Abstract
Brunfelsia calycina flowers lose anthocyanins rapidly and are therefore well suited for the study of anthocyanin degradation mechanisms, which are unclear in planta. Here, we isolated an anthocyanin-β-glycosidase from B. calycina petals. The MS/MS (Mass Spectrometry) peptide sequencing showed that the enzyme (72 kDa) was a β-xylosidase (BcXyl). The enzyme showed high activity to p-Nitrophenyl-β-d-galactopyranoside (pNPGa) and p-Nitrophenyl-β-d-xylopyranoside (pNPX), while no activity to p-Nitrophenyl-β-d-glucopyranoside (pNPG) or p-Nitrophenyl-β-D-mannopyranoside (pNPM) was seen. The optimum temperature of BcXyl was 40 °C and the optimum pH was 5.0. The enzyme was strongly inhibited by 1 mM D-gluconate and Ag+. HPLC (High Performance Liquid Chromatography) analysis showed that BcXyl catalyzed the degradation of an anthocyanin component of B. calycina, and the release of xylose and galactose due to hydrolysis of glycosidic bonds by BcXyl was detected by GC (Gas Chromatography) /MS. A full-length mRNA sequence (2358 bp) of BcXyl (NCBI No. MK411219) was obtained and the deduced protein sequence shared conserved domains with two anthocyanin-β-glycosidases (Bgln and BadGluc, characterized in fungi). BcXyl, Bgln and BadGluc belong to AB subfamily of Glycoside hydrolase family 3. Similar to BcPrx01, an anthocyanin-degradation-related Peroxidase (POD), BcXyl was dramatically activated at the stage at which the rapid anthocyanin degradation occurred. Taken together, we suggest that BcXyl may be the first anthocyanin-β-glycosidase identified in higher plants.
Highlights
Anthocyanins are some of the most important plant natural pigments, responsible for a wide range of colors from red to purple and blue
In the alignment of BcXyl with Bgln, BadGluc and Arabidopsis Xylosidases (AtXyl 1 and 4), we found that the catalytic nucleophile Asp and four amino acid residues which might be involved in substrate binding, were conserved in these AB clad members of glycoside hydrolase family 3 (GH3) [22] (Figure 6D)
Rapid anthocyanin degradation occurs during flower development of Brunfelsia calycina
Summary
Anthocyanins are some of the most important plant natural pigments, responsible for a wide range of colors from red to purple and blue. One possible pathway for enzymatic anthocyanin degradation is PPO-phenols-anthocyanins system, in which PPOs first catalyzed the oxidation of phenols to form quinones, which were unstable molecules prone to form polymers with anthocyanins, leading to decoloration, browning and reduced anthocyanin content. Another possibility is two-step degradation, first deglycosylation of sugar moieties by β-glycosidases and oxidation of the aglycone by PPOs [4]. Some β-glycosidases were found to be located in the vacuoles and were suggested to be involved in in planta anthocyanin degradation [4]
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