VabHLH137 promotes proanthocyanidin and anthocyanin biosynthesis and enhances resistance to Colletotrichum gloeosporioides in grapevine

Abstract

Proanthocyanidins (PAs) and anthocyanins are involved in the response of plants to various environmental stresses. However, the mechanism behind defense-induced PA biosynthetic regulation is still not completely elucidated, also in grapevine. This study performed a transcriptome sequencing analysis of grape berries infected with Colletotrichum gloeosporioides to highlight the induction of the VabHLH137 factor from the basic helix–loop–helix (bHLH) XII subfamily by the fungus, which appeared to be significantly co-expressed with PA-related genes. The functional analysis of VabHLH137 overexpression and knockdown in transgenic grape calli showed that it positively regulated PA and anthocyanin biosynthesis. Moreover, VabHLH137 overexpression in the grape calli significantly increased resistance to C. gloeosporioides. A yeast one-hybrid and electrophoretic mobility shift assay revealed that VabHLH137 directly bound to the VaLAR2 promoter, enhancing its activity and interacting with VaMYBPAR, a transcriptional activator of PA biosynthesis. Furthermore, transient experiments showed that although the VabHLH137 + VaMYBPAR complex activated VaLAR2 expression, it failed to further enhance VaLAR2 expression compared to VaMYBPAR alone. The findings indicated that VabHLH137 enhanced PA biosynthesis by activating of VaLAR2 expression, providing new insight into the transcriptional regulation of defense-induced PA biosynthesis in grapevine.

Acknowledgements

We thank Prof. Daqi Fu, Prof. Benzhong Zhu, Prof. Junping Gao (China Agricultural University) and Prof. Jianye Chen (South China Agricultural University) for providing vectors; and Dr Yi Wang (IBCAS) for help with transcriptome data analysis. This project was supported by the National Natural Science Foundation of China (No. 31171942 and No. 31471835).

Author Contributions

J.Z. conceived of the project, designed the experiments and supervised the research. D.Y. contributed to the experimental designs, performed experiments, analysed the results, and wrote the manuscript. W.W. and Z.F. performed EMSA and CoIP experiments. J.C. provided plasmids and experimental assistance. Y.Y. and W.H. discussed and reviewed the manuscript.

Data availability

The authors confirm that all data that are needed to replicate this study and to draw conclusions are within the paper.

Conflict of interests

The authors declare no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Winkel-Shirley B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 2001; 126: 485-93.

[2] Dixon RA, Xie DY, Sharma SB . Proanthocyanidins - a final frontier in flavonoid research? New Phytol. 2005; 165: 9-28.

[3] Hernandez I, Alegre L, Munne-Bosch S . Plant aging and excess light enhance flavan-3-ol content in Cistus clusii. J Plant Physiol. 2011; 168: 96-102.

[4] Wen PF, Ji W, Gao MY et al. Accumulation of flavanols and expression of leucoanthocyanidin reductase induced by postharvest UV-C irradiation in grape berry. Genet Mol Res. 2015; 14: 7687-95.

[5] Gourlay G, Constabel CP . Condensed tannins are inducible antioxidants and protect hybrid poplar against oxidative stress. Tree Physiol. 2019; 39: 345-55.

[6] Shen YX, Sun T, Pan Q et al. RrMYB5-and RrMYB10-regulated flavonoid biosynthesis plays a pivotal role in feedback loop responding to wounding and oxidation in Rosa rugosa. Plant Biotechnol J. 2019; 17: 2078-95.

[7] An JP, Li R, Qu FJ et al. R2R3-MYB transcription factor MdMYB23 is involved in the cold tolerance and proanthocyanidin accumulation in apple. Plant J. 2018; 96: 562-77.

[8] Yuan L, Wang L, Han Z et al. Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants . J Exp Bot. 2012; 63: 2513-24.

[9] Wang L, Ran L, Hou Y et al. The transcription factor MYB115 contributes to the regulation of proanthocyanidin biosynthesis and enhances fungal resistance in poplar. New Phytol 2017; 215: 351-67.

[10] Mellway RD, Tran LT, Prouse MB et al. The wound-, pathogen-, and ultraviolet B-responsive MYB134 gene encodes an R2R3 MYB transcription factor that regulates proanthocyanidin synthesis in poplar . Plant Physiol. 2009; 150: 924-41.

[11] Ullah C, Unsicker SB, Fellenberg C et al. Flavan-3-ols are an effective chemical defense against rust infection. Plant Physiol. 2017; 175: 1560-78.

[12] Lesschaeve I, Noble AC . Polyphenols: factors influencing their sensory properties and their effects on food and beverage preferences. Am J Clin Nutr. 2005; 81: 330S-5S.

[13] Gonzalo-Diago A, Dizy M, Fernandez-Zurbano P . Taste and mouthfeel properties of red wines proanthocyanidins and their relation to the chemical composition. J Agr Food Chem. 2013; 61: 8861-70.

[14] Smeriglio A, Barreca D, Bellocco E, Trombetta D . Proanthocyanidins and hydrolysable tannins: occurrence, dietary intake and pharmacological effects. Brit J Pharmacol. 2017; 174: 1244-62.

[15] Lepiniec L, Debeaujon I, Routaboul JM et al. Genetics and biochemistry of seed flavonoids. Annu Rev Plant Biol. 2006; 57: 405-30.

[16] Tohge T, de Souza LP, Fernie AR . Current understanding of the pathways of flavonoid biosynthesis in model and crop plants. J Exp Bot. 2017; 68: 4013-28.

[17] Almeida JRM, D’Amico E, Preuss A et al. Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria × ananassa). Arch Biochem Biophys. 2007; 465: 61-71.

[18] Pang YZ, Peel GJ, Wright E et al. Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula. Plant Physiol. 2007; 145: 601-15.

[19] Boss PK, Davies C, Robinson SP . Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv shiraz grape berries and the implications for pathway regulation . Plant Physiol. 1996; 111: 1059-66.

[20] Bogs J, Downey MO, Harvey JS et al. Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiol. 2005; 139: 652-63.

[21] Yu KJ, Jun JH, Duan CQ, Dixon RA . VvLAR1 and VvLAR2 are bifunctional enzymes for proanthocyanidin biosynthesis in grapevine. Plant Physiol. 2019; 180: 1362-74.

[22] Poudel PR, Koyama K, Goto-Yamamoto N . Evaluating the influence of temperature on proanthocyanidin biosynthesis in developing grape berries (Vitis vinifera L.). Mol Biol Rep. 2020; 47: 3501-10.

[23] Winkel-Shirley B. Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Physiol Plantarum. 1999; 107: 142-9.

[24] Cheng J, Shi Y, Wang J et al. Transcription factor VvibHLH93 negatively regulates proanthocyanidin biosynthesis in grapevine. Front Plant Sci. 2022; 13: 1007895.

[25] Li XJ, Cao L, Jiao B et al. The bHLH transcription factor AcB2 regulates anthocyanin biosynthesis in onion (Allium cepa L.). Hortic Res. 2022; 9: 128.

[26] Fan YX, Peng J, Wu J et al. NtbHLH1, a JAF13-like bHLH, interacts with NtMYB6 to enhance proanthocyanidin accumulation in Chinese narcissus. BMC Plant Biol 2021; 21: 275.

[27] Zhao R, Song X, Yang N et al. Expression of the subgroup IIIf bHLH transcription factor CpbHLH1 from Chimonanthus praecox (L.) in transgenic model plants inhibits anthocyanin accumulation . Plant Cell Rep. 2020; 39: 891-907.

[28] Zhao PC, Li X, Jia J et al. bHLH92 from sheepgrass acts as a negative regulator of anthocyanin/proanthocyandin accumulation and influences seed dormancy . J Exp Bot. 2019; 70: 1989-9.

[29] Li M, Sun L, Gu H et al. Genome-wide characterization and analysis of bHLH transcription factors related to anthocyanin biosynthesis in spine grapes (Vitis davidii). Sci Rep. 2021; 11: 6863.

[30] Bailey PC, Martin C, Toledo-Ortiz G et al. Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana. Plant Cell. 2003; 15: 2497-502.

[31] Pires N, Dolan L . Origin and diversification of basic-helix-loop-helix proteins in plants. Mol Biol Evol. 2010; 27: 862-74.

[32] Heim MA, Jakoby M, Werber M et al. The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol Biol Evol. 2003; 20: 735-47.

[33] Hichri I, Heppel SC, Pillet J et al. The basic helix-loop-helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine. Mol Plant. 2010; 3: 509-23.

[34] Matus JT, Poupin MJ, Cañón P et al. Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine (Vitis vinifera L.). Plant Mol Biol 2010; 72: 607-20.

[35] Liu JY, Osbourn A, Ma PD . MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Mol Plant. 2015; 8: 689-708.

[36] Ren YR, Yang YY, Zhao Q et al. MdCIB1, an apple bHLH transcription factor, plays a positive regulator in response to drought stress. Environ Exp Bot. 2021; 188: 104523.

[37] Wang N, Qu C, Jiang S et al. The proanthocyanidin-specific transcription factor MdMYBPA1 initiates anthocyanin synthesis under low-temperature conditions in red-fleshed apples. Plant J. 2018; 96: 39-55.

[38] Zou Q, Xu H, Yang G et al. MdbHLH106-like transcription factor enhances apple salt tolerance by upregulating MdNHX1 expression. Plant Cell Tiss Org. 2021; 145: 333-45.

[39] Yang YY, Zheng PF, Ren YR et al. Apple MdSAT1 encodes a bHLHm1 transcription factor involved in salinity and drought responses. Planta. 2021; 253: 46.

[40] Gao M, Zhu Y, Yang J et al. Identification of the grape basic helix-loop-helix transcription factor family and characterization of expression patterns in response to different stresses. Plant Growth Regul. 2019; 88: 19-39.

[41] Meng FW, Yang C, Cao J et al. A bHLH transcription activator regulates defense signaling by nucleo-cytosolic trafficking in rice. J Integr Plant Biol. 2020; 62: 1552-73.

[42] Cao Y, Liu L, Ma K et al. The Jasmonate-induced bHLH gene SlJIG functions in terpene biosynthesis and resistance to insects and fungus. J Integr Plant Biol. 2022; 64: 1102-15.

[43] O’Connell RJ, Thon MR, Hacquard S et al. Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses. Nat Genet. 2012; 44: 1060-5.

[44] Deng WP, Yang M, Du F et al. Identification of the pathogen causing grape anthracnose in Yunnan and its pathogenicity. Acta Phytophylacica Sinica. 2013; 40: 61-7.

[45] Wei LL, Zheng H, Zhang P et al. Molecular and biochemical characterization of Colletotrichum gloeosporioides isolates resistant to azoxystrobin from grape in China . Plant Pathol. 2021; 70: 1300-9.

[46] Liu LY, Li H . Review: research progress in Amur grape, Vitis amurensis Rupr. Can J Plant Sci. 2013; 93: 565-75.

[47] Feucht W, Treutter D . Flavan-3-Ols in trichomes, pistils and phelloderm of some tree species. Ann Bot-London. 1990; 65: 225-30.

[48] Ullah C, Tsai CJ, Unsicker SB et al. Salicylic acid activates poplar defense against the biotrophic rust fungus Melampsora larici-Populina via increased biosynthesis of catechin and proanthocyanidins . New Phytol. 2019; 221: 960-75.

[49] Aslam M, Jakada BH, Fakher B et al. Genome-wide study of pineapple (Ananas comosus L.) bHLH transcription factors indicates that cryptochrome-interacting bHLH2 (AcCIB2) participates in flowering time regulation and abiotic stress response. BMC Genomics. 2020; 21: 735.

[50] Wan R, Guo C, Hou X et al. Comparative transcriptomic analysis highlights contrasting levels of resistance of Vitis vinifera and Vitis amurensis to Botrytis cinerea. Hortic Res. 2021; 8: 103.

[51] Gong PJ, Kang J, Sadeghnezhad E et al. Transcriptional profiling of resistant and susceptible cultivars of grapevine (Vitis L.) reveals hypersensitive responses to Plasmopara viticola. Front Microbiol. 2022; 13: 846504.

[52] Liu WW, Tai H, Li S et al. bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism . New Phytol. 2014; 201: 1192-204.

[53] Wang PF, Su L, Gao H et al. Genome-wide characterization of bHLH genes in grape and analysis of their potential relevance to abiotic stress tolerance and secondary metabolite biosynthesis. Front Plant Sci. 2018; 9: 64.

[54] Cheng Q, Dong L, Gao T et al. The bHLH transcription factor GmPIB1 facilitates resistance to Phytophthora sojae in Glycine max. J Exp Bot. 2018; 69: 2527-41.

[55] Li P, Chen BB, Zhang GY et al. Regulation of anthocyanin and proanthocyanidin biosynthesis by Medicago truncatula bHLH transcription factor MtTT8 . New Phytol. 2016; 210: 905-21.

[56] Su K, Guo Y, Zhao Y et al. Candidate genes for grape white rot resistance based on SMRT and Illumina sequencing. BMC Plant Biol. 2019; 19: 501.

[57] Wang YH, Liu S, Wang H et al. Identification of the regulatory genes of UV-B-induced anthocyanin biosynthesis in pepper fruit. Int J Mol Sci. 2022; 23: 1960.

[58] Constabel CP . Molecular controls of proanthocyanidin synthesis and structure: prospects for genetic engineering in crop plants. J Agr Food Chem. 2018; 66: 9882-8.

[59] Debeaujon I, Nesi N, Perez P et al. Proanthocyanidin-accumulating cells in Arabidopsis testa: regulation of differentiation and role in seed development. Plant Cell. 2003; 15: 2514-31.

[60] Baudry A, Heim MA, Dubreucq B et al. TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J. 2004; 39: 366-80.

[61] Lin YL, Fan L, He J et al. Anthocyanins contribute to fruit defense against postharvest green mold. Postharvest Biol Tec. 2021; 181: 111661.

[62] James AM, Ma D, Mellway R et al. Poplar MYB115 and MYB134 transcription factors regulate proanthocyanidin synthesis and structure. Plant Physiol. 2017; 174: 154-71.

[63] Coombe BG . Growth stages of the grapevine: adoption of a system for identifying grapevine growth stages. Aust J Grape & Wine Res. 1995; 1: 104-10.

[64] Wang Y, Xin H, Fan P et al. The genome of Shanputao (Vitis amurensis) provides a new insight into cold tolerance of grapevine . Plant J. 2021; 105: 1495-506.

[65] Chen CJ, Chen H, Zhang Y et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020; 13: 1194-202.

[66] Hellemans J, Mortier G, De Paepe A et al. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007; 8: R19.

[67] Liu JT, Fan Y, Zou J et al. A RhABF2/ferritin module affects rose (Rosa hybrida) petal dehydration tolerance and senescence by modulating iron levels . Plant J. 2017; 92: 1157-69.

[68] Han YC, Kuang JF, Chen JY et al. Banana transcription factor MaERF11 recruits histone deacetylase MaHDA1 and represses the expression of MaACO1 and expansins during fruit ripening . Plant Physiol. 2016; 171: 1070-84.

[69] An XH, Tian Y, Chen KQ et al. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant Cell Physiol. 2015; 56: 650-62.

[70] Shan W, Chen JY, Kuang JF, Lu WJ . Banana fruit NAC transcription factor MaNAC5 cooperates with MaWRKYs to enhance the expression of pathogenesis-related genes against Colletotrichum musae. Mol Plant Pathol. 2016; 17: 330-8.