Udp u 14c glucose

For callose synthase assays, the CW, MF, PEF fractions (Figure 1) were incubated for 16 h at 24°C in 100 mM Tris-HCl buffer pH 7, containing 8 mM CaCl₂, 0.8 mM UDP-glucose and 0.76 μM UDP-[U-¹⁴C]glucose (263 mCi/mmol; Perkin Elmer, Boston, MA, United States) (Brown et al., 2012 (link)). To test the effect of calcium on callose synthase activity, assays were repeated in the same reaction mixtures as above, except for the addition of 10 mM EDTA and the omission of CaCl₂. All assays were performed in triplicates on at least three BRs for each sample type. The insoluble product formed in the callose synthase reaction mixtures was characterized by enzymatic hydrolysis using the specific endo-1,3-β-glucanase from Trichoderma sp. (50 U/ml) (Megazyme, Ireland). For this purpose, the reaction mixtures were centrifuged at 21,000 × g for 15 min and the resulting pellets containing insoluble radioactive polysaccharides were resuspended in 50 mM sodium acetate buffer pH 4.5, and subjected to enzymatic hydrolysis for 5 h at 40°C in the presence of 0.026 enzyme unit. In these conditions, one unit of enzyme releases 1 μmol of glucose from carboxymethylated curdlan. Negative controls were prepared by replacing the hydrolytic enzyme with acetate buffer.

Reagents used were analytical grade and were obtained from the following sources: quercetin, FeSO₄, CuSO₄, and ZnCl₂ were from MilliporeSigma (St. Louis, MO, USA); kaempferol was from Indofine (Hillsborough, NJ, USA), UDP-glucose was from Calbiochem (Gibbstown, NJ, USA), UDP- [U-14C] glucose was from PerkinElmer (Boston, MA, USA); Midiprep plasmid extraction kit was obtained from Promega (Madison, WI, USA). Miniprep plasmid extraction kit, MgCl₂, MnCl₂, KCl, and NaCl were purchased from Fisher Scientific (Waltham, MA, USA); CaCl₂ was from Acros Organics (Morris Plains, Morris, NJ, USA); Na₂SO₄ was from Merk (Kenilworth, NJ, USA). Bovine thrombin was obtained from MP Biomedicals (Solon, OH, USA).