The WRKY transcription factor HpWRKY44 regulates CytP450-like1 expression in red pitaya fruit (Hylocereus polyrhizus)

Abstract

Red pitaya (Hylocereus polyrhizus) fruit is a high-value, functional food, containing a high level of betalains. Several genes potentially related to betalain biosynthesis, such as cytochrome P450-like (CytP450-like), have been identified in pitaya fruit, while their transcriptional regulation remains unclear. In this work, the potential involvement of a WRKY transcription factor, HpWRKY44, in regulating CytP450-like1 expression in pitaya fruit was examined. HpWRKY44, a member of the Group 1 WRKY family, contains two conserved WRKY motifs and is localized in the nucleus. HpWRKY44 also exhibits trans-activation ability. Gene expression analysis showed that the expression of HpCytP450-like1 and HpWRKY44 increased steadily during pitaya fruit coloration, which corresponded with the production of elevated betalain levels in the fruit. HpWRKY44 was also demonstrated to directly bind to and activate the HpCytP450-like1 promoter via the recognition of the W-box element present in the promoter. Collectively, our findings indicate that HpWRKY44 transcriptionally activates HpCytP450-like1, which perhaps, at least in part, contributes to betalain biosynthesis in pitaya fruit. The information provided in the current study provides novel insights into the regulatory network associated with betalain biosynthesis during pitaya fruit coloration.

References

[1] Gandía-Herrero F, García-Carmona F . Biosynthesis of betalains: yellow and violet plant pigments. Trends Plant Sci 2013; 18: 334-343.

[2] Khan MI, Giridhar P . Plant betalains: chemistry and biochemistry. Phytochem 2015; 117: 267-295.

[3] Gandía-Herrero F, Escribano J, García-Carmona F . Biological activities of plant pigments betalains. Crit Rev Food Sci Nutr 2016; 56: 937-945.

[4] Gandía-Herrero F, Escribano J, García-Carmona F . Purification and antiradical properties of the structural unit of betalains. J Nat Prod 2012; 75: 1030-1036.

[5] Stafford HA . Anthocyanins and betalains: evolution of the mutually exclusive pathways. Plant Sci 1994; 101: 91-98.

[6] Clifford T, Howatson G, West DJ, Stevenson EJ . The potential benefits of red beetroot supplementation in health and disease. Nutrients 2015; 7: 2801-2822.

[7] Azeredo HMC . Betalains: properties, sources, applications, and stability-a review. Int J Food Sci Technol 2009; 44: 2365-2376.

[8] Han XH, Gao ZJ, Xiao XG . Enzymes and genes involved in the betalain biosynthesis in higher plants. Afr J Biotechnol 2009; 8: 6735-6744.

[9] Hatlestad GJ, Sunnadeniya RM, Akhavan NA et al. The beet R locus encodes a new cytochrome P450 required for red betalain production. Nat Genet 2012; 44: 816-820.

[10] Hua QZ, Chen CJ, Chen Z et al. Transcriptomic analysis reveals key genes related to betalain biosynthesis in pulp coloration of Hylocereus polyrhizus. Front Plant Sci 2016; 6: 1179.

[11] DeLoache WC, Russ ZN, Narcross L, Gonzales AM, Martin VJ, Dueber JE . An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nat Chem Biol 2015; 11: 465-471.

[12] Polturak G, Breitel D, Grossman N et al. Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants. New Phytol 2016; 210: 269-283.

[13] Sunnadeniya R, Bean A, Brown M et al. Tyrosine hydroxylation in betalain pigment biosynthesis is performed by cytochrome P450 enzymes in beets (Beta vulgaris). PLoS ONE 2016; 11: e0149417.

[14] Hatlestad GJ, Akhavan NA, Sunnadeniya RM et al. The beet Y locus encodes an anthocyanin MYB-like protein that activates the betalain red pigment pathway. Nat Genet 2015; 47: 92-96.

[15] Ishiguro S, Nakamura K . Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5′ upstream regions of genes coding for sporamin and beta-amylase from sweet potato. Mol Gen Genet 1994; 244: 563-571.

[16] Rushton PJ, Somssich IE, Ringler P, Shen QJ . WRKY transcription factors. Trends Plant Sci 2010; 15: 247-258.

[17] Rushton DL, Tripathi P, Rabara RC et al. WRKY transcription factors: key components in abscisic acid signalling. Plant Biotechnol J 2012; 10: 2-11.

[18] Chen L, Song Y, Li S, Zhang L, Zou C, Yu D . The role of WRKY transcription factors in plant abiotic stresses. Biochim Biophys Acta 2012; 1819: 120-128.

[19] Phukan UJ, Jeena GS, Shukla RK . WRKY transcription factors: molecular regulation and stress responses in plants. Front Plant Sci 2016; 7: 760.

[20] Jiang J, Ma S, Ye N, Jiang M, Cao J, Zhang J . WRKY transcription factors in plant responses to stresses. J Integr Plant Biol 2017; 59: 86-101.

[21] Eulgem T, Rushton PJ, Robatzek S, Somssich IE . The WRKY superfamily of plant transcription factors. Trends Plant Sci 2000; 5: 199-206.

[22] Song Y, Ai CR, Jing SJ, Yu DQ . Research progress on functional analysis of rice WRKY genes. Rice Sci 2010; 17: 60-72.

[23] Zhou X, Jiang Y, Yu D . WRKY22 transcription factor mediates dark-induced leaf senescence in Arabidopsis. Mol Cells 2011; 31: 303-313.

[24] Bakshi M, Oelmüller R . WRKY transcription factors. Plant Signal Behav 2014; 9: e27700.

[25] Kato N, Dubouzet E, Kokabu Y et al. Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica. Plant Cell Physiol 2007; 48: 8-18.

[26] Ma D, Pu G, Lei C et al. Isolation and characterization of AaWRKY1, an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene, a key gene of artemisinin biosynthesis. Plant Cell Physiol 2009; 50: 2146-2161.

[27] Suttipanta N, Pattanaik S, Kulshrestha M, Patra B, Singh SK, Yuan L . The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol 2011; 157: 2081-2093.

[28] Schluttenhofer C, Yuan L . Regulation of specialized metabolism by WRKY transcription factors. Plant Physiol 2015; 167: 295-306.

[29] García-Cruz L, Valle-Guadarrama S, Salinas-Moreno Y, Joaquín-Cruz E . Physical, chemical, and antioxidant activity characterization of pitaya (Stenocereus pruinosus) fruits. Plant Foods Hum Nutr 2013; 68: 403-410.

[30] Suh DH, Lee S, Heo do Y et al. Metabolite profiling of red and white pitayas (Hylocereus polyrhizus and Hylocereus undatus) for comparing betalain biosynthesis and antioxidant activity. J Agric Food Chem 2014; 62: 8764-8771.

[31] Wybraniec S, Stalica P, Jerz G et al. Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed countercurrent chromatography. J Chromatogr A 2009; 1216: 6890-6899.

[32] Sainsbury F, Thuenemann EC, Lomonossoff GP . pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 2009; 7: 682-693.

[33] Fan ZQ, Kuang JF, Fu CC et al. The banana transcriptional repressor MaDEAR1 negatively regulates cell wall-modifying genes involved in fruit ripening. Front Plant Sci 2016; 7: 1021.

[34] Healey A, Furtado A, Cooper T, Henry RJ . Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods 2014; 10: 21.

[35] Fu CC, Han YC, Qi XY et al. Papaya CpERF9 acts as a transcriptional repressor of cell-wall-modifying genes CpPME1/2 and CpPG5 involved in fruit ripening. Plant Cell Rep 2016; 35: 2341-2352.

[36] Hellens RP, Allan AC, Friel EN et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 2005; 1: 13.

[37] Johnson CS, Kolevski B, Smyth DR . TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell 2002; 14: 1359-1375.

[38] Mao G, Meng X, Liu Y, Zheng Z, Chen Z, Zhang S . Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell 2011; 23: 1639-1653.

[39] Gong X, Zhang J, Hu J et al. FcWRKY70, a WRKY protein of Fortunella crassifolia, functions in drought tolerance and modulates putrescine synthesis by regulating arginine decarboxylase gene. Plant Cell Environ 2015; 38: 2248-2262.

[40] Ye YJ, Xiao YY, Han YC et al. Banana fruit VQ motif-containing protein5 represses cold-responsive transcription factor MaWRKY26 involved in the regulation of JA biosynthetic genes. Sci Rep 2016; 6: 23632.

[41] Ulker B, Somssich IE . WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol 2004; 7: 491-498.

[42] Chen M, Yan T, Shen Q et al. GLANDULAR TRICHOME-SPECIFIC WRKY 1 promotes artemisinin biosynthesis in Artemisia annua. New Phytol 2017; 214: 304-316.

[43] Chi Y, Yang Y, Zhou Y et al. Protein-protein interactions in the regulation of WRKY transcription factors. Mol Plant 2013; 6: 287-300.

[44] 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: 330-338.