Molecular cloning and functional characterization of the anthocyanidin reductase gene from Vitis bellula

Abstract

Anthocyanidin reductase (ANR) is an NADPH-/NADH-dependent enzyme that transfers two hydrides to anthocyanidins to produce three types of isomeric flavan-3-ols. This reductase forms the ANR pathway toward the biosynthesis of proanthocyanidins (PAs, which are also called condensed tannins). Here, we report cloning and functional characterization of an ANR (called VbANR) homolog from the leaves of Vitis bellula, a newly developed grape crop in southern China. The open reading frame (ORF) of VbANR is 1,017 bp in length and encodes 339 amino acids. A phylogenetic analysis and an alignment using 17 sequences revealed that VbANR is approximately 99.9 % identical to the ANR homolog from Vitis vinifera. The VbANR ORF is fused to the Trx gene containing a His-tag in the pET32a(+) vector to obtain a pET32a(+)-VbANR construct for expressing the recombinant VbANR. In vitro enzyme assays show that VbANR converts cyanidin, delphinidin, and pelargonidin to their corresponding flavan-3-ols. Enzymatic products include 2S,3R-trans- and 2R,3R-cis-flavan-3-ols isomers, such as (-)-catechin and (-)-epicatechin. In addition, the third compound that is observed from the enzymatic products is most likely a 2S,3S-cis-flavan-3-ol. To analyze the kinetics and optimize pH and temperature values, a UV spectrometry method was developed to quantify the concentrations of total enzymatic products. The optimum pH and temperature values are 4.0 and 40 °C, respectively. The K m , K cat, V max, and K cat/K m values for pelargonidin and delphinidin were similar. In comparison, VbANR exhibits a slightly lower affinity to cyanidin. VbANR uses both NADPH and NADH but prefers to employ NADPH. GFP fusion and confocal microscopy analyses revealed the cytosolic localization of VbANR. The overexpression of VbANR in ban mutants reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate that VbANR forms the ANR pathway, leading to the formation of three types of isomeric flavan-3-ols and PAs in the leaves of V. bellula.

Proanthocyanidin accumulation and transcriptional responses in the seed coat of cranberry beans (Phaseolus vulgaris L.) with different susceptibility to postharvest darkening

“…For Pv ANR1, the apparent V max for epicatechin formation from cyanidin is within the range of those detected for other ANRs [19, 55]. Similarly, recombinant ANRs from Arabidopsis, Gossypium hirsutum , M. truncatula , Vitis bellula and Camellia sinensis form both flavan-3-ol products in vitro [29, 55–58]. Moreover, the intrinsic epimerase activity of a V. vinifera ANR promotes the stereospecific reduction of cyanidin at the C2 and C4 positions to form both (+)-epicatechin and (−)-catechin [59].…”

“…This is not without precedent as reduced specific activities are evident for Gm ANR1 at cyanidin concentrations in excess of 100 μM [18]. Moreover, non-hyperbolic kinetic relationships have been described for a recombinant V. bellula ANR enzyme [58]. In terms of the biological significance, this could represent a mechanism for feed-forward inhibition of this enzyme as a means of limiting the over-accumulation of proanthocyanidins, and allowing ample substrate for simultaneous anthocyanin formation in cranberry bean seed coats.…”

Proanthocyanidin Synthesis in Chinese Bayberry (Myrica rubra Sieb. et Zucc.) Fruits

“…Intriguingly, in addition to EC and EGC, the MrANR protein in our study also produced C and GC from the substrates of cyanidin and delphinidin, respectively, suggesting that the MrANR protein may have an epimerase activity in vitro . Such epimerase activity inherently associated with ANR has been reported in Gossypium hirsutum (Zhu et al, 2015 ), Vitis bellula (Zhu et al, 2014 ), Vitis vinifere (Gargouri et al, 2010 ) and Camellia sinensis (Pang et al, 2013 ), producing cis-flavan-3-ols in vitro as well as trans-flavan-3-ols.…”

BANYULS genes from Brassica juncea and Brassica nigra: cloning, evolution and involvement in seed coat colour

“…The promoter of the Arabidopsis thaliana BAN gene was also activated by similar components in B. napus seed, suggesting that rapeseed proteins could bind to the promoter of the Arabidopsis BAN and regulate its expression (Nesi et al 2009). In Vitis bellula, ANR is an NADPH-/NADH-dependent enzyme, leading to the formation of flavan-3-ols and PAs in the leaves (Zhu et al 2014). The apple MdMYB9 could activate the ANR promoters of apple and poplar, which could be employed for metabolic engineering of the PA pathway in apple (Gesell et al 2014).…”