The vacuolar transporters MaMATE11 and MaMATE14 affect blue flower coloration in grape hyacinth (Muscari)

Abstract

The development of blue flower coloration involves the biosynthesis, transport, and accumulation of flavonoids in petal epidermal cells. Although the mechanisms of flavonoid biosynthesis and regulation are well understood, much less is known about the molecular basis of vacuolar anthocyanin/flavonoid sequestration. Here, we identified two tonoplast-localized MATE transporters, MaMATE11 and MaMATE14, that participate in flavonoid transport and influence the blue color of grape hyacinth petals. In vitro transport experiments revealed that both proteins transported a range of flavonoid substrates, with a preference for malonylated anthocyanins, but differed in their substrate specificity and kinetic parameters. Both MaMATE11 and MaMATE14 could complement the anthocyanin-deficient phenotype of the Arabidopsis AtDTX35 mutant, and silencing of either gene by RNA interference significantly reduced anthocyanin accumulation in petals of grape hyacinth. Expression of MaMATE11 and MaMATE14 was directly activated by the anthocyanin-biosynthesis-related transcription factors MaMybA and MaAN2, respectively, establishing a coordinated anthocyanin synthesis-transport module. These findings provide insight into mechanisms of floral coloration and flavonoid translocation in blue-pigmented species and identify valuable target genes for molecular breeding of ornamental flower colors.

Acknowledgements

We sincerely thank Professor Y. Wang (Northwest A&F University) for providing the research platform. This study was supported by the National Natural Science Foundation of China (grant no. 32171863), Shaanxi Province Key Research and Development Program (grant no. 2024NC-YBXM-076), and the Fundamental Research Funds for the Central Universities (Z1090323181).

Authors contributions

L.D. conceived and designed the research. X.C., J.X., X.G., W.P., and J.G. conducted the experiments and analyzed the data. L.D. wrote the manuscript. L.D., X.C., and J.X. modified the manuscript. All authors read and approved the manuscript.

Data availability

All relevant data can be found within the manuscript and its supporting materials.

Conflicts of interest statement

The authors declare that they have no conflicts of interests.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Liu WX, Feng Y, Yu SH. et al. The flavonoid biosynthesis net-work in plants. Int J Mol Sci. 2021; 22:12824

[2] Pucker B, Selmar D. Biochemistry and molecular basis of intracellular flavonoid transport in plants. Plants. 2022; 11:963

[3] Wong DCJ, Wang ZM, Perkins J. et al. The road less taken: dihy-droflavonol 4-reductase inactivation and delphinidin antho-cyanin loss underpins a natural intraspecific flower colour variation. Mol Ecol. 2024; 34:e17334

[4] Francisco RM, Regalado A, Ageorges A. et al. ABCC1, an ATP binding cassette protein from grape berry, transports antho-cyanidin 3-O-glucosides. Plant Cell. 2013; 25:1840-54

[5] Grotewold E, Davies K. Trafficking and sequestration of antho-cyanins. Nat Prod Commun. 2008; 3:1251-8

[6] Gomez C, Terrier N, Torregrosa L. et al. Grapevine MATE-type proteins act as vacuolar H+-dependent acylated anthocyanin transporters. Plant Physiol. 2009; 150:402-15

[7] Marinova K, Pourcel L, Weder B. et al. The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+-antiporter active in proanthocyanidin-accumulating cells of the seed coat. Plant Cell. 2007; 19:2023-38

[8] Kaur S, Natasha S, Payal K. et al. Spotlight on the overlapping routes and partners for anthocyanin transport in plants. Phys-iol Plantarum. 2021; 171:868-81

[9] Wang R, Lu N, Liu C. et al. MtGSTF7, a TT19-like GST gene, is essential for accumulation of anthocyanins, but not proanthocyanins in Medicago truncatula. J Exp Bot. 2022; 73:4129-46

[10] Gani U, Vishwakarma RA, Misra P. Membrane transporters: the key drivers of transport of secondary metabolites in plants. Plant Cell Rep. 2021; 40:1-18

[11] Pourcel L, Irani NG, Lu YH. et al. The formation of anthocyanic vacuolar inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments. Mol Plant. 2010; 3:78-90

[12] Kulich I, Pecenková T, Sekeres J. et al. Arabidopsis exocyst subcomplex containing subunit EXO70B1 is involved in autophagy-related transport to the vacuole. Traffic. 2013; 14:1155-65

[13] Chanoca A, Kovinich N, Burkel B. et al. Anthocyanin vacuolar inclusions form by a microautophagy mechanism. Plant Cell. 2015; 27:2545-59

[14] Eichenberger M, Schwander T, Hüppi S. et al. The catalytic role of glutathione transferases in heterologous anthocyanin biosynthesis. Nat Catal. 2023; 6:927-38

[15] Gomez C, Conejero G, Torregrosa L. et al. In vivo grapevine anthocyanin transport involves vesicle-mediated trafficking and the contribution of anthoMATE transporters and GST. Plant J. 2011; 67:960-70

[16] Goodman CD, Casati P, Walbot V. A multidrug resistance-associated protein involved in anthocyanin transport in Zea mays. Plant Cell. 2004; 16:1812-26

[17] Behrens CE, Smith KE, Iancu CV. et al. Transport of antho-cyanins and other flavonoids by the Arabidopsis ATP-binding cassette transporter AtABCC2. Sci Rep. 2019; 9:437

[18] Zhao J, Huhman D, Shadle G. et al. MATE2 mediates vacuolar sequestration of flavonoid glycosides and glycoside malonates in Medicago truncatula. Plant Cell. 2011; 23: 1536-55

[19] Xu H, Yang PP, Cao YW. et al. Cloning and functional character-ization of a flavonoid transport-related MATE gene in Asiatic hybrid lilies (Lilium spp.). Genes. 2020; 11:418

[20] Yuan JW, Qiu ZJ, Long Y. et al. Functional identification of PhMATE1 in flower color formation in petunia. Physiol Plan-tarum. 2023; 175:e13949

[21] Zhao J, Dixon RA. MATE transporters facilitate vacuolar uptake of epicatechin 3’-O-glucoside for proanthocyanidin biosyn-thesis in Medicago truncatula and Arabidopsis. Plant Cell. 2010; 21:991

[22] Liu Y, Wu X, Sun CF. et al. Preferential transport activity of DkDTX5/MATE5 affects the formation of different astringency in persimmon. J Integr Plant Biol. 2023; 65:2304-19

[23] Gani U, Nautiyal AK, Kundan M. et al. Two homeologous MATE transporter genes, NtMATE21 and NtMATE22, are involved in the modulation of plant growth and flavonol transport in Nicotiana tabacum. J Exp Bot. 2022; 73:6186-206

[24] Ng MS, Ku YS, Yung WS. et al. MATE-type proteins are respon-sible for isoflavone transportation and accumulation in soy-bean seeds. Int J Mol Sci. 2021; 22:12017

[25] Lloyd A, Brockman A, Aguirre L. et al. 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; 58:1431-41

[26] Zhang H, Gong J, Chen K. et al. A novel R3 MYB transcriptional repressor, MaMYBx, finely regulates anthocyanin biosynthesis in grape hyacinth. Plant Sci. 2020; 298:110588

[27] Duan AQ, Deng YJ, Tan SS. et al. DcGST1, encoding a glu-tathione S-transferase activated by DcMYB7, is the main contributor to anthocyanin pigmentation in purple carrot. Plant J. 2023; 117:1069-83

[28] Chai QC, Wang XL, Gao MW. et al. A glutathione S-transferase GhTT19 determines flower petal pigmentation via regulat-ing anthocyanin accumulation in cotton. Plant Biotechnol J. 2023; 21:433-48

[29] Hu DG, Sun CH, Ma QJ. et al. MdMYB1 regulates antho-cyanin and malate accumulation by directly facilitating their transport into vacuoles in apples. Plant Physiol. 2016; 170: 1315-30

[30] Yoshida K, Mori M, Kondo T. Blue flower color development by anthocyanins: from chemical structure to cell physiology. Nat Prod Rep. 2009; 26:884-915

[31] Sasaki N, Nakayama T. Achievements and perspectives in biochemistry concerning anthocyanin modification for blue flower coloration. Plant Cell Physiol. 2015; 56:28-40

[32] Lou Q, Wang L, Liu HL. et al. Anthocyanin profiles in flowers of grape hyacinth. Molecules. 2017; 22:688

[33] Chen K, Du L, Liu H. et al. A novel R2R3-MYB from grape hyacinth, MaMybA, which is different from MaAN2, confers intense and magenta anthocyanin pigmentation in tobacco. BMC Plant Biol. 2019; 19:390

[34] Chen K, Liu H, Lou Q. et al. Ectopic expression of the grape hyacinth (Muscari armeniacum) R2R3-MYB transcrip-tion factor gene, MaAN2, induces anthocyanin accumulation in tobacco. Front Plant Sci. 2017; 8:965

[35] Ku YS, Cheng SS, Cheung MY. et al. The roles of Multidrug and Toxic Compound Extrusion (MATE) transporters in regulating agronomic traits. Agronomy. 2022; 12:878

[36] Nelson BK, Cai X, Nebenführ A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007; 51:1126-36

[37] Thompson EP, Wilkins C, Demidchik V. et al. An Arabidopsis flavonoid transporter is required for anther dehiscence and pollen development. J Exp Bot. 2010; 61:439-51

[38] Lou Q, Liu HL, Luo W. et al. Creating a novel petal regenera-tion system for function identification of colour gene of grape hyacinth. Plant Methods. 2021; 17:94

[39] Wang JY, Zhang H, Tian ST. et al. The R2R3MYB transcrip-tion factors MaMYBF and MaMYB1 regulate flavonoid biosyn-thesis in grape hyacinth. Plant Physiol Biochem. 2023; 194: 85-95

[40] Hui Z, Kui LW, Wang FR. et al. Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation. New Phy-tol. 2019; 221:1919-34

[41] Tanaka Y, Sasaki N, Ohmiya A. Biosynthesis of plant pig-ments: anthocyanins, betalains and carotenoids. Plant J. 2008; 54:733-49

[42] Matsuba Y, Sasaki N, Tera M. et al. A novel glucosylation reac-tion on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium. Plant Cell. 2010; 22:3374-89

[43] Nishizaki Y, Yasunaga M, Okamoto E. et al. p-Hydroxybenzoyl-glucose is a zwitter donor for the biosynthesis of 7-polyacylated anthocyanin in delphinium. Plant Cell. 2013; 25:4150-65

[44] Cao XY, Hao WH, Pan WQ. et al. A vacuolar protein MaSCPL1 mediates anthocyanin acylation modifications in blue-flowered grape hyacinth. Plant Sci. 2024; 349:112273

[45] Cao XY, Chen MK, Hao WH. et al. Two glycoside hydrolase family 1 proteins mediate glycosylated modification at the

[46] - position of anthocyanin in grape hyacinth. Int J Biol Macro-mol. 2025; 297:139813

[47] Kallam K, Appelhagen I, Luo J. et al. Aromatic decoration determines the formation of anthocyanic vacuolar inclusions. Curr Biol. 2017; 27:945-57

[48] Huang CB, Zhao T, Li JH. et al. Glutathione transferase VvGSTU60 is essential for proanthocyanidin accumulation and cooperates synergistically with MATE in grapes. Plant J. 2024; 121:e17197

[49] Mathews H, Clendennen SK, Caldwell CG. et al. Activation tag-ging in tomato identifies a transcriptional regulator of antho- cyanin biosynthesis, modification, and transport. Plant Cell. 2003; 15:1689-703

[50] Nakanishi Y, Saijo T, Wada Y. et al. Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase. J Biol Chem. 2001; 276:7654-60

[51] Clough SJ, Bent AF. Floral dip::a simplifed method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998; 16:735-43

[52] Li T, Jiang Z, Zhang L. et al. Apple (Malus domestica) MdERF2 negatively affects ethylene biosynthesis during fruit ripen-ing by suppressing MdACS1 transcription. Plant J. 2016; 88: 735-48