AcGLK2 involves anthocyanin biosynthesis via bidirectionally modulating expression of MYB transcription factors in Actinidia chinensis

Abstract

The GOLDEN2-LIKE (GLK) gene family, known for its role in chloroplast development, has recently been implicated in involvement of anthocyanin biosynthesis in kiwifruit (Actinidia spp.), but the underlying regulatory mechanism remains unclear. Here we report the characterization of a kiwifruit GLK homolog AcGLK2 in regulating anthocyanin accumulation. We found that expression of AcGLK2 is much higher exclusively in the red pigment-accumulated fruit tissue. Overexpression of AcGLK2 in Arabidopsis and kiwifruit significantly enhanced anthocyanin content, whereas its RNAi-mediated silencing compromised anthocyanin accumulation. RNA-Seq analysis revealed significant upregulation of many structural genes and transcription factors (TFs) associated with the flavonoid pathway in AcGLK2-overexpressing kiwifruit. ChIP-Seq analysis indicated that AcGLK2 directly binds to AcMYB5 and AcTRY. We showed AcMYB5, an R2R3-MYB TF, promotes anthocyanin accumulation by interacting with the bHLH protein AcbHLH42, while AcTRY, an R3-MYB protein, competitively inhibits the interaction between key MYBs and AcbHLH42, thereby repressing anthocyanin biosynthesis. Transgenic and molecular assays in tobacco, tomato, and kiwifruit demonstrated that AcGLK2 positively participates into regulation of anthocyanin accumulation through transcriptionally activating AcMYB5 expression and concomitantly suppressing AcTRY expression. This study reveals the dual regulatory mechanism of AcGLK2 in anthocyanin biosynthesis, broadening the understanding of GLK gene functions and providing a valuable genetic resource for molecular breeding of nutritional quality in kiwifruit and other crops.

References

[1] Fitter DW, Martin DJ, Copley MJ, et al. GLK gene pairs regulate chloroplast development in diverse plant species. Plant J. 2002; 31: 713-27

[2] Waters MT, Moylan EC, Langdale JA . GLK transcription factors regulate chloroplast development in a cell-autonomous manner. Plant J. 2008; 56: 432-44

[3] Wang P, Fouracre J, Kelly S, et al. Evolution of GOLDEN2-LIKE gene function in C3 and C4 plants. Planta. 2013; 237: 481-95

[4] Riechmann JL, Heard J, Martin G, et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science. 2000; 290: 2105-10

[5] Nagatoshi Y, Mitsuda N, Hayashi M, et al. GOLDEN 2-LIKE transcription factors for chloroplast development affect ozone tolerance through the regulation of stomatal movement. Proc Natl Acad Sci USA. 2016; 113: 4218-23

[6] Zeng X, Ye L, Zhang R, et al. GLK2, a GOLDEN2-LIKE transcription factor, directly regulates anthocyanin accumulation by binding with promoters of key anthocyanin biosynthetic genes in Arabidopsis. Plant Cell Environ. 2025; 48: 7055-71

[7] Liu D, Zhao D, Li X, et al. AtGLK2, an Arabidopsis GOLDEN2-LIKE transcription factor, positively regulates anthocyanin biosynthesis via AtHY5-mediated light signaling. Plant Growth Regul. 2022; 96: 79-90

[8] Li Y, Lei W, Zhou Z, et al. Transcription factor GLK1 promotes anthocyanin biosynthesis via an MBW complex-dependent pathway in Arabidopsis thaliana. J Integr Plant Biol. 2023; 65: 1521-35

[9] Nakabayashi R, Yonekura-Sakakibara K, Urano K, et al. Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids. Plant J. 2014; 77: 367-79

[10] Chalker-Scott L . Environmental significance of anthocyanins in plant stress responses. Photochem Photobiol. 1999; 70: 1-9

[11] Flavonoid W-SB, Biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 2001; 126: 485-93

[12] Feng X, Gao G, Yu C, et al. Transcriptome and metabolome analysis reveals anthocyanin biosynthesis pathway associated with ramie (Boehmeria nivea (L.) Gaud.) leaf color formation. BMC Genomics. 2021; 22: 684

[13] Dubos C, le Gourrierec J, Baudry A, et al. MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. Plant J. 2008; 55: 940-53

[14] Gonzalez A, Zhao M, Leavitt JM, et al. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 2008; 53: 814-27

[15] Henry-Kirk RA, Plunkett B, Hall M, et al. Solar UV light regulates flavonoid metabolism in apple (Malus x domestica). Plant Cell Environ. 2018; 41: 675-88

[16] Tao R, Bai S, Ni J, et al. The blue light signal transduction pathway is involved in anthocyanin accumulation in ‘red Zaosu’ pear. Planta. 2018; 248: 37-48

[17] Bursch K, Toledo-Ortiz G, Pireyre M, et al. Identification of BBX proteins as rate-limiting cofactors of HY5. Nat Plants. 2020; 6: 921-8

[18] Wang Y, Wang Y, Song Z, et al. Repression of MYBL2 by both microRNA858a and HY5 leads to the activation of anthocyanin biosynthetic pathway in Arabidopsis. Mol Plant. 2016; 9: 1395-405

[19] Li Y-Y, Mao K, Zhao C, et al. MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple. Plant Physiol. 2012; 160: 1011-22

[20] He M, Kong X, Jiang Y, et al. MicroRNAs: emerging regulators in horticultural crops. Trends Plant Sci. 2022; 27: 936-51

[21] Gou J-Y, Felippes FF, Liu C-J, et al. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell Online. 2011; 23: 1512-22

[22] Tirumalai V, Swetha C, Nair A, et al. MiR828 and miR858 regulate VvMYB114 to promote anthocyanin and flavonol accumulation in grapes. J Exp Bot. 2019; 70: 4775-92

[23] Cong L, Qu Y, Sha G, et al. PbWRKY75 promotes anthocyanin synthesis by activating PbDFR, PbUFGT, and PbMYB10b in pear. Physiol Plant. 2021; 173: 1841-9

[24] Martín-Pizarro C, Vallarino JG, Osorio S, et al. The NAC transcription factor FaRIF controls fruit ripening in strawberry. Plant Cell. 2021; 33: 1574-93

[25] Zhang Z, Shi Y, Ma Y, et al. The strawberry transcription factor FaRAV1 positively regulates anthocyanin accumulation by activation of FaMYB10 and anthocyanin pathway genes. Plant Biotechnol J. 2020; 18: 2267-79

[26] Amato A, Cavallini E, Walker AR, et al. The MYB5-driven MBW complex recruits a WRKY factor to enhance the expression of targets involved in vacuolar hyper-acidification and trafficking in grapevine. Plant J. 2019; 99: 1220-41

[27] Yasumura Y, Moylan EC, Langdale JA . A conserved transcription factor mediates nuclear control of organelle biogenesis in anciently diverged land plants. Plant Cell. 2005; 17: 1894-907

[28] Kobayashi K, Baba S, Obayashi T, et al. Regulation of root greening by light and auxin/cytokinin signaling in Arabidopsis. Plant Cell. 2012; 24: 1081-95

[29] Wang L, Tang X, Zhang S, et al. Tea GOLDEN2-LIKE genes enhance catechin biosynthesis through activating R2R3-MYB transcription factor. Hortic Res. 2022; 9: uhac117

[30] 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: e108849

[31] Li G, Chen D, Tang X, et al. Heterologous expression of kiwifruit (Actinidia chinensis) GOLDEN2-LIKE homolog elevates chloroplast level and nutritional quality in tomato (Solanum lycopersicum). Planta. 2018; 247: 1351-62

[32] Fang X, Zhao L, Li J, et al. AcGLK1 promotes chloroplast division through regulating AcFtsZ1 in Actinidia chinensis. Planta. 2025; 261: 17

[33] Li Y, Song Z, Zhan X, et al. Chromosome-level genome assembly assisting for dissecting mechanism of anthocyanin regulation in kiwifruit (Actinidia arguta). Mol Hortic. 2025; 5: 18

[34] Pilkington SM, Crowhurst R, Hilario E, et al. A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants. BMC Genomics. 2018; 19: 257

[35] Yue J, Chen Q, Wang Y, et al. Telomere-to-telomere and gap-free reference genome assembly of the kiwifruit Actinidia chinensis. Hortic Res. 2023; 10: uhac264

[36] Faiz MU, Liu X, Sun J, et al. Kiwifruit genomics and applications: recent advances, current challenges and future prospects. Hortic Res. 2026; 10: uhag024

[37] Wang L, Tang W, Hu Y, et al. A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang. Plant J. 2019; 99: 359-78

[38] Li S, Wang W, Gao J, et al. MYB75 phosphorylation by MPK4 is required for light-induced anthocyanin accumulation in Arabidopsis. Plant Cell. 2016; 28: 2866-83

[39] Xu W, Dubos C, Lepiniec L . Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci. 2015; 20: 176-85

[40] Wang XC, Wu J, Guan ML, et al. Arabidopsis MYB4 plays dual roles in flavonoid biosynthesis. Plant J. 2020; 101: 637-52

[41] Zhang D, Tan W, Yang F, et al. A BIN2-GLK1 signaling module integrates brassinosteroid and light signaling to repress chloroplast development in the dark. Dev Cell. 2021; 56: 310-324.e7

[42] Waters MT, Wang P, Korkaric M, et al. GLK transcription factors coordinate expression of the photosynthetic apparatus in Arabidopsis. Plant Cell. 2009; 21: 1109-28

[43] Li Y, Shan X, Zhou L, et al. The R2R3-MYB factor FhMYB5 from Freesia hybrida contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis. Front Plant Sci. 2019; 9: 1935

[44] Jiang L, Yue M, Liu Y, et al. A novel R2R3-MYB transcription factor FaMYB5 positively regulates anthocyanin and proanthocyanidin biosynthesis in cultivated strawberries (Fragaria × ananassa). Plant Biotechnol J. 2023; 21: 1140-58

[45] Yang K, Hou Y, Wu M, et al. DoMYB5 and DobHLH24, transcription factors involved in regulating anthocyanin accumulation in Dendrobium officinale. Int J Mol Sci. 2023; 24: 7552

[46] Deluc L, Bogs J, Walker AR, et al. The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries. Plant Physiol. 2008; 147: 2041-53

[47] Kirik V, Simon M, Huelskamp M, et al. The ENHANCER of TRY and CPC1 gene acts redundantly with TRIPTYCHON and CAPRICE in trichome and root hair cell patterning in Arabidopsis. Dev Biol. 2004; 268: 506-13

[48] Wakamatsu J, Nagao K, Sumida Y, et al. Function of the TRY C-terminal region artificially fused with its homologous transcription factors inducing root hair differentiation in Arabidopsis. Biosci Biotechnol Biochem. 2021; 85: 1114-20

[49] Schellmann S, Schnittger A, Kirik V, et al. TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis. EMBO J. 2002; 21: 5036-46

[50] Powell ALT, Nguyen CV, Hill T, et al. Uniform ripening encodes a Golden 2-like transcription factor regulating tomato fruit chloroplast development. Science. 2012; 336: 1711-5

[51] Wang P, Khoshravesh R, Karki S, et al. Re-creation of a key step in the evolutionary switch from C3 to C4 leaf anatomy. Curr Biol. 2017; 27: 3278-3287.e6

[52] Nguyen CV, Vrebalov JT, Gapper NE, et al. Tomato GOLDEN2-LIKE transcription factors reveal molecular gradients that function during fruit development and ripening. Plant Cell. 2014; 26: 585-601

[53] Savitch LV, Subramaniam R, Allard GC, et al. The GLK1 ‘regulon’ encodes disease defense related proteins and confers resistance to Fusarium graminearum in Arabidopsis. Biochem Biophys Res Commun. 2007; 359: 234-8

[54] Ali N, Chen H, Zhang C, et al. Ectopic expression of AhGLK1b (GOLDEN2-like transcription factor) in Arabidopsis confers dual resistance to fungal and bacterial pathogens. Genes (Basel). 2020; 11: 343

[55] Han XY, Li PX, Zou LJ, et al. GOLDEN2-LIKE transcription factors coordinate the tolerance to cucumber mosaic virus in Arabidopsis. Biochem Biophys Res Commun. 2016; 477: 626-32

[56] Liu X, Cheng X, Cao J, et al. GOLDEN 2-LIKE transcription factors regulate chlorophyll biosynthesis and flavonoid accumulation in response to UV-B in tea plants. Hortic Plant J. 2023; 9: 1055-66

[57] Li Y, Song Z, Zhan X, et al. Combination of 3D chromatin architecture and omics analysis provides insight into anthocyanin regulation in Actinidia arguta. Hortic Res. 2025; 12: uhaf183

[58] Wang Y, Li P, Zhu Y, et al. Graph-based pangenome of Actinidia chinensis reveals structural variations mediating fruit degreening. Adv Sci. 2024; 11: e2400322

[59] Han X, Zhang Y, Zhang Q, et al. Two haplotype-resolved, gap-free genome assemblies for Actinidia latifolia and Actinidia chinensis shed light on the regulatory mechanisms of vitamin C and sucrose metabolism in kiwifruit. Mol Plant. 2023; 16: 452-70

[60] Yu X, Chen B, Li J, et al. Genomic analyses reveal dead-end hybridization between two deeply divergent kiwifruit species rather than homoploid hybrid speciation. Plant J. 2023; 115: 1528-43

[61] Li X, Huo L, Li X, et al. Genomes of diverse Actinidia species provide insights into cis-regulatory motifs and genes associated with critical traits. BMC Biol. 2024; 22: 20

[62] Xu L, Miao T, Cui Z, et al. Synteny-based comparative pan-genome reveals a male-specific FT gene underlying flowering time dimorphism in kiwifruit. Plant J. 2026; 125: e70664

[63] Luo H, Dai C, Li Y, et al. Reduced anthocyanins in petioles codes for a GST anthocyanin transporter that is essential for the foliage and fruit coloration in strawberry. J Exp Bot. 2018; 69: 2595-608

[64] Chai Q, Wang X, Gao M, et al. A glutathione S-transferase GhTT19 determines flower petal pigmentation via regulating anthocyanin accumulation in cotton. Plant Biotechnol J. 2023; 21: 433-48

[65] Wu Z, Bian R, Zhang Z, et al. Transcription factors repressing anthocyanin biosynthesis in horticultural crops. Fruit Res. 2025; 5: e007

[66] Yanhui C, Xiaoyuan Y, Kun H, et al. The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol Biol. 2006; 60: 107-24

[67] Simon M, Lee MM, Lin Y, et al. Distinct and overlapping roles of single-repeat MYB genes in root epidermal patterning. Dev Biol. 2007; 311: 566-78

[68] Esch JJ, Chen MA, Hillestad M, et al. Comparison of TRY and the closely related At1g01380 gene in controlling Arabidopsis trichome patterning. Plant J. 2004; 40: 860-9

[69] Matsui K, Umemura Y, Ohme-Takagi M . AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. Plant J. 2008; 55: 954-67

[70] Zhu HF, Fitzsimmons K, Khandelwal A, et al. CPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis. Mol Plant. 2009; 2: 790-802

[71] Tominaga R, Iwata M, Okada K, et al. Functional analysis of the epidermal-specific MYB genes CAPRICE and WEREWOLF in Arabidopsis. Plant Cell. 2007; 19: 2264-77

[72] Ni J, Wang S, Yu W, et al. The ethylene-responsive transcription factor PpERF9 represses PpRAP2.4 and PpMYB114 via histone deacetylation to inhibit anthocyanin biosynthesis in pear. Plant Cell. 2023; 35: 2271-92

[73] Zhang B, Wang N, Chen X, et al. Effect of fruit debagging before harvest on the accumulation of anthocyanins in peach (Prunus persica L.) fruit based on transcriptome analysis. Sci Hortic (Amsterdam). 2023; 308: 111582

[74] Schmittgen TD, Livak KJ . Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008; 3: 1101-8

[75] Liu L, Qu J, Wang C, et al. An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion. Plant Biotechnol J. 2024; 22: 2093-103

[76] Aggarwal PR, Nag P, Choudhary P, et al. Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies. Plant Methods. 2018; 14: 55

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

[78] Fillatti JJ, Kiser J, Rose R, et al. Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. BioTechnology. 1987; 5: 726-30

[79] Han Y, Lu M, Yue S, et al. Comparative methylomics and chromatin accessibility analysis in Osmanthus fragrans uncovers regulation of genic transcription and mechanisms of key floral scent production. Hortic Res. 2022; 9: uhac096

[80] Yue J, Liu J, Tang W, et al. Kiwifruit genome database (KGD): a comprehensive resource for kiwifruit genomics. Hortic Res. 2020; 7: 117

[81] Li B, Li X, Wang Y, et al. KPGD: a kiwifruit pangenome database for comprehensive mining of genetic diversity in the genus Actinidia. Plant Commun. 2025; 6: 101373-6

[82] Ren W, Cao L, Liu L, et al. The transcription factor MYB78 negatively regulates cold stress tolerance in Actinidia chinensis. Plant Physiol. 2025; 199: kiaf586

[83] Liu F, Guo X, Yao Y, et al. Cloning and function characterization of two dehydroascorbate reductases from kiwifruit (Actinidia chinensis L.). Plant Mol Biol Report. 2016; 34: 815-26