Identification of UDP-rhamnosyltransferases and UDP-galactosyltransferase involved in flavonol glycosylation in Morella rubra

Abstract

Flavonol glycosides are health-promoting phytochemicals important for human nutrition and plant defense against environmental stresses. Glycosylation modification greatly enriches the diversity of flavonols. Morella rubra, a member of the Myricaceae, contains high amounts of myricetin 3- O-rhamnoside (M3Rha), quercetin 3- O-rhamnoside (Q3Rha), and quercetin 3- O-galactoside (Q3Gal). In the present study, MrUGT78R1 and MrUGT78R2 were identified as two functional UDP-rhamnosyltransferases, while MrUGT78W1 was identified as a UDP-galactosyltransferase. Site-directed mutagenesis identified Pro143 and Asn386 as important residues for rhamnosyl transfer activity of MrUGT78R1, while the two corresponding positions in MrUGT78W1 (i.e. Ser147 and Asn370) also play important roles in galactosyl transfer activity. Transient expression data for these three MrUGTs in Nicotiana benthamiana tested the function of MrUGT78R1 and MrUGT78R2 as rhamnosyltransferases and MrUGT78W1 as a galactosyltransferase in glycosylation of flavonols. This work enriches knowledge of the diversity of UDP-rhamnosyltransferase in planta and identifies two amino acid positions important for both rhamnosyltransferase and galactosyltransferase.

References

[1] Jung SK, Lee KW, Byun S et al. Myricetin suppresses UVB-induced skin cancer by targeting Fyn. Cancer Res. 2008; 68: 6021-9.

[2] Perez-Vizcaino F, Duarte J . Flavonols and cardiovascular disease. Mol Asp Med. 2010; 31: 478-94.

[3] Bondonno NP, Dalgaard F, Kyrø C et al. Flavonoid intake is associated with lower mortality in the Danish diet cancer and health cohort. Nat Commun. 2019; 10: 3651.

[4] Yin R, Han K, Heller W et al. Kaempferol 3-O-rhamnoside-7-O-rhamnoside is an endogenous flavonol inhibitor of polar auxin transport in Arabidopsis shoots. New Phytol. 2014; 201: 466-75.

[5] Onkokesung N, Reichelt M, van Doorn A et al. Modulation of flavonoid metabolites in Arabidopsis thaliana through overexpression of the MYB75 transcription factor: role of kaempferol-3,7-dirhamnoside in resistance to the specialist insect herbivore Pieris brassicae. J Exp Bot. 2014; 65: 2203-17.

[6] Silva-Navas J, Moreno-Risueno MA, Manzano C et al. Flavonols mediate root phototropism and growth through regulation of proliferation-to-differentiation transition. Plant Cell. 2016; 28: 1372-87.

[7] Vogt T, Jones P . Glycosyltransferases in plant natural product synthesis: characterization of a supergene family. Trends Plant Sci. 2000; 5: 380-6.

[8] Bowles D, Lim EK, Poppenberger B et al. Glycosyltransferases of lipophilic small molecules. Annu Rev Plant Biol. 2006; 57: 567-97.

[9] Plaza M, Pozzo T, Liu J et al. Substituent effects on in vitro antioxidizing properties, stability, and solubility in flavonoids. J Agric Food Chem. 2014; 62: 3321-33.

[10] Naeem A, Ming Y, Pengyi H et al. The fate of flavonoids after oral administration: a comprehensive overview of its bioavailability. Crit Rev Food Sci Nutr. 2022; 62: 6169-86.

[11] Xie L, Cao Y, Zhao Z et al. Involvement of MdUGT75B1 and MdUGT71B1 in flavonol galactoside/glucoside biosynthesis in apple fruit. Food Chem. 2020; 312: 126124.

[12] Tohge T, Nishiyama Y, Hirai MY et al. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. Plant J. 2005; 42: 218-35.

[13] Griesser M, Vitzthum F, Fink B et al. Multi-substrate flavonol O-glucosyltransferases from strawberry (Fragaria × ananassa) achene and receptacle. J Exp Bot. 2008; 59: 2611-25.

[14] Cui L, Yao S, Dai X et al. Identification of UDP-glycosyltransferases involved in the biosynthesis of astringent taste compounds in tea (Camellia sinensis). J Exp Bot. 2016; 67: 2285-97.

[15] Miller KD, Guyon V, Evans JN et al. Purification, cloning, and heterologous expression of a catalytically efficient flavonol 3-O-galactosyltransferase expressed in the male gametophyte of Petunia hybrida. J Biol Chem. 1999; 274: 34011-9.

[16] Zhang X, Huang H, Zhang Q et al. Phytochemical characterization of Chinese bayberry (Myrica rubra Sieb. et Zucc.) of 17 cultivars and their antioxidant properties. Int J Mol Sci. 2015; 16: 12467-81.

[17] Sun C, Huang H, Xu C et al. Biological activities of extracts from Chinese bayberry (Myrica rubra Sieb. et Zucc.): a review. Plant Food Hum Nutr. 2013; 68: 97-106.

[18] Yan S, Zhang X, Wen X et al. Purification of flavonoids from Chinese bayberry (Morella rubra Sieb. et Zucc.) fruit extracts and α-glucosidase inhibitory activities of different fractionations. Molecules. 2016; 21: 1148.

[19] Ahangarpour A, Oroojan AA, Khorsandi L et al. Solid lipid nanoparticles of myricitrin have antioxidant and antidiabetic effects on streptozotocin-nicotinamide-induced diabetic model and myotube cell of male mouse. Oxid Med Cell Longev. 2018; 2018: 7496936.

[20] Liu Y, Zhang X, Zhan L et al. LC-Q-TOF-MS characterization of polyphenols from white bayberry fruit and its antidiabetic effect in KK-Ay mice. ACS Omega. 2020; 5: 17839-49.

[21] Liu Y, Zhan L, Xu C et al. α-Glucosidase inhibitors from Chinese bayberry (Morella rubra Sieb. et Zucc.) fruit: molecular docking and interaction mechanism of flavonols with different B-ring hydroxylations. RSC Adv. 2020; 10: 29347-61.

[22] Qi S, Feng Z, Li Q et al. Myricitrin modulates NADPH oxidase-dependent ROS production to inhibit endotoxin-mediated inflammation by blocking the JAK/STAT1 and NOX2/p47phox pathways. Oxid Med Cell Longev. 2017;2017:1-20.

[23] Qin M, Luo Y, Meng X et al. Myricitrin attenuates endothelial cell apoptosis to prevent atherosclerosis: an insight into PI3K/Akt activation and STAT3 signaling pathways. Vasc Pharmacol. 2015; 70: 23-34.

[24] Xing M, Cao Y, Ren C et al. Elucidation of myricetin biosynthesis in Morella rubra of the Myricaceae. Plant J. 2021; 108: 411-25.

[25] Gachon CMM, Langlois-Meurinne M, Saindrenan P . Plant secondary metabolism glycosyltransferases: the emerging functional analysis. Trends Plant Sci. 2005; 10: 542-9.

[26] Yonekura-Sakakibara K, Hanada K . An evolutionary view of functional diversity in family 1 glycosyltransferases. Plant J. 2011; 66: 182-93.

[27] Caputi L, Malnoy M, Goremykin V et al. A genome-wide phylogenetic reconstruction of family 1 UDP-glycosyltransferases revealed the expansion of the family during the adaptation of plants to life on land. Plant J. 2012; 69: 1030-42.

[28] Yin R, Messner B, Faus-Kessler T et al. Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation. J Exp Bot. 2012; 63: 2465-78.

[29] Osmani SA, Bak S, Møller BL . Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling. Phytochemistry. 2009; 70: 325-47.

[30] Feng C, Chen M, Xu CJ et al. Transcriptomic analysis of Chinese bayberry (Myrica rubra) fruit development and ripening using RNA-Seq. BMC Genomics. 2012; 13: 19.

[31] Jia H, Jia H, Cai Q et al. The red bayberry genome and genetic basis of sex determination. Plant Biotechnol J. 2019; 17: 397-409.

[32] Niu S, Xu C, Zhang W et al. Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry (Myrica rubra) fruit by a R2R3 MYB transcription factor. Planta. 2010; 231: 887-99.

[33] Irmisch S, Ruebsam H, Jancsik S et al. Flavonol biosynthesis genes and their use in engineering the plant antidiabetic metabolite montbretin A. Plant Physiol. 2019; 180: 1277-90.

[34] Cao Y, Jia H, Xing M et al. Genome-wide analysis of MYB gene family in Chinese bayberry (Morella rubra) and identification of members regulating flavonoid biosynthesis. Front Plant Sci. 2021; 12: 691384.

[35] Irmisch S, Jo S, Roach CR et al. Discovery of UDP-glycosyltransferases and BAHD-acyltransferases involved in the biosynthesis of the antidiabetic plant metabolite montbretin A. Plant Cell. 2018; 30: 1864-86.

[36] Jones P, Messner B, Nakajima J et al. UGT73C6 and UGT78D1, glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana. J Biol Chem. 2003; 278: 43910-8.

[37] Ono E, Homma Y, Horikawa M et al. Functional differentiation of the glycosyltransferases that contribute to the chemical diversity of bioactive flavonol glycosides in grapevines (Vitis vinifera). Plant Cell. 2010; 22: 2856-71.

[38] Zong G, Fei S, Liu X et al. Crystal structures of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana reveal the molecular basis of sugar donor specificity for UDP-β-l-rhamnose and rhamnosylation mechanism. Plant J. 2019; 99: 257-69.

[39] Kubo A, Arai Y, Nagashima S et al. Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation. Arch Biochem Biophys. 2004; 429: 198-203.

[40] Fu R, Martin C, Zhang Y . Next-generation plant metabolic engineering, inspired by an ancient Chinese irrigation system. Mol Plant. 2018; 11: 47-57.

[41] Xie L, Guo Y, Ren C et al. Unravelling the consecutive glycosylation and methylation of flavonols in peach in response to UV-B irradiation. Plant Cell Environ. 2022; 45: 2158-75.

[42] Irmisch S, Jancsik S, Man Saint Yuen M et al. Complete biosynthesis of the anti-diabetic plant metabolite montbretin A. Plant Physiol. 2020; 184: 97-109.

[43] Cao Y, Xie L, Ma Y et al. PpMYB15 and PpMYBF1 transcription factors are involved in regulating flavonol biosynthesis in peach fruit. J Agric Food Chem. 2019; 67: 644-52.

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

[45] Sheikh MO, Halmo SM, Patel S et al. Rapid screening of sugar-nucleotide donor specificities of putative glycosyltransferases. Glycobiology. 2017; 27: 206-12.

[46] Trott O, Olson AJ . AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2009; 31: 455-61.