For the temperature test, 50 mM HEPES (pH7.0) was added plus the reactions have been performed at four different temperature values (20uC, 30uC, 37uC and 45uC). For the buffer and pH test, 50 mM Tris buffer (pH six.0?.0), 50 mM HEPES buffer (pH 5.0?.0),50 mM MES buffer (pH five.0?.0) or 50 mM phosphate buffer (pH six.0?.0) was added plus the reactions were performed at 37uC. All of the reactions had been carried out for 30 min and after that stopped by adding ten ml trichloroacetic acid (240 mg/ml), quickfrozen, and stored at 220uC just before reverse-phase HPLC analysis.Assays of Glycosyltransferase Activity and Glucosylated Metabolites of Auxin in Transgenic PlantsThe full-length cDNA on the UGT74D1 gene was subcloned in the pBluescriptSK into the plant overexpression vector pBI121 and replaced the glucuronidase (GUS) gene. The overexpression construct was transferred into Agrobacterium tumefaciens GV3101 and after that transformed into Arabidopsis (Col-0) viaPLOS One particular | plosone.orgUGT74D1 Novel Auxin GlycosyltransferaseFigure 5. Analyses on components affecting the activity of recombinant UGT74D1. (A) The effects of temperature. (B) The effects of buffer and pH worth. Each of the reaction mix (one hundred ml) contained 0.2 ug of recombinant UGT74D1, five mM UDP-glucose, 1 mM IBA, 2.five mM MgSO4, 10 mM KCl, 14.four mM 2-mercaptoethanol, 50 mM buffer and was incubated for 30 min as described in “Materials and Procedures ”. The outcomes represent means6SD from three replicates. The precise enzyme activity was defined as nmol of substrates converted into glucose conjugates per second (nanokatal, nkat) by 1 mg of protein. doi:10.1371/journal.pone.0061705.gfloral dip approach [44]. At least four homozygous transgenic lines had been selected by kanamycin resistance and also the overexpression of UGT74D1 was determined by RT-PCR.Buy2-Chloro-1,3,4-thiadiazole Total crude protein was extracted from 2-week-old transgenic seedlings as described previously [42].DBCO-acid Price To investigate the glycosyltransferase activity with the crude protein extracts prepared from plant tissues, 50 ml crude protein extracts (containing ,0.PMID:23659187 1 mg of total protein) have been mixed 1 mM auxin, five mM UDP-glucose, 50 mM HEPES (pH7.0), two.5 mM MgSO4, ten mM KCl, and 14.four mM 2-mercaptoethanol, in a 100 ml reaction. The reactions had been incubated at 37uC for 1 h and were stopped by the addition of ten ml of trichloroacetic acid (240 mg/ml). The reaction mix was analyzed subsequently making use of reverse-phase HPLC following the system described above. To analyze the level of the glucose conjugates of interest within the transgenic plants, the wild-type and UGT74D1 transgenicplants (line 23, line 24) were grown on the MS agar plates for 12 days, and removed very carefully to immersed in MS liquid culture technique with or with no one hundred mM IBA. Right after incubation for 24 h, 1 g of plant tissues from each line was collected, frozen in liquid nitrogen, and stored at 280uC before the extraction. The extraction of IBA glucose conjugates was carried out following the method described previously [42]. 0.1 mM picloram was added as internal control at the starting from the extraction to monitor the recovery rate. The amounts of IBA glucose conjugates in extraction buffers of different transgenic lines were analyzed by HPLC as described above.Leaf-flattening Experiments of Transgenic PlantsArabidopsis plants had been grown within the greenhouse on Nutrition Soil (Shangdao Biotech Co. Ltd., Shandong, China) with vermiculite (Nutrition Soil:vermiculite = 2:1) at 2262uC beneath a 16/8 h light/PLOS 1 | plosone.orgUGT74D1 Novel Auxin Glyco.
