BACKGROUND: Gaseous phytohormone ethylene levels are directly influenced by the production of its immediate non-volatile precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Owing to the strongly acidic character of the ACC molecule, its quantification has been difficult to perform. Here, we present a simple and straightforward validated method for accurate quantification of not only ACC levels, but also major members of other important phytohormonal classes - auxins, cytokinins, jasmonic acid, abscisic acid and salicylic acid from the same biological sample. RESULTS: The presented technique facilitates the analysis of 15 compounds by liquid chromatography coupled with tandem mass spectrometry. It was optimized and validated for 10 mg of fresh weight plant material. The extraction procedure is composed of a minimal amount of necessary steps. Accuracy and precision were the basis for evaluating the method, together with process efficiency, recovery and matrix effects as validation parameters. The examined compounds comprise important groups of phytohormones, their active forms and some of their metabolites, including six cytokinins, four auxins, two jasmonates, abscisic acid, salicylic acid and 1-aminocyclopropane-1-carboxylic acid. The resulting method was used to examine their contents in selected Arabidopsis thaliana mutant lines. CONCLUSION: This profiling method enables a very straightforward approach for indirect ethylene study and explores how it interacts, based on content levels, with other phytohormonal groups in plants.

Department of Forest Genetics and Plant Physiology Umeå Plant Science Centre Swedish University of Agricultural Sciences Umeå SE 901 83 Sweden

Department of Plant Physiology Umeå Plant Science Centre Umeå University Umeå SE 901 87 Sweden

Laboratory of Growth Regulators Institute of Experimental Botany Palacký University The Czech Academy of Sciences and Faculty of Science Olomouc CZ 783 71 Czechia

Zobrazit více v PubMed

Vanderstraeten L, Van Der Straeten D. Accumulation and Transport of 1-Aminocyclopropane-1-Carboxylic Acid (ACC) in Plants: Current Status, Considerations for Future Research and Agronomic Applications. 2017;8:1–18. PubMed PMC

Sauter M, Moffatt B, Saechao MC, Hell R, Wirtz M. Methionine salvage and S-adenosylmethionine: essential links between sulfur, ethylene and polyamine biosynthesis. Biochem J. 2013;451:145–54. doi: 10.1042/BJ20121744. PubMed DOI

Polko JK, Kieber JJ. 1-Aminocyclopropane 1-Carboxylic acid and its emerging role as an Ethylene-Independent Growth Regulator. Front Plant Sci. 2019;10:1–9. doi: 10.3389/fpls.2019.01602. PubMed DOI PMC

Adams DO, Yang SF. Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc Natl Acad Sci USA. 1979;76:170–4. doi: 10.1073/pnas.76.1.170. PubMed DOI PMC

Van De Poel B, Van Der Straeten D. 1-aminocyclopropane-1-carboxylic acid (ACC) in plants: more than just the precursor of ethylene! 2014;5:1–11. PubMed PMC

Li D, Mou W, Van de Poel B, Chang C. Something old, something new: conservation of the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid as a signaling molecule. Curr Opin Plant Biol. 2022;65:102116. doi: 10.1016/j.pbi.2021.102116. PubMed DOI

Friml J. Fourteen stations of Auxin. Cold Spring Harb Perspect Biol. 2022;14. PubMed PMC

Brunoni F, Collani S, Šimura J, Schmid M, Bellini C, Ljung K. A bacterial assay for rapid screening of IAA catabolic enzymes. Plant Methods. 2019;15. PubMed PMC

Stepanova AN, Alonso JM. From ethylene-auxin interactions to auxin biosynthesis and signal integration. Plant Cell. 2019;31:1393–4. doi: 10.1105/tpc.19.00339. PubMed DOI PMC

Romanov GA, Schmülling T. On the biological activity of cytokinin free bases and their ribosides. Planta. 2021;255. PubMed PMC

Sakakibara H. Cytokinin biosynthesis and transport for systemic nitrogen signaling. Plant J. 2021;105:421–30. doi: 10.1111/tpj.15011. PubMed DOI

Lievens L, Pollier J, Goossens A, Beyaert R, Staal J. Abscisic acid as pathogen effector and immune regulator. Front Plant Sci. 2017;8:587. doi: 10.3389/fpls.2017.00587. PubMed DOI PMC

Clarke SM, Cristescu SM, Miersch O, Harren FJM, Wasternack C, Mur LAJ. Jasmonates act with salicylic acid to confer basal thermotolerance in Arabidopsis thaliana. New Phytol. 2009;182:175–87. doi: 10.1111/j.1469-8137.2008.02735.x. PubMed DOI

Munemasa S, Hauser F, Park J, Waadt R, Brandt B, Schroeder JI. Mechanisms of abscisic acid-mediated control of stomatal aperture. Curr Opin Plant Biol. 2015;28:154–62. doi: 10.1016/j.pbi.2015.10.010. PubMed DOI PMC

Zhang Y, Li X. Salicylic acid: biosynthesis, perception, and contributions to plant immunity. Curr Opin Plant Biol. 2019;50:29–36. doi: 10.1016/j.pbi.2019.02.004. PubMed DOI

Wasternack C, Hause B. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in annals of Botany. Ann Bot. 2013;111:1021–58. doi: 10.1093/aob/mct067. PubMed DOI PMC

Tarkowská D, Novák O, Floková K, Tarkowski P, Turečková V, Grúz J, et al. Quo vadis plant hormone analysis? Planta. 2014;240:55–76. doi: 10.1007/s00425-014-2063-9. PubMed DOI

Cristescu SM, Mandon J, Arslanov D, De Pessemier J, Hermans C, Harren FJM. Current methods for detecting ethylene in plants. Ann Bot. 2013;111:347–60. doi: 10.1093/aob/mcs259. PubMed DOI PMC

Concepcion M, Lizada C, Yang SF. A simple and sensitive assay for 1-aminocyclopropane-1-carboxylic acid. Anal Biochem. 1979;100:140–5. doi: 10.1016/0003-2697(79)90123-4. PubMed DOI

Bulens I, Van De Poel B, Hertog ML, De Proft MP, Geeraerd AH, Nicolaï BM. Protocol: an updated integrated methodology for analysis of metabolites and enzyme activities of ethylene biosynthesis. Plant Methods. 2011;7:17. doi: 10.1186/1746-4811-7-17. PubMed DOI PMC

Novák O, Napier R, Ljung K. Zooming in on plant hormone analysis: tissue- and cell-specific approaches. Annu Rev Plant Biol. 2017;68:323–48. doi: 10.1146/annurev-arplant-042916-040812. PubMed DOI

Chen Y, Wang Y, Liang X, Zhang Y, Fernie AR. Mass spectrometric exploration of phytohormone profiles and signaling networks. Trends Plant Sci. 2023;28:399–414. doi: 10.1016/j.tplants.2022.12.006. PubMed DOI

Penrose DM, Moffatt BA, Glick BR. Determination of 1-aminocycopropane-1-carboxylic acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings. Can J Microbiol. 2001;47:77–80. doi: 10.1139/w00-128. PubMed DOI

Petritis K, Koukaki G, Koussissi E, Elfakir C, Dreux M, Dourtoglou V. The simultaneous determination of 1-aminocyclopropane-1-carboxylic acid and cyclopropane-1,1-dicarboxylic acid in Lycopersicum esculentum by high-performance liquid chromatography-electrospray tandem mass spectrometry. Phytochem Anal. 2003;14:347–51. doi: 10.1002/pca.725. PubMed DOI

Smets R, Claes V, Van Onckelen HA, Prinsen E. Extraction and quantitative analysis of 1-aminocyclopropane-1-carboxylic acid in plant tissue by gas chromatography coupled to mass spectrometry. J Chromatogr A. 2003;993:79–87. doi: 10.1016/S0021-9673(02)01817-4. PubMed DOI

Liu X, Li D-F, Wang Y, Lu Y-T. Determination of 1-aminocyclopropane-1-carboxylic acid in apple extracts by capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A. 2004;1061:99–104. doi: 10.1016/j.chroma.2004.10.067. PubMed DOI

Ziegler J, Qwegwer J, Schubert M, Erickson JL, Schattat M, Bürstenbinder K, et al. Simultaneous analysis of apolar phytohormones and 1-aminocyclopropan-1-carboxylic acid by high performance liquid chromatography/electrospray negative ion tandem mass spectrometry via 9-fluorenylmethoxycarbonyl chloride derivatization. J Chromatogr A. 2014;1362:102–9. doi: 10.1016/j.chroma.2014.08.029. PubMed DOI

Müller M, Munné-Bosch S. Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Plant Methods. 2011;7. PubMed PMC

Zlobin IE, Vankova R, Pashkovskiy PP, Dobrev P, Kartashov AV, Ivanov YV, et al. Profiles of endogenous phytohormones and expression of some hormone-related genes in scots pine and Norway spruce seedlings under water deficit. Plant Physiol Biochem. 2020;151:457–68. doi: 10.1016/j.plaphy.2020.03.056. PubMed DOI

Kretzler B, Sales CRG, Karady M, Carmo-Silva E, Dodd IC. Maintenance of photosynthesis as leaves age improves whole plant water use efficiency in an Australian wheat cultivar. Agronomy. 2020;10.

Hirayama T, Mochida K. Plant Hormonomics: a Key Tool for deep physiological phenotyping to Improve Crop Productivity. Plant Cell Physiol. 2023;63:1826–39. doi: 10.1093/pcp/pcac067. PubMed DOI PMC

Schäfer M, Brütting C, Baldwin IT, Kallenbach M. High-throughput quantification of more than 100 primary- and secondary-metabolites, and phytohormones by a single solid-phase extraction based sample preparation with analysis by UHPLC-HESI-MS/MS. Plant Methods. 2016;12:1–18. doi: 10.1186/s13007-016-0130-x. PubMed DOI PMC

Zheng Z, Guo Y, Novák O, Dai X, Zhao Y, Ljung K, et al. Coordination of auxin and ethylene biosynthesis by the aminotransferase VAS1. Nat Chem Biol. 2013;9:244–46. doi: 10.1038/nchembio.1178. PubMed DOI PMC

Qin H, He L, Huang R. The coordination of ethylene and other hormones in primary root development. Front Plant Sci. 2019;10. PubMed PMC

Žuvela P, Skoczylas M, Jay Liu J, Baczek T, Kaliszan R, Wong MW, et al. Column characterization and Selection systems in reversed-phase high-performance liquid chromatography. Chem Rev. 2019;119:3674–729. doi: 10.1021/acs.chemrev.8b00246. PubMed DOI

Liu Z, Rochfort S. Recent progress in polar metabolite quantification in plants using liquid chromatography–mass spectrometry. J Integr Plant Biol. 2014;56:816–25. doi: 10.1111/jipb.12181. PubMed DOI

Jain A, Verma KK. Strategies in liquid chromatographic methods for the analysis of biogenic amines without and with derivatization. TrAC - Trends Anal Chem. 2018;109:62–82. doi: 10.1016/j.trac.2018.10.001. DOI

Svoboda T, Parich A, Güldener U, Schöfbeck D, Twaruschek K, Václavíková M et al. Biochemical characterization of the Fusarium graminearum candidate ACC-Deaminases and virulence testing of knockout mutant strains. Front Plant Sci. 2019;10. PubMed PMC

Laborda P, Chen X, Wu G, Wang S, Lu X, Ling J, et al. Lysobacter gummosus OH17 induces systemic resistance in Oryza sativa ‘Nipponbare’. Plant Pathol. 2020;69:838–48. doi: 10.1111/ppa.13167. DOI

Chauvaux N, Van Dongen W, Esmans EL, Van Onckelen HA. Quantitative analysis of 1-aminocyclopropane-1-carboxylic acid by liquid chromatography coupled to electrospray tandem mass spectrometry. J Chromatogr A. 1997;775:143–50. doi: 10.1016/S0021-9673(97)00307-5. DOI

Boughton BA, Callahan DL, Silva C, Bowne J, Nahid A, Rupasinghe T, et al. Comprehensive profiling and quantitation of amine group containing metabolites. Anal Chem. 2011;83:7523–30. doi: 10.1021/ac201610x. PubMed DOI

Gray N, Zia R, King A, Patel VC, Wendon J, McPhail MJW, et al. High-speed quantitative UPLC-MS analysis of multiple amines in Human plasma and serum via Precolumn Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate: application to Acetaminophen-Induced Liver failure. Anal Chem. 2017;89:2478–87. doi: 10.1021/acs.analchem.6b04623. PubMed DOI

Gaudin Z, Cerveau D, Marnet N, Bouchereau A, Delavault P, Simier P, et al. Robust method for investigating nitrogen metabolism of 15N labeled amino acids using AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry: application to a parasitic plant-plant interaction. Anal Chem. 2014;86:1138–45. doi: 10.1021/ac403067w. PubMed DOI

Guan L, Tayengwa R, Cheng ZM, Peer WA, Murphy AS, Zhao M. Auxin regulates adventitious root formation in tomato cuttings. BMC Plant Biol. 2019;19:1–16. doi: 10.1186/s12870-019-2002-9. PubMed DOI PMC

Jiroutová P, Mikulík J, Novák O, Strnad M, Oklestkova J. Brassinosteroids induce strong, dose-dependent inhibition of etiolated pea seedling growth correlated with Ethylene Production. Biomol. 2019;9:849. PubMed PMC

Svačinová J, Novák O, Plačková L, Lenobel R, Holík J, Strnad M, et al. A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction. Plant Methods. 2012;8:1–14. doi: 10.1186/1746-4811-8-17. PubMed DOI PMC

Široká J, Brunoni F, Pěnčík A, Mik V, Žukauskaitė A, Strnad M, et al. High-throughput interspecies profiling of acidic plant hormones using miniaturised sample processing. Plant Methods. 2022;18:1–15. doi: 10.1186/s13007-022-00954-3. PubMed DOI PMC

Novák O, Hényková E, Sairanen I, Kowalczyk M, Pospíšil T, Ljung K. Tissue-specific profiling of the Arabidopsis thaliana auxin metabolome. Plant J. 2012;72:523–36. doi: 10.1111/j.1365-313X.2012.05085.x. PubMed DOI

Pěnčík A, Casanova-Sáez R, Pilařová V, Žukauskaite A, Pinto R, Micol JL, et al. Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis. J Exp Bot. 2018;69:2569–79. doi: 10.1093/jxb/ery084. PubMed DOI PMC

Cao Z, Sun L, Mou R, Zhang L, Lin X, Zhu Z, et al. Profiling of phytohormones and their major metabolites in rice using binary solid-phase extraction and liquid chromatography-triple quadrupole mass spectrometry. J Chromatogr A. 2016;1451:67–74. doi: 10.1016/j.chroma.2016.05.011. PubMed DOI

Cao D, He M, Editorial Methods in phytohormone detection and quantification: 2022. Front Plant Sci. 2023;14:1235688. doi: 10.3389/fpls.2023.1235688. PubMed DOI PMC

Šimura J, Antoniadi I, Široká J, Tarkowská D, Strnad M, Ljung K, et al. Plant hormonomics: multiple phytohormone profiling by targeted metabolomics. Plant Physiol. 2018;177:476–89. doi: 10.1104/pp.18.00293. PubMed DOI PMC

Thevenet D, Pastor V, Baccelli I, Balmer A, Vallat A, Neier R, et al. The priming molecule β-aminobutyric acid is naturally present in plants and is induced by stress. New Phytol. 2017;213:552–9. doi: 10.1111/nph.14298. PubMed DOI

Floková K, Tarkowská D, Miersch O, Strnad M, Wasternack C, Novák O. UHPLC-MS/MS based target profiling of stress-induced phytohormones. Phytochemistry. 2014;105:147–57. doi: 10.1016/j.phytochem.2014.05.015. PubMed DOI

Lu W, Su X, Klein MS, Lewis IA, Fiehn O, Rabinowitz JD. Metabolite Measurement: pitfalls to avoid and practices to follow. Annu Rev Biochem. 2017;86:277–304. doi: 10.1146/annurev-biochem-061516-044952. PubMed DOI PMC

Ljung K, Sandberg G, Moritz T. Methods of plant hormone analysis. Signal Transduction, Action!: Plant Horm Biosynthesis; 2010. pp. 717–40.

Chen Y, Guo Z, Wang X, Qiu C. Sample preparation. J Chromatogr A. 2008;1184:191–219. doi: 10.1016/j.chroma.2007.10.026. PubMed DOI

Keunchkarian S, Reta M, Romero L, Castells C. Effect of sample solvent on the chromatographic peak shape of analytes eluted under reversed-phase liquid chromatogaphic conditions. J Chromatogr A. 2006;1119:20–8. doi: 10.1016/j.chroma.2006.02.006. PubMed DOI

Greer B, Chevallier O, Quinn B, Botana LM, Elliott CT. Redefining dilute and shoot: the evolution of the technique and its application in the analysis of foods and biological matrices by liquid chromatography mass spectrometry. TrAC Trends Anal Chem. 2021;141:116284. doi: 10.1016/j.trac.2021.116284. DOI

Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003;75:3019–30. doi: 10.1021/ac020361s. PubMed DOI

Böttcher C, Roepenack-Lahaye EV, Willscher E, Scheel D, Clemens S. Evaluation of matrix effects in metabolite profiling based on capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. Anal Chem. 2007;79:1507–13. doi: 10.1021/ac061037q. PubMed DOI

Vogel JP, Woeste KE, Theologis A, Kieber JJ. Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively. Proc Natl Acad Sci USA. 1998;95:4766–71. doi: 10.1073/pnas.95.8.4766. PubMed DOI PMC

Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR. EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science. 1999;284:2148–52. doi: 10.1126/science.284.5423.2148. PubMed DOI

Plačková L, Oklestkova J, Pospíšková K, Poláková K, Buček J, Stýskala J, et al. Microscale magnetic microparticle-based immunopurification of cytokinins from Arabidopsis root apex. Plant J. 2017;89:1065–75. doi: 10.1111/tpj.13443. PubMed DOI

Huang J, Zhao X, Bürger M, Chory J, Wang X. The role of ethylene in plant temperature stress response. Trends Plant Sci. 2023;28:808–24. doi: 10.1016/j.tplants.2023.03.001. PubMed DOI

Thain SC, Vandenbussche F, Laarhoven LJJ, Dowson-Day MJ, Wang ZY, Tobin EM, et al. Circadian rhythms of Ethylene Emission in Arabidopsis. Plant Physiol. 2004;136:3751. doi: 10.1104/pp.104.042523. PubMed DOI PMC

Vandenbussche F, Vriezen WH, Smalle J, Laarhoven LJJ, Harren FJM, Van Der Straeten D. Ethylene and Auxin Control the Arabidopsis response to decreased light intensity. Plant Physiol. 2003;133:517. doi: 10.1104/pp.103.022665. PubMed DOI PMC

Depaepe T, Van Der Straeten D. Tools of the Ethylene Trade: a Chemical Kit to Influence Ethylene responses in plants and its use in Agriculture. Small Methods. 2020;4:1–18. doi: 10.1002/smtd.201900267. DOI

Sun X, Li Y, He W, Ji C, Xia P, Wang Y, et al. Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase. Nat Commun. 2017;8:15758. doi: 10.1038/ncomms15758. PubMed DOI PMC

Schellingen K, Van Der Straeten D, Vandenbussche F, Prinsen E, Remans T, Vangronsveld J, et al. Cadmium-induced ethylene production and responses in Arabidopsis thaliana rely on ACS2 and ACS6 gene expression. BMC Plant Biol. 2014;14:1–14. doi: 10.1186/s12870-014-0214-6. PubMed DOI PMC

Chae HS, Faure F, Kieber JJ. The eto1, eto2, and eto3 Mutations and Cytokinin Treatment Increase Ethylene Biosynthesis in Arabidopsis by Increasing the Stability of ACS Protein. Plant Cell. 2003;15:545. PubMed PMC

Vaseva II, Qudeimat E, Potuschak T, Du Y, Genschik P, Vandenbussche F, et al. The plant hormone ethylene restricts Arabidopsis growth via the epidermis. Proc Natl Acad Sci USA. 2018;115:E4130–9. doi: 10.1073/pnas.1717649115. PubMed DOI PMC

Pattyn J, Vaughan-Hirsch J, Van de Poel B. The regulation of ethylene biosynthesis: a complex multilevel control circuitry. New Phytol. 2020;229:770–82. doi: 10.1111/nph.16873. PubMed DOI PMC

Muday GK, Rahman A, Binder BM. Auxin and ethylene: collaborators or competitors? Trends Plant Sci. 2012;17:181–95. doi: 10.1016/j.tplants.2012.02.001. PubMed DOI

Huang P, Dong Z, Guo P, Zhang X, Qiu Y, Li B, et al. Salicylic acid suppresses apical hook formation via NPR1-Mediated repression of EIN3 and EIL1 in Arabidopsis. Plant Cell. 2020;32:612–29. doi: 10.1105/tpc.19.00658. PubMed DOI PMC

Hansen M, Chae HS, Kieber JJ. Regulation of ACS protein stability by cytokinin and brassinosteroid. Plant J. 2009;57:606–14. doi: 10.1111/j.1365-313X.2008.03711.x. PubMed DOI PMC

Rittenberg D, Foster GL. A new procedure for quantitative analysis by isotope dilution, with application to the determination of amino acids and fatty acids. J Biol Chem. 1940;133:737–44. doi: 10.1016/S0021-9258(18)73304-8. DOI

Modification of xylan in secondary walls alters cell wall biosynthesis and wood formation programs and improves saccharification