| MS Description
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For the measurement of the inorganic ion c … For the measurement of the inorganic ion concentrations, whole shoots were harvested and frozen in liquid nitrogen before extraction. Frozen tissues (approximately 50 mg fresh weight) were homogenized in ten times volumes of 80% ethanol using the mixer mill MM300 (Retsch, Haan, Germany) and zirconia beads, and vacuum-evaporated at 38 °C to dryness. The dried samples were reconstituted in ten times volumes of milli-Q water (Millipore, Billerica, MA), vortexed for 10 min, and ultrafiltrated through a 0.22 μm pore filter (Millipore, Billerica, MA). The filtrates were diluted 10-fold in milli-Q water and the ion concentrations were determined using a Hewlett-Packard HP 3D capillary electrophoresis photodiode array detection system and an inorganic anion analysis kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol. Four biological replicates for each treatment were used for analysis.<br />
For the measurement of the amino acid concentrations, whole shoots were dried in the same way as for the analysis of inorganic ions, then reconstituted in four times volumes of 10 mM HCl, vortexed for 10 min, and ultrafiltrated through a 0.22 μm pore filter (Millipore, Billerica, MA). The filtrates were immediately used for the reaction with an AccQ-Fluor reagent kit (Waters, Milford, MA). Ten μl of the filtrate was mixed with 10 μl of AccQ-Fluor borate buffer, and 5 μl of AccQ-Fluor reagent solution was added, then vortexed immediately for 10 s. The mixture was incubated at 55 °C for 10 min, and centrifuged at 14 000 g at 4 °C for 5 min. The supernatants were placed into glass vials and 2 μl of the sample was applied to the ACQUITY UPLC system (Waters, Milford, MA) and separated on ACQUITY UPLC BEH C18 column (φ 2.1×50 mm). Solvent A (0.2% acetic acid, 0.1% dibutylamine, pH 5.02 with NH4OH) and solvent B (55% acetonitrile) were used for elution at 0.5 ml min−1 at 40 °C and metabolites were detected by an ACQUITY UPLC tunable UV detector at 250 nm. The gradient program was as follows: 100–96.0% solvent A (0–0.1 min, curve 6), 96.0–92.5% solvent A (0.1–2.0 min, curve 6), 92.5–60.0% solvent A (2.0–7.5 min, curve 7), 60.0–40.0% solvent A (7.5–8.0 min, curve 6), 40.0% solvent A (8.0–9.5 min), 40.0–100% solvent A (9.5–9.6 min, curve 6), 100% solvent A (9.6–10 min). The remaining portion of solvent A was solvent B. For standards, the amino acid mixture standard solution, type H (Wako, Osaka, Japan) was diluted and the standard solution for Gln, Asn, Trp, O-acetylserine (OAS), and homoserine were prepared separately before use. The chromatography data were analysed using Empower software (Waters, Milford, MA). Eight biological replicates for each treatment were used for analysis.<br />
Metabolites were also analysed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS) as described previously (Watanabe et al., 2008). In each analysis, 10 biological replicates were used. lysis, 10 biological replicates were used.
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