Orbance of ABA-GE and ABA was monitored at a wavelength of
Orbance of ABA-GE and ABA was monitored at a wavelength of 270 nm using a photodiode array detector (Dionex PDA-100). Authentic (six)-cis,trans-ABA-GE (OlChemIM) and (six)-ABA have been utilized as reference compounds. The mobile phase Coccidia medchemexpress containing the eluted peak corresponding to ABA-GE was collected into a glass vial and evaporated to dryness beneath a N2 stream at roughly 50 . Lastly, the tube was filled with argon, sealed, and stored at 220 with desiccant up to three months. To verify the purity and identity of the ABA-GE synthesized with this process, four enzymatic ABA-GE synthesis reactions with 30 nmol of nonradiolabeled UDP-Glc (Sigma) were performed. The purifications have been carried out as described, along with the obtained dried ABA-GEs have been redissolved in 100 mL of water and pooled. Aliquots of 100 mL have been mixed with 11 mL of water or 10 N NaOH. Following incubation for 1 h at 30 , one hundred mL of each mix was injected in to the previously described HPLC technique, which was utilized for the purification.Expression of the Recombinant UDP-Glucosyltransferase AtUGT71BThe expression and purification of your recombinant ABA UDPglucosyltransferase AtUGT71B6 (Lim et al., 2005) was performed together with the GST Gene Fusion System (GE Healthcare) with modifications. The intron-free AtUGT71B6 gene was directly amplified from Arabidopsis genomic DNA with all the primers 59-CCGGAATTCATGAAAATAGAGCTAGTATTCATTCCCTC-39 and 59-CCCGCTCGAGCTAGCTTTCAGTTTCCGACCAA-39 and ligated into the glutathione S-transferase gene fusion vector pGEX-4T-1 making use of the BamHIXhoI restriction internet sites. The resulting plasmid was transformed in to the Escherichia coli BL21-CodonPlus(DE3)-RIL strain (Agilent Technologies). An overnight preculture from a fresh transformant colony was grown in 20 mL of Luria-Bertani medium containing one hundred mg mL21 ampicillin. A 4-mL aliquot of this preculture was inoculated in 400 mL of prewarmed 23 yeast extract tryptone medium (16 g L21 tryptone, 10 g L21 yeast extract, 5 g L21 NaCl, adjusted to pH 7.0 with NaOH) containing 100 mg mL21 ampicillin and grown at 30 with vigorous shaking to an optical density at 600 nm of 1.0 to 1.two. This culture was then cooled on ice to around 14 to 18 , and isopropylthio-b-galactoside was added at a final concentration of 0.4 mM. Immediately after incubation at 14 for 16 h, cells had been harvested by centrifugation at 7,700g for ten min at 4 , resuspended in 20 mL of ice-cold 13 phosphate-buffered saline containing 1 (wv) Triton X-100, and frozen overnight at 220 . The following day, the suspension was thawed on ice, briefly sonicated with five bursts of three s, and centrifuged at 12,000g for ten min at four . The supernatant was incubated with 400 mL of a 50 slurry of Glutathione Sepharose 4B beads (GE Healthcare Life Sciences) for 30 min at room temperature. Following washing three times with ice-cold 13 phosphate-buffered saline, fusion proteins were eluted three instances with 200 mL of 20 mM reduced glutathione and 120 mM NaCl in one hundred mM Tris-HCl, pH 8.0, for ten min at room temperature. Pooled eluates were concentrated with a 30-kD Amicon Ultra 0.5-mL centrifugation ultrafilter (Millipore) to a protein concentration greater than five mg mL21, determined with the IL-17 Biological Activity Bradford protein assay (Bio-Rad) working with bovine serum albumin (BSA) asIsolation of Arabidopsis Mesophyll VacuolesThe preparation of intact Arabidopsis mesophyll vacuoles was according to previously described procedures (Frangne et al., 2002; Song et al., 2003), which had been further optimized. All experimental measures had been perfor.
