Biochemical and functional characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L

Authors: TAN, LIN - Chinese Academy Of Tropical Agricultural Sciences; WANG, MEI - University Of Mississippi; KANG, YOUFA - Hainan University; AZEEM, FARRUKH - Chinese Academy Of Tropical Agricultural Sciences; ZHOU, ZHAOXI - Chinese Academy Of Tropical Agricultural Sciences; TUO, DECAI - Chinese Academy Of Tropical Agricultural Sciences; PRECIADO-ROJO, LINA MARIA - University Of Antioquia; KHAN, IKHLAS - University Of Mississippi; Pan, Zhiqiang - Peter

Submitted to: Molecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/30/2018
Publication Date: 11/5/2018
Publication URL: http://handle.nal.usda.gov/10113/6471037
Citation: Tan, L., Wang, M., Kang, Y., Azeem, F., Zhou, Z., Tuo, D., Preciado-Rojo, L., Khan, I.A., Pan, Z. 2018. Biochemical and functional characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L. Molecules. 23(11):2876-2896. https://doi.org/10.3390/molecules23112876.
DOI: https://doi.org/10.3390/molecules23112876

Interpretive Summary: Proanthocyanidins (PAs), a group of chemical compounds found in many types of plants, have been shown to have potential health benefits and are an important part of the human diet. PAs belong to a larger category of plant chemicals called flavonoids. Mango fruits, one of the most important fruit commodities, are a rich source of flavonoids and xanthones. Understanding the biosynthetic pathway could contribute to improving the quality of the mango fruits. In this report, cloning and functional characterization of mango anthocyanidin reductase (ANR), a key enzyme that convert anthocyanidins to catechin and epicatechin in the PA biosynthesis pathway, were described. Three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango. Their functions, enzymatic kinetics, and substrate specificity were determined by in vitro enzyme analyses using proteins purified by expressing these genes in E.coli. Their functions in planta were further confirmed by overexpressing these genes in Arabidopsis ANR mutant plants, indicating that these genes can complement the PA deficiency pathway. The isolation of these genes provided a tool for increasing the levels of PAs in the fruits.

Technical Abstract: Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (-)-catechin and (-)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.