Ja ile

Samples were prepared using a slightly modified crude extraction procedure reported by Liu et al. (2012 (link)). The standard IAA, JA were purchased from Sigma-Aldrich (St. Louis, MO, USA), and ABA, OPDA (JA biosynthetic precursor 12-oxophytodienoic) and JA-Ile (the isoleucine conjugate of JA) were from OlChemIm (OlChemIm, Olomouc, Czech Republic). The internal standards were ²H₆ABA (OlChemIm) for ABA, 10-dihydro-JA (DHJA; OlChemIm) for JA, OPDA and JA-Ile, and D₂-IAA (Sigma-Aldrich) for IAA. The modified steps were using internal standards, 30 ng ²H₆ABA, 40 ng DHJA, and 50 ng D₂-IAA for each sample, and the filtrate was dried then dissolved in 200 μL 50% methanol. Total IAA, JA, OPDA, JA-Ile, and ABA were determined by ultra-fast liquid chromatography-electrospray ionization tandem mass spectrometry (UFLC-ESI-MS).

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.

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).