Polymerization of proanthocyanidins under the catalysis of miR397a-regulated laccases in Salvia miltiorrhiza and Populus trichocarpa

Abstract

Proanthocyanidins (PAs) play significant roles in plants and are bioactive compounds with health benefits. The polymerization mechanism has been debated for decades. Here we show that laccases (LACs) are involved in PA polymerization and miR397a is a negative regulator of PA biosynthesis in Salvia miltiorrhiza and Populus trichocarpa. Elevation of miR397a level causes significant downregulation of LACs, severe reduction of polymerized PAs, and significant increase of flavan-3-ol monomers in transgenic S. miltiorrhiza and P. trichocarpa plants. Enzyme activity analysis shows that miR397a-regulated SmLAC1 catalyzes the polymerization of flavan-3-ols and the conversion of B-type PAs to A-type. Both catechin and epicatechin can serve as the starter unit and the extension unit during PA polymerization. Overexpression of SmLAC1 results in significant increase of PA accumulation, accompanied by the decrease of catechin and epicatechin contents. Consistently, CRISPR/Cas9-mediated SmLAC1 knockout shows the opposite results. Based on these results, a scheme for LAC-catalyzed PA polymerization is proposed. The work provides insights into PA polymerization mechanism.

Fig. 1. Overexpression of miR397a in S. miltiorrhiza.

a Generation of miR397a-overexpressed S. miltiorrhiza transgenics. b MiR397a levels in three wild type plants (WT1–WT3) and seven transgenic lines (n = 3 biologically independent experiments). Expression levels are relative with wild type (set as 1). Data are means ± SD of three biologically independent experiments (n = 3). ** indicates P < 0.01 (One-way ANOVA). c Relative expression levels of target genes, including SmLAC1, SmLAC23, SmLAC24, and SmLAC32, in the flowers of wild type and transgenic plants. The expression levels were analyzed using qRT-PCR. SmUBQ10 was used as a control. Expression levels in wild type (WT) were set to 1. Data are means ± SD of three biologically independent experiments (n = 3). d Enzyme activity of LACs in the flowers of wild type and transgenic plants. ** indicates P < 0.01 (One-way ANOVA). Source data are provided in the Source Data file.

Fig. 2. PA contents in the flowers of wild type and transgenic S. miltiorrhiza plants.

a Extractable PA contents determined using the dimethylaminocinnamaldehyde (DMACA) method and shown as epicatechin equivalents. b Non-extractable PA contents measured using the butanol/HCl method and shown as procyanidin B1 equivalents. c Coloration of PA extracts from wild type and transgenic S. miltiorrhiza flowers. d–h Procyanidins A1, A2, B1, B2, and B3 contents analyzed using UPLC. i, j Catechin and epicatechin contents analyzed using UPLC. All data are the average of three independent biological replicates (n = 3). Error bars show the standard deviations. The mean contents were analyzed using one-way ANOVA test. * and ** indicate P < 0.05 and P < 0.01, respectively. Source data are provided in the Source Data file.

Fig. 3. Identification of procyanidin A1, A2, B1, B2 and B3 from S. miltiorrhiza flowers.

a Extracted ion chromatogram (EIC) at m/z = 575 showing two peaks of procyanidin A1 and A2. b Extracted ion chromatogram (EIC) at m/z = 575 showing two peaks of procyanidin B1, B2, and B3. c MS/MS spectra of the m/z = 575 peaks of procyanidin A1 and A2 in (b). d MS/MS spectra of the m/z = 577 peaks from procyanidin B1, B2, and B3 in (b). e Fragmentation pattern of procyanidin A1 and A2 in (a). f Fragmentation patterns of procyanidin B1, B2, and B3 in (a). Source data for Fig. 3a–d are provided in the Source Data file.

Fig. 4. PA and flavan-3-ol contents in wild type and transgenic P. trichocarpa leaves.

a Extractable PA contents determined using the dimethylaminocinnamaldehyde (DMACA) method and shown as epicatechin equivalents. b Non-extractable PA contents determined using the butanol/HCl method and shown as procyanidin B1 equivalents. c, d Detection of procyanidins A1, A2, B1, B2, and B3 in leaf extracts of P. trichocarpa through elective ion chromatography (EIC) analysis. e–k The contents of procyanidins A1, A2, B1, B2, B3, catechin and epicatechin in wild type and transgenic P. trichocarpa leaves. All data are the average of three independent biological replicates (n = 3). Error bars show the standard deviations. The mean contents were analyzed using one-way ANOVA test. * and ** indicates P < 0.05 and P < 0.01, respectively. Source data are provided in the Source Data file.

Fig. 5. Recombinant SmLAC1 catalyzed the polyermization of catechin and epicatechin in vitro.

a An SDS-PAGE gel image shows the purified recombinant MBP-SmLAC1 protein. b MBP-SmLAC1 protein catalyzed the conversion of catechin (C) and epicatechin (EC) to yellowish compounds. The reactions without SmLAC1 were used as the controls. c UPLC analysis of the products from catechin and epicatechin under the catalysis of SmLAC1. d MS/MS spectra of the products C1, E1, and CE1. e MS/MS spectra of the products C2, E2, and CE2. Source data for Fig. 5a–c are provided in the Source Data file.

Fig. 6. Structures of products C1, E1, CE1, C2, E2, and CE2 determined by NMR analysis.

a–c ¹H NMR assignments of 15 protons on the six rings and 10 hydroxyl groups in products C1, E1, and CE1, respectively. d–f ¹³C NMR assignments of 30 carbons in products C1, E1, and CE1, respectively. g, h ¹H NMR assignments of 14 protons on the six rings of products C2 and E2, respectively. i, j ¹³C NMR assignments of 30 carbons in products C2 and E2, respectively.

Fig. 7. Recombinant SmLAC1 catalyzed the oxidation of procyanidins B1, B2, and B3 in vitro.

a–c UPLC analysis of the products from procyanidins B1, B2, and B3 under the catalysis of SmLAC1. d MS/MS spectra of the products 1, 2, and 3. e–g ¹H NMR assignments of 14 protons on the six rings of products 1, 2, and 3, respectively. h–j ¹³C NMR assignments of 30 carbons in products 1, 2, and 3, respectively.

Fig. 8. Transcript levels and PA contents in leaves of the control and SmLAC1-overexpressed transgenic S. miltiorrhiza.

a Transcript levels of SmLAC1 in different transgenic lines. Transcripts were determined by qPCR and normalized relative to the expression of SmUBQ10. b Extractable PA levels in leaves of the control and SmLAC1 transgenic lines quantified using DMACA reagent and expressed as epicatechin equivalents. c Non-extractable PA levels evaluated by the butanol-HCl method and expressed as procyanidin B1 equivalents. d–f Elective ion chromatography (EIC) analysis of procyanidins A1, A2, B1, B2, B3, catechin and epicatechin in leaf extracts of the control and SmLAC1-overexpressed transgenic S. miltiorrhiza. g–k. The contents of procyanidins A1, A2, B1, B2, and B3 analyzed using UPLC. l, m Catechin and epicatechin contents in leaves of the control and SmLAC1-overexpressed transgenic S. miltiorrhiza. All data are the average of three independent biological replicates (n = 3). * and ** indicates P < 0.05 and P < 0.01 (One-way ANOVA), respectively. Source data are provided in the Source Data file.

Fig. 9. PA contents in leaves of the control and smlac1 mutants.

a The sgRNA-edited nucleotide sequences in smlac1 mutants. b Extractable PA levels in leaves of the control and smlac1 mutants quantified using DMACA reagent and expressed as epicatechin equivalents. c Non-extractable PA levels quantified by the butanol-HCl method and expressed as procyanidin B1 equivalents. d, e EIC profiles of procyanidin A1, A2, B1, B2, and B3 contents in the control and smlac1 mutants. f EIC profiles of catechin and epicatechin contents in the control and smlac1 mutants. g, k UPLC analysis of procyanidin A1, A2, B1, B2, and B3 contents in the control and five smlac1 mutants. l, m Catechin and epicatechin contents in leaves of the control and five smlac1 mutants. Values represent means ± SD from three biological replicates (n = 3). * and ** indicate P < 0.05 and P < 0.01 (One-way ANOVA), respectively. Source data are provided in the Source Data file.

Fig. 10. Scheme for the polymerization of PAs.

MiR397a-regulated LACs (e.g., SmLAC1) initiate the polymerization reaction. Both (+)-catechin and (−)-epicatechin can serve as the starter and the extension units.