13c5 proline

All standard reagents were of analytical grade or equivalent and obtained from Sigma-Aldrich (St Louis, MO, USA), Merck (Darmstadt, Germany) and J.T. Baker (Phillipsburg, NJ, USA). N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) plus 1% trimethylchlorosilane (TMCS) and pyridine were obtained from Thermo Fisher Scientific (Rockford, IL, USA). The stable isotope-labelled internal standards, [¹³C₅]-proline, [²H₄]-succinic acid, [¹³C₅,¹⁵N]-glutamic acid, [1,2,3-¹³C₃]-myristic acid, [²H₇]-cholesterol and [13C4]-disodium α-ketoglutarate were purchased from Cambridge Isotope Laboratories (Andover, MA); [¹³C₆]-glucose, [¹³C₁₂]-sucrose, [¹³C₄]-hexadecanoic acid and [²H₄]-putrescine were purchased from Campro (Veenendaal, The Netherlands) and [²H₆]-salicylic acid was obtained from Icon (Summit, NJ). Stock solutions of each internal standard were prepared in either Milli-Q water or methanol to a concentration of 0.5 μg/μL and stored at 4°C.

The chemicals used for sample preparation were all of analytical grade, except as otherwise stated. The following were purchased from Cambridge Isotope Laboratories, (Andover, MA, USA): stable isotope labelled IS compound 13C5-proline, 2H4-succinic acid, 13C5, 15 N-glutamic acid, 1,2,3-13C3 myristic acid, 2H7-cholesterol and 13C4 disodium alpha-ketoglutarate. The following were purchased from Campro (Veenendaal, The Netherlands): 13C12-sucrose, 13C4-palmitic acid and 2H4-butanediamine. 2HCl and 13C6-glucose from Aldrich (Steinheim, Germany) and 2H6-salicylic acid from Icon (Summit, NJ, USA). Stock solutions of the IS were prepared either in purified and deionised water (Milli-Q, Millipore, Billerica, MA, USA) or in methanol (J.T.Baker, Deventer, Holland) at the same concentration, 0.5 μg/μl. Metyl stearate was purchased from Sigma (St. Louis, MO, USA). N-Metyl-N-trimethylsilyltriflouroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) and pyridine (silylation grade) were purchased from Pierce Chemical Co, Dallas,TX, USA). Heptane was purchased from Fisher Scientific (Loughborough, UK).

Quantification of proline content was conducted as described in Kusano et al. (2007) (link). Briefly, each frozen sample was extracted with a concentration of 25 mg fresh weight (FW) of whole seedling per milliliter of extraction medium (methanol/chloroform/water, 3:1:1 v/v/v) containing 10 stable isotope reference compounds. An equivalent of the 27.8 µg FW of the derivatized sample was injected to Leco Pegasus HT gas chromatography–time-of-flight mass spectrometry (LECO, St. Joseph, MI, USA). Data processing was performed using ChromaTOF version 4.72.0.0 (LECO, St. Joseph, MI, USA). A calibration curve was generated by analyzing the derivatized proline at concentrations of 0.1, 0.5, 1.0, 3.0, 5.0, and 10 ng/µl of injection buffer, respectively. We applied specific ions at m/z 142 and 216 to quantify proline content. For normalization, 15 ng/µl of [¹³C₅]-proline (Cambridge Isotope Laboratories, Andover, MA, USA) was used. We chose m/z 146 and 220 for it.