant Physiology 169, 1862–1935.

Matsui K, Umemura Y, Ohme-Takagi M (2008) AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. The Plant Journal 55, 954–967.
AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis.Crossref | GoogleScholarGoogle Scholar |

Matsumoto H, Nakamura Y, Tachibanaki S, Kawamura S, Hirayama M (2003) Stimulatory effect of cyanidin 3-glycosides on the regeneration of rhodopsin. Journal of Agricultural and Food Chemistry 51, 3560–3563.
Stimulatory effect of cyanidin 3-glycosides on the regeneration of rhodopsin.Crossref | GoogleScholarGoogle Scholar |

Nakamura N, Nakamae H, Maekawa L (1980) Effects of light and kinetin on anthocyanin accumulation in the petals of Rosa hybrida Hort cv. Ehigasa. Zeitschrift für Pflanzenphysiologie 98, 263–270.
Effects of light and kinetin on anthocyanin accumulation in the petals of Rosa hybrida Hort cv. Ehigasa.Crossref | GoogleScholarGoogle Scholar |

Nesi N, Debeaujon I, Jond C, Pelletier G, Caboche M, Lepiniec L (2000) The TT8 gene encodes a basic helix–loop–helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques. The Plant Cell 12, 1863–1878.
The TT8 gene encodes a basic helix–loop–helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques.Crossref | GoogleScholarGoogle Scholar |

Ohashi-Ito K, Saegusa M, Iwamoto K, Oda Y, Katayama H, Kojima M, Sakakibara H, Fukuda H (2014) A bHLH complex activates vascular cell division via cytokinin action in root apical meristem. Current Biology 24, 2053–2058.
A bHLH complex activates vascular cell division via cytokinin action in root apical meristem.Crossref | GoogleScholarGoogle Scholar |

Paolocci F, Robbins MP, Passeri V, Hauck B, Morris P, Rubini A, Arcioni S, Damiani F (2011) The strawberry transcription factor FaMYB1 inhibits the biosynthesis of proanthocyanidins in Lotus corniculatus leaves. Journal of Experimental Botany 62, 1189–1200.
The strawberry transcription factor FaMYB1 inhibits the biosynthesis of proanthocyanidins in Lotus corniculatus leaves.Crossref | GoogleScholarGoogle Scholar |

Pecket RC, Bassim TAH (1974) The effect of kinetin in relation to photocontrol of anthocyanin biosynthesis in Brassica oleracea. Phytochemistry 13, 1395–1399.
The effect of kinetin in relation to photocontrol of anthocyanin biosynthesis in Brassica oleracea.Crossref | GoogleScholarGoogle Scholar |

Pérez-Díaz JR, Pérez-Díaz J, Madrid-Espinoza J, González-Villanueva E, Moreno Y, Ruiz-Lara S (2016) New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco. Plant Molecular Biology 90, 63–76.
New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco.Crossref | GoogleScholarGoogle Scholar |

Quattrocchio F, Wing JF, Leppen H, Mol J, Koes RE (1993) Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. The Plant Cell 5, 1497–1512.
Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes.Crossref | GoogleScholarGoogle Scholar |

Rossi A, Serraino I, Dugo P, Di Paola R, Mondello L, Genovese T, Morabito D, Dugo G, Sautebin L, Caputi AP, Cuzzocrea S (2003) Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation. Free Radical Research 37, 891–900.
Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation.Crossref | GoogleScholarGoogle Scholar |

Rubin G, Tohge T, Matsuda F, Saito K, Scheible WR (2009) Members of the LBD family of transcription factors repress anthocyanin synthesis and affect additional nitrogen responses in Arabidopsis. The Plant Cell 21, 3567–3584.
Members of the LBD family of transcription factors repress anthocyanin synthesis and affect additional nitrogen responses in Arabidopsis.Crossref | GoogleScholarGoogle Scholar |

Sawa S (2002) Overexpression of the AtMYBL2 gene represses trichome development in Arabidopsis. DNA Research 9, 31–34.
Overexpression of the AtMYBL2 gene represses trichome development in Arabidopsis.Crossref | GoogleScholarGoogle Scholar |

Sevimli-Gur C, Cetin B, Akay S, Gulceiz S, Yesilceliktas O (2013) Extracts from black carrot tissue culture as potent anticancer agents. Plant Foods for Human Nutrition 68, 293–298.
Extracts from black carrot tissue culture as potent anticancer agents.Crossref | GoogleScholarGoogle Scholar |

Shulman Y, Lavee S (1973) The effect of cytokinins and auxins on anthocyanin accumulation in green manzanillo olives. Journal of Experimental Botany 24, 655–661.
The effect of cytokinins and auxins on anthocyanin accumulation in green manzanillo olives.Crossref | GoogleScholarGoogle Scholar |

Sonbol FM, Fornale S, Capellades M, Encina A, Tourino S, Torres JS, Rovira P, Ruel K, Puigdomenech P, Rigau J, Caparros-Ruiz D (2009) The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana. Plant Molecular Biology 70, 283–296.
The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana.Crossref | GoogleScholarGoogle Scholar |

Stintzing FC, Carle R (2004) Functional properties of anthocyanins and betalains in plants, food, and in human nutrition. Trends in Food Science & Technology 15, 19–38.
Functional properties of anthocyanins and betalains in plants, food, and in human nutrition.Crossref | GoogleScholarGoogle Scholar |

Stracke R, Ishihara H, Huep G, Barsch A, Mehrtens F, Niehaus K, Weisshaar B (2007) Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. The Plant Journal 50, 660–677.
Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling.Crossref | GoogleScholarGoogle Scholar |

Sun JJ, Wang YC, Chen XS, Gong XJ, Wang N, Ma L, Qiu YF, Wang YL, Feng SQ (2017) Effects of methyl jasmonate and abscisic acid on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell, Tissue and Organ Culture 130, 227–237.
Effects of methyl jasmonate and abscisic acid on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana).Crossref | GoogleScholarGoogle Scholar |

Takos AM, Jaffe’ FW, Jacob SR, Jochen B, Robinson SP, Walker AR (2006) Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiology 142, 1216–1232.
Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples.Crossref | GoogleScholarGoogle Scholar |

Teng S, Keurentjes J, Bentsink L, Koornneef M, Smeekens S (2005) Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene. Plant Physiology 139, 1840–1852.
Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene.Crossref | GoogleScholarGoogle Scholar |

Toyama T, Teramoto H, Ishiguro S, Tanaka A, Okada K, Takeba G (1999) A cytokinin-repressed gene in cucumber for a bHLH protein homologue is regulated by light. Plant & Cell Physiology 40, 1087–1092.
A cytokinin-repressed gene in cucumber for a bHLH protein homologue is regulated by light.Crossref | GoogleScholarGoogle Scholar |

Tsuda T, Horio F, Uchida K, Aoki H, Osawa T (2003) Dietary cyanidin 3-O-beta-D-glucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice. Journal of Nutrition 133, 2125–2130.
Dietary cyanidin 3-O-beta-D-glucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice.Crossref | GoogleScholarGoogle Scholar |

Tuan PA, Bai S, Yaegaki H, Tamura T, Hihara S, Moriguchi T, Oda K (2015) The crucial role of PpMYB10.1 in anthocyanin accumulation in peach and relationships between its allelic type and skin color phenotype. BMC Plant Biology 15, 280–294.
The crucial role of PpMYB10.1 in anthocyanin accumulation in peach and relationships between its allelic type and skin color phenotype.Crossref | GoogleScholarGoogle Scholar |

Wang N, Zhang Z, Jiang S, Xu H, Wang Y, Feng S, Chen XS (2016) Synergistic effects of light and temperature on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell, Tissue and Organ Culture 127, 217–227.
Synergistic effects of light and temperature on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana).Crossref | GoogleScholarGoogle Scholar |

Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology 126, 485–493.
Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology.Crossref | GoogleScholarGoogle Scholar |

Xie XB, Shen LI, Zhang RF, Zhao J, Chen YC, Zhao Q, Yao YX, You CX, Zhang XS, Hao YJ (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.
The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples.Crossref | GoogleScholarGoogle Scholar |

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, e65680–e65688.
The heterologous expression of the chrysanthemum R2R3-MYB transcription factor CmMYB1 alters lignin composition and represses flavonoid synthesis in Arabidopsis thaliana.Crossref | GoogleScholarGoogle Scholar |

Zimmermann IM, Heim MA, Weisshaar B, Uhrig JF (2004) Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like bHLH proteins. The Plant Journal 40, 22–34.
Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like bHLH proteins.Crossref | GoogleScholarGoogle Scholar |