Udp glucose

Reactions were performed at 25 °C in 100 mM Tris-Cl pH 7, and 2 different concentrations of DON in the reactions were tested. Low DON concertation reactions contained 30 µM DON (Sigma Aldrich, Lyon, France), 1 mM UDP-glucose (Uridine-diphosphate-glucose disodium salt) (Sigma Aldrich, Lyon, France) and 1 mg/mL purified enzyme. High DON concertation reactions contained 1 mM DON, 10 mM UDP-glucose and 1 mg/mL purified enzyme. Preparations of proteins expressed by E. coli transformed with the empty vector were used as controls. All reactions were performed in three repetitions. Samples from the reactions were taken at 0, 1, 2, 4, 6, 24 h after the start, and the reactions were stopped by adding 10 volumes of 99.8% methanol (Sigma-Aldrich, Lyon, France). Products of the reactions were analysed with quantitative LC-MS/MS.

Ginkgo leaves, seed coats, and seeds at different developmental stages from June 15th to September 15th were collected in Beijing botanical gardens. Mature seeds from 58 cultivars were collected in October at the Pizhou Resource Nursery (Jiangsu, China). These samples were immediately frozen in liquid nitrogen and stored at −80 °C for further use. The substrates tested in this study were purchased from Xili Limited Co. (Shanghai, China) and Indofine (Hillsborough, NJ, USA). UDP-glucose, UDP-galactose, and UDP-glucuronic acid were purchased from Sigma-Aldrich (Oakville, CA, USA). UDP-rhamnose was enzymatically synthesized using methods published by Rautengarten et al.^{39 (link)}. All chemicals used in this study were of analytical or HPLC grade.

Enzyme activity was assayed as previously described [51 (link)], using 200 ng α-1,6-mannobiose (Dextra Laboratories, Reading, UK) as the rhamnose acceptor and 500 µM UDP-L-rhamnose (Chemily Glycoscience; Peachtree Corners, GA, USA). The reaction products were analyzed by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) with a Dionex system (Thermo Fisher Scientific), using a CarboPac PA-100 column (4.6 × 250 mm) and a linear gradient of 10–100 mM sodium acetate in 100 mM NaOH at a flow rate of 0.8 mL min⁻¹ for 30 min. [52 (link)]. For assays including treatment with α-L-rhamnosidase, the reaction product of rhamnosyltransferase activity was mixed with 1 U enzyme (Megazyme, Bre, Ireland) and incubated for 60 min at 50 °C. The reactions were stopped by boiling for 10 min and subjected to monosaccharide separation by HPAEC-PAD using the following conditions: a CarboPac PA-200 analytical column (3 × 250 mm) with a CarboPac PA-200 guard column (3 × 50 mm) and an isocratic gradient of 3.2 mM NaOH with a flux rate of 0.15 mL min⁻¹ for 25 min [53 (link)]. Fluorescence-assisted carbohydrate analysis (FACE) was performed essentially as previously described [54 (link)]. UDP-glucose, GDP-mannose, UDP-N-acetylglucosamine, and UDP-galactose were from Sigma-Aldrich.

Following electrophoresis, gel lane was cut into sections containing sample lane and rinsed in 100 mL of 10 mM Tris-HCL (pH 7.5) for 30 min and buffer was changed after 15 min. Each gel section was then placed in a plastic petri dish and incubated for enzyme activity at room temperature in 50 mM Tris-HCL (pH 7.5), 3 mM NaN₃, and UDP-glucose (Sisco Research Laboratories, India). Unreacted substrate was removed from gel sections by rinsing twice for 20 min each in 100 mL of 5 mM EDTA, 10 mM Tris-HCL (pH 7.5). The β-glucan product in the gel was visualized with Congo red after staining in 50 mL of 0.1% Congo red for 30 min and destaining for 20 min in 100 mL of 10 mM Tris-HCL (pH 7.5) buffer.
An alternative method was employed to detect the β-glucan product. After reaction with the substrate UDP-glucose, the β-glucan product was visualized by its fluorescence under UV light staining with Tinopal (4,4′-distyrylbiphenyl sodium sulfonate salt, Sigma-Aldrich) in solution of 50 mL of 0.01% Tinopal blue in K₂PO₄ buffer, pH 8.2, for 10 min. The gel was then washed in water and stored in the same solution.

UDP-glucose, UDP-galactose, UDP-rhamnose, UDP-arabinose, cyanidin, delphinidin, and malvidin were purchased from Sigma-Aldrich (USA), and pelargonidin, petunidin, peonidin, cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, pelargonidin 3-O-glucoside, malvidin 3-O-glucoside, petunidin 3-O-glucoside, and peonidin 3-O-glucoside were obtained from Phytolab (Germany).

Tea samples (Camellia sinensis cultivar Shuchazao) for qRT-PCR were all collected from the Horticultural Research Station of Anhui Agricultural University. The leaves for heat treatment were collected and then immediately put into the MS liquid medium at 50°C for 0.5 h, 1 h and 6 h, and then recovered to 20°C for 0.5 h and 1 h, and 20°C was used as control. The leaves were then rapidly frozen in liquid nitrogen and stored at -80°C until use.
Substrates tested in the present study were purchased from Tongtian Limited Co., Ltd (Shanghai, China) and Indofine (Hillsborough, NJ, USA). UDP-glucose was purchased from Sigma-Aldrich (Oakville, CA, USA). Chemicals used in this study were of analytical or HPLC grade.