Uhplc esi tofms

The transition leaf (position 0) on rosette-stage plants was treated with W+OS and excised at the petiole 72 h later by which time treated leaves had grown to position +2 or +3. Midveins were excised, and leaf tissue was flash-frozen in liquid nitrogen. Tissue was ground over liquid nitrogen to a find powder and kept at −80°C until extraction and analysis. Extracts of leaves in acidified 40% methanol (soluble metabolome) were analyzed on a Bruker UHPLC-ESI/TOFMS in positive ionization mode as previously described (Gaquerel et al., 2010 (link)). Chromatograms were exported as netCDF files and peak detection, picking and integration was performed using the R package XCMS (Smith et al., 2006 (link); Tautenhahn et al., 2008 (link)), and then ion traces were deconvoluted and putative in-source pseudo-spectra reconstructed with the R package CAMERA (Kuhl et al., 2012 (link)) as previously described (Gaquerel et al., 2010 (link); Stitz et al., 2011a (link)). The data matrix was imported to Microsoft Excel and isotopic peaks and multi-charged m/z signals detected by CAMERA were excluded to reduce the redundancy within the data matrix. Consistent mass features—present (for a single plant genotype) in four out of the five biological replicates—with a retention time >50 s were considered for further statistical analysis.

UNIVERSAL Hood gel imaging system was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA USA) for gel imaging. UV1000 spectrophotometer was purchased from Lairui Scientific Instrument Co, Ltd. (Shanghai, China) for photometric measurements. Protein electrophoresis system PowerPac™/ Mini-PROTEAN® and HC/Mini-PROTEAN® were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA USA) for SDS-PAGE analysis of expression products. A Hypersil C18 column was purchased from Thermo Fisher Scientific Inc. (Waltham, MA USA) for the quantitative analysis of L-threonine and 2,5-DMP. Avance III HD 600 MHz and UHPLC-ESI-TOF-MS were purchased from Bruker Corporation (Karlsruhe, Germany) for the qualitative analysis of L-threonine and 2,5-DMP.