Fig 1.
1-O-sinapoylglucose biosynthesis pathway.
Sinapic acid and UDP-glucose act as potential substrates for the UDP-glucose: sinapic acid glucosyltransferase, which catalyzes the transfer of glucose moiety from UDP-glucose to the 1-O-position of sinapic acid.
Table 1.
Primer sequences of DcUSAGT1 gene and the primer sequences used for Quantitative Real time PCR amplification of Dcactin and DcUSAGT1 gene.
Fig 2.
Structure-based domain sequence alignments of DcUSAGT1 with UDP-glucose: glucosyltransferases in other plants.
Fig 3.
Predicted 3D structure of UGT72B1 and DcUSAGT1.
(A) 3D structure of UGT72B1. (B) 3D structure of DcUSAGT1. N represents the N-terminal, C represents the C-terminal. Red-helix represents α-helix, blue-sheet represents β-sheet. The long deep cleft in the middle of the structure is used for substrate binding.
Fig 4.
Chromatograms of product formation by the purified enzyme of UDP-glucose: sinapic acid glucosyltransferase in carrots by HPLC.
Peak area A represents the reaction mix without enzyme as the blank control. Peak area B represents the reaction volume with deactivated enzyme. Peak area C represents the reaction system with active enzyme. The retention time of the product: 1-O-sinapoyl-glucose was about 5.6 min, and the peaks for sinapic acid were 9.1 min in average.
Fig 5.
Effects of temperature and optimal pH value of rDcUSAGT1 activity with sinapic acid as substrate.
(A) Effect of temperature. (B) Optimal pH value. The values are the means of three separate experiments with error bars showing ± SD.
Table 2.
Kinetic analysis data of rDcUSAGT1.
Fig 6.
Photos of purple and non-purple carrot cultivars used in this study.
D represents ‘Deep purple’, T represents ‘Tianzi2hao’, and P represents ‘purple68’; S represents ‘Sanhongliucun’, K represents ‘Kurodagosun’, and J represents ‘Junchuanhong’.
Fig 7.
Expression profiles of DcUSAGT1 gene in three purple carrot cultivars and three other non-purple carrot cultivars.
The mRNA level of Dcactin was defined as 1. The values are the means from triplicate qRT-PCR analysis with error bars showing ± SD. D represents ‘Deep purple’, T represents ‘Tianzi2hao’, and P represents ‘purple68’; S represents ‘Sanhongliucun’, K represents ‘Kurodagosun’, and J represents ‘Junchuanhong’.
Fig 8.
Chromatogram of USAGT activity in vitro by HPLC.
(A) Reaction of crude protein from ‘deep purple’ carrot taproots. (B) Reaction of crude protein from non-purple ‘Kurodagosun’ carrot taproots.