2h4 sa

For the hormonal analysis, fresh material was frozen in liquid N, ground and freeze-dried. Fresh tissue (0.5 g) was immediately homogenized in 2.5 mL of ultrapure water, and 100 ng mL^-1 of a mixture of internal standards [(²H₆-ABA (to quantify ABA), ²H₄-SA (to quantify SA), dihydrojasmonic acid [to quantify OPDA, JA, and JA-Ile) and propylparaben (to quantify ferulic acid), all acquired from Sigma–Aldrich] were added prior to extraction (Flors et al., 2008 (link)). After extraction, a 20-μL aliquot was injected directly into a ultra-high-performance liquid chromatography (UPLC) system. Hormone analyses were carried out in a Waters Alliance 2690 UPLC system (Milford, MA, United States) inside a nucleosil ODS reversed-phase column (100 mm × 2 mm i.d.; 5 μm; Scharlab, Barcelona, Spain⁵). The chromatographic system was interfaced to a Quattro LC (quadrupole–hexapole–quadrupole) mass spectrometer (Micromass⁶). Version 4.1 of the MASSLYNX NT software (Micromass), was used to process the quantitative data from the calibration standards and plant samples. The concentrations of hormones and metabolites were determined in each sample by normalizing the chromatographic area for each compound with the fresh weight of the corresponding sample.

The youngest fully expanded leaf samples (ca 50 mg FW) were purified and analyzed according to Dobrev and Kamínek (2002), Dobrev and Vankova (2012) and Svačinova et al. [94 –96 ]. Frozen samples were homogenized and extracted with cold (− 20 °C) methanol/water/formic acid (15/4/1, v/v/v). The following isotope-labelled internal standards (10 pmol/sample) were added: ¹³C₆-IAA (Cambridge Isotope Laboratories); ²H₄-SA (Sigma-Aldrich); ²H₃-PA, ²H₃-DPA (NRC-PTI); ²H₆-ABA, ²H₅-JA, ²H₅-transZ, ²H₅-transZR, ²H₅-transZ7G, ²H₅-transZ9G, ²H₅-transZOG, ²H₅-transZROG, ²H₅-transZRMP, ²H₃-DZ, ²H₃-DZR, ²H₃-DZ9G, ²H₆-iP, ²H₆-iPR, ²H₆-iP7G, ²H₆-iP9G, ²H₆-iPRMP (Olchemim). Phytohormones were separated with a reverse phase-cation exchange SPE column (Oasis-MCX, Waters) into the acid fraction by elution with methanol [auxins, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA)], and into the basic fraction by elution with 0.35 M NH₄OH in 60% methanol [cytokinins (CKs)]. Fractions were analyzed using HPLC (Ultimate 3000, Dionex) coupled to a 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems). Hormone quantification was performed by the isotope dilution method with multilevel calibration curves (r² > 0.99). Data processing was performed with the Analyst 1.5 software package (Applied Biosystems). Raw data are included in the Supplemented Table 3.

Frozen samples (100 mg FW) were homogenized with liquid nitrogen in a mortar and pestle. The phytohormones were extracted with a cold (−20 °C) methanol/water/formic acid mixture (15/4/1, v/v), as described in [84 ]. Internal isotope-labeled standards (10 pmol per sample) were added for hormone analysis: ¹³C₆-IAA (Cambridge Isotope Laboratories, Tewksbury, MA, USA); ²H₄-SA (Sigma-Aldrich, St. Louis, MO, USA); ²H₃-PA, ²H₃-DPA, ²H₅-ABA-GE (NRC-PBI, Saskatoon, SK, Canada); and ²H₆-ABA, ²H₅-JA, and others (Olchemim, Olomouc, Czech Republic). The extracts were passed through reversed-phase cation exchange SPE columns (Oasis-MCX, Waters, Milford, MA, USA) in a mixed mode (mixed phase–cation exchange). The hormone fraction containing ABA, IAA, SA, and JA was eluted with methanol. Hormone metabolites were analyzed using HPLC (Ultimate 3000, Dionex, Sunnyvale, CA, USA) coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer (3200 Q TRAP, Applied Biosystems, Waltham, MA, USA). The quantification of hormones was carried out using the isotope dilution method with multilevel calibration curves (R² > 0.99). Data processing was carried out with Analyst 1.5 software (Applied Biosystems).

Authentic and stable isotope labelled standards (Table 1) were provided from in-house standard library of JAs: cis-OPDA, OPC-8, OPC-6, OPC-4, dn-OPDA, JA, MeJA, 9,10-dhJA, 12-OHJA, JA-Val, JA-Ile, JA-Phe and [²H₅]-OPDA, [²H₆]-JA, [²H₂]-( − )-JA-Ile were purchased from OlChemIm Ltd. (Olomouc, Czech Republic), JA-Trp was synthetised as described in [64 ], 11-OHJA was kindly provided by Dr. Otto Miersch, [²H₆]-( ±)-MeJA was synthesized by acetyl chloride–Methanol-d₄ esterification; AUXs: IAA was purchased from Sigma Aldrich (St. Louis, MO, USA), oxIAA, IAA-Asp, IAA-Glu, IAA-glc, oxIAA-glc and [¹³C₆]-IAA, [¹³C₆]-IAA-Asp, [¹³C₆]-IAA-Glu were purchased from OlChemIm Ltd. (Olomouc, Czech Republic), [¹³C₆]-oxIAA was synthesized as described in [65 (link)], [¹³C₆]-IAA-glc, [¹³C₆]-oxIAA-glc were synthesized as described in [66 (link), 67 (link)]; ABAs: ABA, PA, DPA, neoPA, 7′-OHABA and [²H₆]-ABA, [²H₃]-PA, [²H₃]-DPA, [²H₃]-neoPA, [²H₄]-7′-OHABA were purchased from National Research Council Canada (Saskatoon, Canada); SAs: SA and [²H₄]-SA were purchased from Sigma Aldrich (St. Louis, MO, USA). Methanol, acetonitrile, formic acid, all LC–MS grade, acetic acid of gradient grade and hydrochloric acid were purchased from Sigma-Aldrich (St. Luis, MO, USA). Purified Milli-Q water from a Simplicity 185 System was used for preparation of aqueous solutions.

Frozen samples (ca. 50 mg FW) were homogenized and extracted with cold (−20 °C) methanol/water/formic acid (15/4/1, v/v/v) as described previously [50 (link),51 ]. The following isotope-labelled internal standards (10 pmol/sample) were added: ¹³C₆-IAA (Cambridge Isotope Laboratories); ²H₄-SA (Sigma-Aldrich); ²H₃-PA, ²H₃-DPA (NRC-PBI); ²H₆-ABA, ²H₅-JA, ²H₅-transZ, ²H₅-transZR, ²H₅-transZ7G, ²H₅-transZ9G, ²H₅-transZOG, ²H₅-transZROG, ²H₅-transZRMP, ²H₃-DZ, ²H₃-DZR, ²H₃-DZ9G, ²H₆-iP, ²H₆-iPR, ²H₆-iP7G, ²H₆-iP9G, ²H₆-iPRMP (Olchemim). Phytohormones were separated with a reverse-phase cation exchange SPE column (Oasis-MCX, Waters) into the acid fraction by elution with methanol (auxins, ABA, SA, JA), and into the basic fraction by elution with 0.35 M NH₄OH in 60% methanol (CKs). Fractions were analyzed using HPLC (Ultimate 3000, Dionex) coupled to a 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems). Hormone quantification was performed by the isotope dilution method with multilevel calibration curves (r² > 0.99). Data processing was performed with the Analyst 1.5 software package (Applied Biosystems).

Cytokinin labeled and nonlabeled standards (see Table 1), [²H₆]-ABA; JA; [²H₆]-JA; JA-Ile; [²H₂]-(-)-JA-Ile; oxIAA; IAA-Asp; IAA-Glu; [¹³C₆]-IAA; [¹³C₆]-IAA-Asp and [¹³C₆]-IAA-Glu were obtained from Olchemim Ltd. (Olomouc, Czech republic). [¹³C₆]-oxIAA came from laboratory library of standards [47 (link)]. ACC; [²H₄]-ACC; SA; [²H₄]-SA; ABA and IAA were acquired from Sigma Aldrich (St. Louis, MO, USA). AccQ-Tag™ Ultra Derivatization Kit for UPLC Amino Acid Analysis is from Waters Inc. (Milford, MA, USA). All other chemicals were of analytical or higher grade from Sigma Aldrich (St. Louis, MO, USA).