Evolutionary and functional characterization of leucoanthocyanidin reductases from Camellia sinensis

Peiqiang Wang,Liping Gao,Tao Xia,Yanzhi Li,Xiaolan Jiang,Lingjie Zhang,Tong Li,Xinlong Dai,Lijuan Xu,Dawei Xing,Mingzhuo Li

doi:10.1007/s00425-017-2771-z

Peiqiang Wang, Liping Gao + Show 9 more

Open Access

https://doi.org/10.1007/s00425-017-2771-z

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Journal: Planta	Publication Date: Sep 8, 2017
Citations: 74	License type: open-access

Affiliation: Anhui Agricultural University

Abstract

Main conclusionLARs promoted the biosynthesis of catechin monomers and inhibited their polymerization. The accumulation of catechin monomers and polymers was increased by up-regulating the expression ofNtLARandNtANRs in CsMYB5b transgenic tobacco.Tea is rich in polyphenolic compounds, and catechins are the major polyphenols in tea. The biosynthesis of polyphenols is closely related to the expression of the leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes. In this paper, an evolutionary analysis and functional characterization of three CsLARs were performed. The phylogenetic tree showed that plant LARs could be grouped into three, including gymnosperms, monocotyledons and dicotyledons (clusters I and II). The eighth amino acid residue in a conserved LAR-specific motif is changeable due to a transversion (G → T) and transition (G → C) that occur in the corresponding codon. Therefore, plant LARs can be classified as G-type, A-type and S-type LARs due to this variable amino acid residue. Although (2R, 3S)-trans-flavan-3-ols were the products of recombinant CsLARs proteins expressed in Escherichia coli, both (2R, 3S)-trans and (2R, 3R)-cis-flavan-3-ols were detected in tobacco overexpressing CsLARs. However, a butanol/HCl hydrolysis assay indicated that overexpression of the CsLARs caused a decrease in polymerized catechins. A hybridization experiment with CsLARc + AtPAP1 also showed that no polymers other than epicatechin, catechin and glycoside were detected, although the accumulation of anthocyanins was markedly decreased. CsMYB5b promoted the biosynthesis of both flavan-3-ols and proanthocyanidins (PAs). Therefore, LARs promoted the biosynthesis of catechin monomers and inhibited their polymerization. The accumulation of catechin monomers and polymers was increased by up-regulating the expression of the NtLAR and NtANRs in CsMYB5b transgenic tobacco.

Highlights

Polyphenolic compounds are a large class of plant secondary metabolites that are ubiquitously present in many plants, and they mainly consist of flavones, flavonols, anthocyanins, isoflavones, condensed tannins (CT; or PAs) and other derivatives
The results showed that the full cDNA lengths of the three CsLARs were 1518, 1401 and 1595 bp with 1029, 984 and 1197 bp open reading frames (ORFs), respectively
The leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) are known to be responsible for the biosynthesis of flavan-3-ols (Tanner et al 2003; Xie et al 2004)

Summary

Introduction

Polyphenolic compounds are a large class of plant secondary metabolites that are ubiquitously present in many plants, and they mainly consist of flavones, flavonols, anthocyanins, isoflavones, condensed tannins (CT; or PAs) and other derivatives (Winkel-Shirley 2001; Tanner et al 2003; Xie1 3 Vol.:(0123456789)Planta (2018) 247:139–154 et al 2003). Different classes of polyphenolic compounds contribute in various ways to plant growth and defense against biotic and abiotic stress, including pathogen infections, ultraviolet radiation and herbivory (Dixon and Sumner 2003; Mellway et al 2009). Flavan-3-ols and PAs greatly contribute to the astringency of tea, wine and certain fruits, such as persimmon (Diospyros kaki) (Kennedy et al 2008; Ma et al 2010; Liu et al 2012). Dissociative flavan-3-ol monomers are mainly distributed in the leaves of the aerial parts of the plant, and polymeric catechins are distributed in the stems and root (Jiang et al 2013, 2015). Dissociative flavon-3-ols are called catechins and include (2R, 3S)trans-flavan-3-ols (catechin, C, and gallocatechin, GC) and (2R, 3R)-cis-flavan-3-ols (epicatechin, EC; epigallocatechin, EGC; epicatechin gallate, ECG; and epigallocatechin gallate, EGCG) (Qian et al 2015). Catechins play a crucial role in the dominating astringent taste of tea and possess a number of effects that are beneficial to human health, the galloylated catechins (Chung et al 2003; Cui et al 2016)

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