Functional analysis of the dihydroflavonol 4-reductase family of Camellia sinensis: exploiting key amino acids to reconstruct reduction activity

Abstract

Anthocyanins and proanthocyanidins (PAs) are important types of flavonoids, plant secondary metabolites with a wide range of industrial and pharmaceutical applications. DFR (dihydroflavonol 4-reductase) is a pivotal enzyme that plays an important role in the flavonoid pathway. Here, four CsDFR genes were isolated from Camellia sinensis, and their overexpression was analyzed in vitro and in vivo. Based on transcription and metabolic analyses, CsDFR expression was closely consistent with catechins and PAs accumulation. Moreover, enzyme activity analyses revealed that the two recombinant proteins CsDFRa and CsDFRc exhibited DFR activity, converting dihydroflavonols into leucoanthocyanins in vitro, but CsDFRb1 and CsDFRb3 did not. CsDFRa and CsDFRc overexpression in AtDFR mutants (tt3) revealed that CsDFRs are involved in the biosynthesis of anthocyanins and PAs, as CsDFRa and CsDFRc restored not only the purple petiole phenotype but also the seed coat color. Site-directed mutagenesis revealed that the two amino acid residues S117 and T123 of CsDFRa play a prominent role in controlling DFR reductase activity. Enzymatic assays indicated that CsDFRa and CsDFRc exhibited a higher affinity for DHQ and DHK, respectively, whereas CsDFRb1^N120S and CsDFRb1^C126T exhibited a higher affinity for DHM. Our findings comprehensively characterize the DFRs from C. sinensis and shed light on their critical role in metabolic engineering.

References

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

[2] Cabrera C, Artacho R, Gimenez R . Beneficial effects of green tea-a review. J Am Coll Nutr. 2006; 25: 79-99.

[3] Chen AY, Chen YC . A review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention. Food Chem. 2013; 138: 2099-107.

[4] Dalgaard F, Bondonno NP, Murray K et al. Associations between habitual flavonoid intake and hospital admissions for atherosclerotic cardiovascular disease: a prospective cohort study. Lancet Planet Health. 2019; 3: e450-9.

[5] Tanaka Y, Brugliera F . Flower colour and cytochromes P450. Philos Trans R Soc Lond Ser B Biol Sci. 2013; 368: 20120432.

[6] Hua C, Linling L, Shuiyuan C et al. Molecular cloning and characterization of three genes encoding dihydroflavonol-4-reductase from Ginkgo biloba in anthocyanin biosynthetic pathway. PLoS One. 2013; 8: e72017.

[7] Jiang X, Liu Y, Li W et al. Tissue-specific, development-dependent phenolic compounds accumulation profile and gene expression pattern in tea plant [Camellia sinensis]. PLoS One. 2013; 8: e62315.

[8] Bowers JE, Dangl GS, Vignani R et al. Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.). Genome. 1996; 39: 628-33.

[9] Helariutta Y, Elomaa P, Kotilainen M et al. Cloning of cDNA coding for dihydroflavonol-4-reductase (DFR) and characterization of dfr expression in the corollas of Gerbera hybrida var. Regina (Compositae). Plant Mol Biol. 1993; 22: 183-93.

[10] Wang J, Wang Y, Wang S et al. Cloning and temporal-spatial expression analysis of dfr gene from Scutellaria baicalensis with different colors. Sheng Wu Gong Cheng Xue Bao. 2021; 37: 1312-23.

[11] Wang L, Zhu Y, Wang P et al. Functional characterization of a Dihydroflavanol 4-reductase from the fiber of upland cotton (Gossypium hirsutum). Molecules. 2016; 21: 32.

[12] Bashandy H, Pietiainen M, Carvalho E et al. Anthocyanin biosynthesis in gerbera cultivar ’Estelle’ and its acyanic sport ’Ivory’. Planta. 2015; 242: 601-11.

[13] Kubasek WL, Shirley BW, Mckillop A et al. Regulation of flavonoid biosynthetic genes in germinating Arabidopsis seedlings. Plant Cell. 1992; 4: 1229-36.

[14] Johnson ET, Yi H, Shin B et al. Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin-type anthocyanins. Plant J. 1999; 19: 81-5.

[15] Johnson ET, Ryu S, Yi H et al. Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase. Plant J. 2001; 25: 325-33.

[16] Alagawany M, Abd El-Hack ME, Saeed M et al. Nutritional applications and beneficial health applications of green tea and l-theanine in some animal species: a review. J Anim Physiol Anim Nutr (Berl). 2020; 104: 245-56.

[17] Yao SS, Guo WF, Lu Y et al. Flavor characteristics of lapsang souchong and smoked lapsang souchong, a special Chinese black tea with pine smoking process. J Agric Food Chem. 2005; 53: 8688-93.

[18] Asakawa T, Hamashima Y, Kan T . Chemical synthesis of tea polyphenols and related compounds. Curr Pharm Des. 2013; 19: 6207-17.

[19] Hu J, Zhou D, Chen Y . Preparation and antioxidant activity of green tea extract enriched in epigallocatechin (EGC) and epigallocatechin gallate (EGCG). J Agric Food Chem. 2009; 57: 1349-53.

[20] Rani A, Singh K, Ahuja PS et al. Molecular regulation of catechins biosynthesis in tea [Camellia sinensis (L.) O. Kuntze]. Gene. 2012; 495: 205-10.

[21] Wang W, Zhou Y, Wu Y et al. Insight into Catechins metabolic pathways of Camellia sinensis based on genome and transcriptome analysis. J Agric Food Chem. 2018; 66: 4281-93.

[22] Katsu K, Suzuki R, Tsuchiya W et al. A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity. BMC Plant Biol. 2017; 17: 239.

[23] Shirley BW, Hanley S, Goodman HM . Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations. Plant Cell. 1992; 4: 333-47.

[24] Pang Y, Abeysinghe IS, He J et al. Functional characterization of proanthocyanidin pathway enzymes from tea and their application for metabolic engineering. Plant Physiol. 2013; 161: 1103-16.

[25] Xie DY, Sharma SB, Paiva NL et al. Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science. 2003; 299: 396-9.

[26] Trabelsi N, Petit P, Manigand C et al. Structural evidence for the inhibition of grape dihydroflavonol 4-reductase by flavonols. Acta Crystallogr D Biol Crystallogr. 2008; 64: 883-91.

[27] Petit P, Granier T, D’estaintot BL et al. Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis. J Mol Biol. 2007; 368: 1345-57.

[28] Benach J, Filling C, Oppermann UC et al. Structure of bacterial 3beta/17beta-hydroxysteroid dehydrogenase at 1.2 a resolution: a model for multiple steroid recognition. Biochemistry. 2002; 41: 14659-68.

[29] Bottoms CA, Smith PE, Tanner JJ . A structurally conserved water molecule in Rossmann dinucleotide-binding domains. Protein science : a publication of the Protein Society. 2002; 11: 2125-37.

[30] Zhao J, Li P, Xia T et al. Exploring plant metabolic genomics: chemical diversity, metabolic complexity in the biosynthesis and transport of specialized metabolites with the tea plant as a model. Crit Rev Biotechnol. 2020; 40: 667-88.

[31] Li H, Qiu J, Chen F et al. Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in Saussurea medusa. Mol Biol Rep. 2012; 39: 2991-9.

[32] Nakatsuka A, Izumi Y, Yamagishi M . Spatial and temporal expression of chalcone synthase and dihydroflavonol 4-reductase genes in the Asiatic hybrid lily. Plant Sci. 2003; 165: 759-67.

[33] Shimada N, Sasaki R, Sato S et al. A comprehensive analysis of six dihydroflavonol 4-reductases encoded by a gene cluster of the Lotus japonicus genome. J Exp Bot. 2005; 56: 2573-85.

[34] Tang LK, Chu H, Yip WK et al. An anther-specific dihydroflavonol 4-reductase-like gene (DRL1) is essential for male fertility in Arabidopsis. New Phytol. 2009; 181: 576-87.

[35] Mamati GE, Liang YR, Lu JL . Expression of basic genes involved in tea polyphenol synthesis in relation to accumulation of catechins and total tea polyphenols. J Sci Food Agr. 2006; 86: 459-64.

[36] Martens S, Teeri T, Forkmann G . Heterologous expression of dihydroflavonol 4-reductases from various plants. FEBS Lett. 2002; 531: 453-8.

[37] Polashock JJ, Griesbach RJ, Sullivan RF et al. Cloning of a cDNA encoding the cranberry dihydroflavonol-4-reductase (DFR) and expression in transgenic tobacco. Plant Sci. 2002; 163: 241-51.

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