Exogenous 24-Epibrassinolide Interacts with Light to Regulate Anthocyanin and Proanthocyanidin Biosynthesis in Cabernet Sauvignon (Vitis vinifera L.)

Abstract

Anthocyanins and proanthocyanidins (PAs) are crucial factors that affect the quality of grapes and the making of wine, which were stimulated by various stimuli and environment factors (sugar, hormones, light, and temperature). The aim of the study was to investigate the influence of exogenous 24-Epibrassinolide (EBR) and light on the mechanism of anthocyanins and PAs accumulation in grape berries. Grape clusters were sprayed with EBR (0.4 mg/L) under light and darkness conditions (EBR + L, EBR + D), or sprayed with deionized water under light and darkness conditions as controls (L, D), at the onset of veraison. A large amount of anthocyanins accumulated in the grape skins and was measured under EBR + L and L treatments, whereas EBR + D and D treatments severely suppressed anthocyanin accumulation. This indicated that EBR treatment could produce overlay effects under light, in comparison to that in dark. Real-time quantitative PCR analysis indicated that EBR application up-regulated the expression of genes (VvCHI1, VvCHS2, VvCHS3, VvDFR, VvLDOX, VvMYBA1) under light conditions. Under darkness conditions, only early biosynthetic genes of anthocyanin biosynthesis responded to EBR. Furthermore, we also analyzed the expression levels of the BR-regulated transcription factor VvBZR1 (Brassinazole-resistant 1) and light-regulated transcription factor VvHY5 (Elongated hypocotyl 5). Our results suggested that EBR and light had synergistic effects on the expression of genes in the anthocyanin biosynthesis pathway.

Keywords: 24-Epibrassinolide; VvBZR1; VvHY5; anthocyanin biosynthesis; grape.

Figure 1

Biosynthetic pathway of anthocyanins and proanthocyanins in grape. Notes: Transcription regulators: VvMYBPA1, VvMYBPA2, VvMYBA1, VvMYBA2, VvMYB5a, VvMYB5b. PAL: phenylalanine ammonia-lyase, CHI: chalcone isomerase, DFR: dihydroflavonol 4-reductase, LAR: leucoanthocyanin reductase, ANR anthocyanin reductase, UFGT UDP-glucose: flavonoid 3-O-glucosyltransferase, LDOX: leucoanthocyandin dioxygenase.

Figure 2

Effects of the four treatments on 100-berry weight (a); reducing sugar (b) and total acidity (c) in grape berry during fruit development. Data represent the mean of three replicates ± standard deviation (error bars). The different letters (a, b, c, d) indicate significant differences between treatments at p < 0.05 (Duncan’s multiple range test). DAT: Days after treatment. The same as belows.

Figure 3

(a) Accumulation of anthocyanins per gram of skins dry weight in berry skins (cv. Cabernet Sauvignon) during development. The amounts are expressed as milligrams of cyanidin-3-monoglucoside equivalence (ME) per gram of dry berry skins (mg ME/g); (b) Accumulation of proanthocyanidins (PAs) per gram of skins dry weight in grape berries (cv. Cabernet Sauvignon) during development. The amounts are expressed as milligrams (+)-catechin equivalence (CE) per gram of dry berry skins (mg CE/g) (mean ± SE; n = 3). The different letters (a, b, c, d) indicate significant differences between treatments at p < 0.05 (Duncan’s multiple range test).

Figure 4

Individual anthocyanins content identified in Cabernet Sauvignon skins in 15 DAT and 46 DAT. Notes: Pe: 3’-Petunidin-3-O-glucoside; Po: Peonidin-3-O-glucoside; Ma: malvidin-3-O-glucoside; Po-Ac: Peonidin-3-O-(6-O-Acetyl)-glucoside; Ma-Ac: malvidin-3-O-(6-O-Acetyl)-glucoside; Pe-Co.: Petunidin-3-O-(6-O-Coumaryl)-glucoside; Ma-Co.: malvidin-3-O-(6-O-Coumaryl)-glucoside; De: Delphinidin-3-O-glucoside; Cy: Cyanidin-3-O-glucoside; D: dark; L: light; EBR + D: EBR + dark; EBR + L: EBR + light; Data represent the mean of three replicates ± standard deviation (error bars); FW: fresh weight.

Figure 5

Transcript profiles of VvCHI1, VvCHS2, VvCHS3, VvF3’5’H, VvDFR, VvLDOX, VvUFGT and VvMYBA1 as the molar ratio of the mRNA level to that of VvGAPDH in each sample (mean ± SE; n = 3). The different letters (a, b, c, d) indicate significant differences between treatments at p < 0.05 (Duncan’s multiple range test).

Figure 5

Transcript profiles of VvCHI1, VvCHS2, VvCHS3, VvF3’5’H, VvDFR, VvLDOX, VvUFGT and VvMYBA1 as the molar ratio of the mRNA level to that of VvGAPDH in each sample (mean ± SE; n = 3). The different letters (a, b, c, d) indicate significant differences between treatments at p < 0.05 (Duncan’s multiple range test).

Figure 6

Transcript profiles of VvMYB5a, VvMYB5b, VvLAR1, VvLAR2, VvANR, VvMYBPA1 and VvMYBPA2 as the molar ratio of the mRNA level to that of VvGAPDH in each sample (mean ± SE; n = 3). The different letters (a, b, c, d) indicate significant differences between treatments at p < 0.05 (Duncan’s multiple range test).

Figure 7

Transcript profiles of VvBRI1, VvBZR1 and VvHY5 as the molar ratio of the mRNA level to that of VvGAPDH in each sample (mean ± SE; n = 3). The different letters (a, b, c, d) indicate significant differences between treatments at p < 0.05 (Duncan’s multiple range test).

Figure 8

Hierarchical clustering of the transcript profiles of all genes. The relative expression levels of the genes after the four treatments compared to the reference gene, used in the original rank-based algorithm, were used for the hierarchical cluster analysis with Genesis. Red colors represent relatively higher transcript abundances, and green colors represent relatively lower transcript abundances. Sampling times and treatments are indicated at the top; the numbers A, B, C and D represent treatments D, L, EBR + D, EBR + L, respectively, and the numbers 3, 5, 15, 40 and 46 are days after treatment.