Gene specific primers used in the qRT-PCRs

Gene specific primers used in the qRT-PCRs. Table S3. acid biosynthesis. and are among the core hypoxia-responsive genes described by Mustroph and colleagues (2009) and shown to be controlled by members of the group VII ethylene response factor (ERF-VII) family, whose activity is directly linked to oxygen availability. In brief, in the presence of oxygen, ERF-VII transcription factors are degraded by the 26S proteasome following the N-degron pathway, while in low-oxygen conditions they are stabilized and can activate the hypoxic genes (Gibbs (described in Abbas (2015) and kindly provided to TERT us by Michael Holdsworth (University of Nottingham)), and a chimeric reporter line (Weits and genes (gene (and and (gene (lines were surface sterilized with 70% ethanol for 30 s and rinsed six times with sterile deionized water. Seedlings were grown in sterile six-well plates in liquid medium under continuous shaking. The liquid medium was half-strength Murashige and Skoog medium (pH 5.7) (Sigma-Aldrich Co., St Louis, MO, USA) enriched with 1% (w/v) sucrose. In each well about 10 sterile seeds were placed and incubated for 3 d at 4 C in darkness. Later, plates were moved to a growth chamber and seedlings were grown under 80 mol NOD-IN-1 m?2 s?1 photosynthetically active radiation, at 23 C/18 C day/night temperature and at a 12/12 h day/night photoperiod. When plants were 6 d old, the old growth medium was replaced with fresh sucrose-free half-strength Murashige and Skoog medium (pH 5.7), and the desired herbicide dose where needed. Commercial formulations of imazamox and glyphosate were used at a final concentration in the growth medium of 1 1.5 mg active ingredient l?1 (4.9 M) of Pulsar?40 (BASF Espa?ola SA, Barcelona, Spain), in the case of imazamox, or 50 mg active ingredient l?1 (219.12 M) of Fortin Green? (Industrial Qumica NOD-IN-1 Key, SA, Trrega, Lleida, Spain), in the case of glyphosate (Gil-Monreal mutants was maintained under herbicide treatment until death was observed. Untreated plants were used as controls. In addition, recovery of wild-type and mutants was evaluated by transferring the 5-d treated seedlings to herbicide-free half-strength Murashige and Skoog medium (pH 5.7) enriched with 1% (w/v) sucrose. The nutrient solution, with or without herbicides, was renewed every 4 d. In both lethality and recovery experiments, visual inspection was used as a marker of lethality and plates were scanned using a GS-800 densitometer (Bio-Rad Laboratories Inc., Hercules, CA, USA). Chlorophyll extraction and quantification Total chlorophyll was extracted from homogenized seedlings using 80% (v/v) acetone buffered with 2.5 mM Na-phosphate at pH 7.8. Samples were incubated for 30 min in the dark at 4 oC with shaking. The debris was pelleted by centrifugation at 5000 for 10 min. The chlorophyll content was spectrophotometrically measured in the supernatant according to Porra (1989). Four biological replicates were used for each experimental condition. Lipid peroxidation assay The extent of lipid peroxidation was estimated spectrophotometrically by the amount of malondialdehyde in seedlings as described by Hodges (1999). This method takes into account the possible interference generated by non-specific turbidity, thiobarbituric acidCsugar complexes and other non-thiobarbituric acid reactive substances absorbing at 532 nm. Four biological replicates were analysed for each experimental condition. activities of PDC and ADH The activities of PDC and ADH were assayed in ground seedlings as described in NOD-IN-1 Gaston (2002). Briefly, PDC and ADH activities were measured in a spectrophotometer monitoring NADH consumption or NOD-IN-1 formation at 340 nm, respectively. Four biological replicates were analysed for each experimental condition. PDC and ADH immunoblotting PDC and ADH protein immunoblot assay was performed according to standard techniques (as in Zulet values lower than the geNorm threshold of 1 1.5 and SD of online). The geometric mean of the expression ratios of the two most stable reference genes (and seedlings was evaluated with the aid of the Dual-Luciferase? Reporter (DLRTM) Assay System NOD-IN-1 (Promega). Approximately 100 mg fresh tissue was extracted with 400.