Hypocotyls from the wild-type Col-0, single mutants tir1-1 and afb2-3, and double mutant tir1-1afb2-3 were collected from seedlings grown as described by Gutierrez et al. (2012) . Samples were prepared from six biological replicates; for each, at least two technical replicates were performed. Endogenous levels of free IAA, SA, and JA as well as JA-Ile, the conjugated form of JA, were determined in 20 mg of hypocotyls according to the method described by Flokova et al. (2014) . The phytohormones were extracted using an aqueous solution of methanol (10% MeOH/H 2 O, v/v). To validate the liquid chromatography-mass spectrometry method, we added a cocktail of stable isotope-labeled standards with the following composition: 5 pmol of [ 13 C 6 ]IAA, 10 pmol of [ 2 H 6 ]JA and [ 2 H 2 ]JA-Ile, and 20 pmol of [ 2 H 4 ]SA (all from Olchemim, Czech Republic) per sample. The extracts were purified using Oasis HLB columns (30 mg/1 ml; Waters, Milford, MA, USA) and targeted analytes were eluted using 80% MeOH. Eluent containing neutral and acidic compounds was gently evaporated to dryness under a stream of nitrogen. Separation was performed on an Acquity UPLC System (Waters) equipped with an Acquity UPLC BEH C18 column (100 3 2.1 mm, 1.7 mm; Waters), and the effluent was introduced into the electrospray ion source of a triple quadrupole mass spectrometer Xevo TQ-S MS (Waters).
13c6 iaa
Manufactured by OlChemIm
Sourced in Czechia
[13C6]-IAA is a stable isotope-labeled form of the plant hormone indole-3-acetic acid (IAA). It is used as a tracer compound in various plant biology and biochemistry studies.
Automatically generated - may contain errors
Lab products found in correlation
2h4 sa, by Merck Group (2 mentions)
Ja ile, by OlChemIm (2 mentions)
2h6 ja, by OlChemIm (2 mentions)
2h2 ja ile, by OlChemIm (2 mentions)
Oxiaa, by OlChemIm (2 mentions)
Iaa asp, by OlChemIm (2 mentions)
Iaa glu, by OlChemIm (2 mentions)
13c6 iaa asp, by OlChemIm (2 mentions)
13c6 iaa glu, by OlChemIm (2 mentions)
2h6 aba, by OlChemIm (2 mentions)
Oasis hlb column, by Waters Corporation (1 mentions)
Xevo tq s ms, by Waters Corporation (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
Lc ms grade, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Cis opda, by OlChemIm (1 mentions)
4 protocols using 13c6 iaa
1
Phytohormone Quantification in Arabidopsis
2
Quantification of Plant Hormones and Auxins
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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.
3
Extraction and Quantification of Plant Hormones
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For extraction, 900 μL ethyl acetate and 100 μL of 50 ng/mL [13C6]-IAA (OlChemIm), 50 ng/mL [15C4]-trans zeatin (OlChemIm), 50 ng/mL 2H6-ABA (OlChemIm), and 50 ng/mL [2H4]-SA (OlChemIm) isotope internal standard were added to each sample (100 mg). The samples were vortexed and sonicated for 20 min at 4°C and centrifuged at 12,000 g for 10 min at 4°C. The supernatant was transferred into a new 2-mL centrifugal tube and then was centrifuged to dry. The precipitation was redissolved with 200 μL of precooled 70% methanol. After filtration through a 0.22-μm PVDF filter, the solution was taken in a sample bottle and then analyzed by UPLC-QqQ MS (ACQUITY TQD).
4
Quantification of Plant Hormones
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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).
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