Volume 10, Issue 7, Pages 1000-1003 (July 2017) Developmentally Regulated Glucosylation of Bitter Triterpenoid in Cucumber by the UDP-Glucosyltransferase UGT73AM3  Yang Zhong, Xiaofeng Xue, Zhiqiang Liu, Yongshuo Ma, Kewu Zeng, Lida Han, Jingjing Qi, Dae-Kyun Ro, Søren Bak, Sanwen Huang, Yuan Zhou, Yi Shang  Molecular Plant  Volume 10, Issue 7, Pages 1000-1003 (July 2017) DOI: 10.1016/j.molp.2017.02.005 Copyright © 2017 The Author Terms and Conditions

Figure 1 Characterization of CuC Glycoside and Functional Analysis of the UGT Involved in CuC Glucosylation. (A) The structure of CuC 3-O-β-glucopyranoside and the potential catalytic reaction to generate CuC 3-O-β-glucopyranoside from CuC. (B and C) The MTT assays reflect the correlation between the contents of CuC or CuC 3-O-β-glucopyranoside and the cell viability of HepG2 (B, hepatoma cells) or A549 (C, lung cancer cells). Untreated cells were treated with conditioned medium. Data are average values ± SD (n = 3 biological replicates). (D) The content of CuC or CuC 3-O-β-glucopyranoside in the leaves of 9930 sampled at six different nodes of the plant. N, node; N-5 to N-25, the node number counts from the bottom to the top of the plant. Developmental statuses of the leaves used in this analysis are shown in Supplemental Figure 6A. Data are average values ± SD (n = 3 biological replicates). (E) RNA-seq data (shown as log2 FPKM) of Bi and the five UGT candidates in 9930 leaves sampled at six different nodes. The numeric FPKM values are shown in Supplemental Table 3. (F) UPLC-qTOF-MS analyses of the product catalyzed by each recombinant UGT candidates during the in vitro assay. A specific peak m/z = 740.4 at the retention time of 5.35 min (indicated with a red arrow) was produced by Csa3G744990. The MS/MS spectrum of this peak is shown in Supplemental Figure 8. The assay without adding the purified protein served as the negative control. ESI+, electrospray ionization in positive mode. (G) Relative gene expression of UGTs, as well as the metabolic level of CuC glucoside and CuC in 9930 cotyledons that were transiently overexpressed with each UGT candidates. 990, Csa3G744990; 410, Csa7G051410; INF 990, the sample infiltrated with Agrobacterium tumefaciens harboring Csa3G744990; INF 410, the sample infiltrated with A. tumefaciens harboring another UGT (Csa7G051410). The ratio between CuC glucoside and CuC is presented as a molar ratio. Data were average values ± SD (n = 6 biological replicates). (H) Kinetic parameters of Csa3G744990 toward CuC and naringenin. Molecular Plant 2017 10, 1000-1003DOI: (10.1016/j.molp.2017.02.005) Copyright © 2017 The Author Terms and Conditions