MdMYB6 regulates anthocyanin formation in apple both through direct inhibition of the biosynthesis pathway and through substrate removal

Abstract

Anthocyanin biosynthesis and sugar metabolism are important processes during plant growth, but the molecular interactions underlying these pathways are still unclear. In this work, we analyzed the anthocyanin and soluble sugar contents, as well as the transcript levels of transcription factors that are known to be related to the biosynthesis of anthocyanin in ‘Hongcui 1’ apple flesh during fruit development. Overexpression of MdMYB6 in red-fleshed calli was found to reduce anthocyanin content and result in downregulated expression of the MdANS and MdGSTF12 proteins. Yeast one-hybrid and electrophoretic mobility shift analyses showed that MdMYB6 could directly bind to the promoters of MdANS and MdGSTF12, indicating that MdMYB6 could inhibit anthocyanin biosynthesis by regulating MdANS and MdGSTF12. Overexpression of MdTMT1 in the Arabidopsis tmt1 mutant restored the glucose and fructose contents to the wild-type levels, while overexpression of MdTMT1 in red-fleshed calli increased the contents of glucose and fructose but reduced the contents of UDP-glucose, UDP-galactose, and anthocyanin. Using a GUS reporter system, yeast one-hybrid, chromatin immunoprecipitation-PCR and electrophoretic mobility shift analyses, we found that MdMYB6 could bind to the promoter of MdTMT1, resulting in increased promoter activity. Overexpression of MdMYB6 in calli overexpressing MdTMT1 increased the expression of MdTMT1, which led to reduced contents of UDP-glucose and UDP-galactose and decreased anthocyanin content compared to those of the calli that overexpressed MdTMT1. This finding suggested that MdMYB6 could also inhibit anthocyanin biosynthesis by regulating MdTMT1 to decrease the contents of UDP-glucose and UDP-galactose. Taken together, these results showed that MdMYB6 and MdTMT1 play key roles in both anthocyanin biosynthesis and sugar transport.

References

[1] Zhang, X. Y., Zhang, S. L., Luo, J., Ye, Z. W. & Li, S. C. Advances in research on fruit anthocyanin synthesis. J. Fruit Sci. 21, 456-460 (2004).

[2] Zhang, W. S. et al. Bioactive components and antioxidant capacity of Chinese bayberry (Myrica rubra Sieb. and Zucc.) fruit in relation to fruit maturity and postharvest storage. Eur. Food Res. Technol. 227, 1091-1097 (2008).

[3] Sun, C. D., Huang, H. Z., Xu, C. J., Li, X. & Chen, K. S. Biological activities of extracts from Chinese bayberry (Myrica rubra Sieb. et Zucc.): A review. Plant Foods Hum. Nutr. 68, 97-106 (2013).

[4] Kondo, S., Hiraoka, K., Kobayashi, S., Honda, C. & Terahara, N. Changes in the expression of anthocyanin biosynthetic genes during apple development. J. Am. Soc. Hortic. Sci. 127, 971-976 (2002).

[5] Kim, S. Y., Lee, J. R. & Kim, S. R. Characterization of an Apple anthocyanidin synthase gene in transgenic Tobacco. J. Plant Biol. 49, 326-330 (2006).

[6] Baudry, A. et al. TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J. 39, 366-380 (2004).

[7] Ballester, A. R. et al. Biochemical molecular analysis of pink tomatoes, deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit color. Plant Physiol. 152, 71-84 (2010).

[8] Albert, N. W. et al. Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning. Plant J. 65, 771-784 (2011).

[9] 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 protein to regulate anthocyanin accumulation. Plant Physiol. 169, 710-717 (2012).

[10] Azuma, A. et al. Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape berry skin. Theor. Appl. Genet 117, 1009-1019 (2008).

[11] Wang, L. K. et al. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biol. 10, 50 (2010).

[12] Feng, S. Q., Wang, Y. L., Song, Y., Xu, Y. T. & Chen, X. S. Anthocyanin bio-synthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10. Planta 232, 245-255 (2010).

[13] Espley, R. V. et al. Red colouration in apple fruit is due to the activity of the MYB transcription factor MdMYB10. Plant J. 49, 414-427 (2007).

[14] Espley, R. V. et al. Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. Plant Cell 21, 168-183 (2009).

[15] Chagné, D., Lin-Wang, K. & Espley, R. V. et al. An Ancient Duplication of Apple MYB Transcription Factors Is Responsible for Novel RedFruit-Flesh Phenotypes. Plant Physiol. 161, 225-239 (2013).

[16] Aharoni, A. et al. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J. 28, 319-332 (2001).

[17] Albert, N. W. et al. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. Plant Cell 26, 962-980 (2014).

[18] Gao, J. J. et al. The myb transcription factor MdMYB6 suppresses anthocyanin biosynthesis in transgenic Arabidopsis. Plant Cell Tiss. Organ Cult. 106, 235-242 (2011).

[19] Xu, H. F. et al. The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes. Plant Mol. Biol. 94, 149-165 (2017).

[20] Koes, R., Verweij, W. & Quattrocchio, F. Flavonoids, a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci. 10, 236-242 (2005).

[21] Solfanelli, C., Bartolini, S., Vitagliano, C. & Lorenzi, R. Immunolocalization and quantification of IAA after self- and freepollination in Olea europaea L. Sci. Hortic. 110, 345-351 (2006).

[22] Xie, X. B. et al. The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apple. Plant Cell Environ. 35, 1884-1897 (2012).

[23] Bai, S. L. et al. An apple B-box protein, MdCOL11, is involved in UV-Band temperature-induced anthocyanin biosynthesis. Planta 240, 1051-1062 (2014).

[24] Fang H. C., et al. The B-box zinc finger protein MdBBX20 integrates antho-cyanin accumulation in response to ultraviolet radiation and low temperature. Plant Cell Environ. 42, 2090-2092 (2019).

[25] An, X. H. et al. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant Cell Physiol. 56, 650-662 (2014).

[26] Sun, Y., Li, H. & Huang, J. R. Arabidopsis TT19 functions as a carrier to transport anthocyanin from the cytosol to tonoplasts. Mol. Plant 5, 387-400 (2012).

[27] Jiang, S. H. et al. MdGSTF6, activated by MdMYB1, plays an essential role in anthocyanin accumulation in apple. Horticulture Res. 6, 40 (2019).

[28] Ruan, Y. L. Sucrose metabolism: gateway to diverse carbon use and sugar signaling. Annu. Rev. Plant Biol. 65, 33-67 (2014).

[29] Rae, A., Perroux, J. M. & Grof, C. P. L. Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: apotential role for the ShSUT1 sucrose transporter. Planta 220, 817-825 (2005).

[30] Sauer, N. Molecular physiology of higher plant sucrose transporters. FEBS Lett. 581, 2309-2317 (2007).

[31] Kühn, C. in Transporters and Pumps in Plant Signaling (eds Geisler, M. & Venema, K.) (Springer-Verlag, Berlin, Heidelberg, 2011) 225-251.

[32] Geiger, D. Plant sucrose transporters from a biophysical point of view. Mol. Plant 4, 395-40 (2011).

[33] Enlder, A. et al. Identification of a Vacuolar Sucrose Transporter in Barley and Arabidopsis Mesophyll Cells by a Tonoplast Proteomic Approach. Plant Physiol. 141, 196-207 (2006).

[34] Wormit, A. et al. Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport. Plant Cell 18, 3476-3490 (2006).

[35] Yamada, K. et al. Monosaccharide absorption activity of Arabidopsis roots depends on expression profiles of transporter genes under high salinity conditions. J. Biol. Chem. 286, 43577-43586 (2011).

[36] Poschet, G. et al. A novel Arabidopsis vacuolar glucose exporter is involved in cellular sugar homeostasis and affects the composition of seed storage compounds. Plant Physiol. 157, 1664-1676 (2011).

[37] Wingenter, K. et al. Increased activity of the vacuolar monosaccharide trans-porter TMT1 alters cellular sugar partitioning, sugar signaling, and seed yield in Arabidopsis. Plant Physiol. 154, 665-677 (2010).

[38] Schulze, W. X., Schneider, T., Starck, S., Martinoia, E. & Trentmann, O. Cold acclimation induces changes in Arabidopsis tonoplast protein abundance and activity and alters phosphorylation of tonoplast monosaccharide transporters. Plant J. 69, 529-541 (2012).

[39] Weiss, D. Regulation of flower pigmentation and growth: multiple signaling pathways control anthocyanin synthesis in expanding petals. Physiol. Plant 110, 152-157 (2000).

[40] Zheng, Y. J. et al. Sugars induce anthocyanin accumulation and flavanone 3-hydroxylase expression in grape berries. Plant Growth Regul. 58, 251-260 (2009).

[41] Sivitz, A. B., Reinders, A. & Ward, J. M. Arabidopsis Sucrose Transporter AtSUC1 Is Important for Pollen Germination and Sucrose-Induced Anthocyanin Accumulation. Plant Physiol. 147, 92-100 (2008).

[42] Ban, Y. S. et al. UDP-sugar biosynthetic pathway: contribution to cyanidin 3-galactoside biosynthesis in apple skin. Planta 230, 871-881 (2009).

[43] Song, Z., Li, T. Z. & Xu, G. X. Studies on the relationship among the antho-cyanin sugar and related enzymes activity during the coloring stage of’F-uji’apple. Chin. Agric. Sci. Bull. 24, 255-260 (2008). (in Chinese).

[44] Sainz, M. B., Grotewold, E. & Chandler, V. L. Evidence for direct activation of an anthocyanin promoter by the maize C1 protein and comparison of DNA binding by related Myb domain proteins. Plant Cell 9, 611-625 (1997).

[45] Solano, R. N. C., Avila, J., Canas, L., Diaz, I. & Paz-Ares, J. Dual DNA binding specificity of a petal epidermis-specific MYB transcription factor (MYB.Ph3) from Petunia hybrida. EMBO J. 14, 1773-1784 (1995).

[46] Hartmann, U., Sagasser, M., Mehrtens, F., Stracke, R. & Weisshaar, B. Differential combinatorial interactions of cis-acting elements recognized by R2R3-MYB, BZIP, and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes. Plant Mol. Biol. 57, 155-171 (2005).

[47] Wang, N. et al. The proanthocyanidin-specific transcription factor MdMYBPA1 initiates anthocyanin synthesis under low-temperature conditions in red-fleshed apples. Plant J. 96, 39-55 (2018).

[48] Wang, K. L. et al. High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex. Plant Cell & Environment 34, 1176-1190 (2011).

[49] Colquhoun, T. A. et al. PhMYB4 fine-tunes the floral volatile signature of Petunia x hybrida through PhC4H. J. Exp. Bot. 62, 1133-1143 (2011).

[50] Cavallini, E. et al. The phenylpropanoid pathway is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine. Plant Physiol. 167, 1448-1470 (2015).

[51] Paolocci, F. et al. The strawberry transcription factor FaMYB1 inhibits the biosynthesis of proanthocyanidins in Lotus corniculatus leaves. J. Exp. Bot. 62, 1189-1200 (2011).

[52] Xu, H. F. et al. Overexpression of a repressor MdMYB15L negatively regulates anthocyanin and cold tolerance in red-fleshed callus. Biochem. Biophys. Res. Commun. 500, 405-410 (2018).

[53] Jin, H. L. et al. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J. 19, 6150-6161 (2000).

[54] Hu, B. et al. LcGST4 is an anthocyanin-related glutathione S-transferase gene in Litchi chinensis Sonn. Plant Cell Rep. 35, 831-843 (2016).

[55] Martinoia, E., Meyer, S., De Angeli, A. & Nagy, R. Vacuolar transporters in their physiological context. Annu. Rev. Plant Biol. 63, 183-213 (2012).

[56] Schulz, A. et al. Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2. Plant J. 68, 129-136 (2011).

[57] Afoufa-Bastien, D. et al. The Vitis vinifera sugar transporter gene family: Phy-logenetic overview and macroarray expression profiling. BMC Plant Biol. 10, 22 (2010).

[58] Vogt, T. & Jones, P. Glycosyltransferases in plant natural product synthesis: characterization of a supergene family. Trends Plant Sci. 5, 1360-1385 (2000).

[59] Ji, X. H. et al. Effect of auxin, cytokinin and nitrogen on anthocyanin bio-synthesis in callus cultures of red-fleshed apple (Malus sieversii f.niedzwetz-kyana). Plant Cell Tiss. Organ Cult. 120, 325-337 (2015).

[60] Kenneth, J. L. & Thomas, D. S. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method . Methods 25, 402-408 (2001).

[61] Zhao, Z. Z., Zhang, S. L., Xu, C. J., Chen, K. S. & Liu, S. T. Roles of activities of sucrose-metabolizing enzymes in accumulation of sugars in satsuma man-darin fruit. Acta Hortic. Sin. 28, 112-118 (2001). (in Chinese).

[62] Wang, N. et al. MYB12 and MYB22 play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant J. 90, 276-292 (2017).

[63] Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. GUS fusions, beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907 (1987).

[64] Bradford, M. M. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976).

[65] He, J. X. et al. BZR1 is a transcriptional repressor with dual roles in brassi-nosteroid homeostasis and growth responses. Science 307, 1634-1638 (2005).