BACKGROUND: Acidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants. RESULTS: A high-throughput sample preparation method for liquid chromatography-tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports. CONCLUSION: The method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

Department of Chemical Biology Faculty of Science Palacký University Šlechtitelů 27 78371 Olomouc Czech Republic

Department of Experimental Biology Faculty of Science Palacký University Šlechtitelů 27 78371 Olomouc Czech Republic

Laboratory of Growth Regulators Institute of Experimental Botany of the Czech Academy of Sciences and Palacký University Šlechtitelů 27 78371 Olomouc Czech Republic

Plant Hormone Biology Group Swammerdam Institute for Life Sciences University of Amsterdam Science Park 904 1098 XH Amsterdam The Netherlands

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Casanova-Saez R, Mateo-Bonmati E, Ljung K. Auxin metabolism in plants. Cold Spring Harb Perspect Biol. 2021;13:a039867. PubMed PMC

Hayashi K, Arai K, Aoi Y, Tanaka Y, Hira H, Guo RP, et al. The main oxidative inactivation pathway of the plant hormone auxin. Nat Commun. 2021;12:6752. PubMed PMC

Porco S, Pencik A, Rashed A, Voss U, Casanova-Saez R, Bishopp A, et al. Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis. Proc Natl Acad Sci. 2016;113:11016–11021. PubMed PMC

Tanaka K, Hayashi K, Natsume M, Kamiya Y, Sakakibara H, Kawaide H, et al. UGT74D1 catalyzes the glucosylation of 2-Oxindole-3-acetic acid in the auxin metabolic pathway in arabidopsis. Plant Cell Physiol. 2014;55:218–228. PubMed PMC

Brunoni F, Collani S, Simura J, Schmid M, Bellini C, Ljung K. A bacterial assay for rapid screening of IAA catabolic enzymes. Plant Methods. 2019;15(1):1–10. PubMed PMC

Muller K, Dobrev PI, Pencik A, Hosek P, Vondrakova Z, Filepova R, et al. dioxygenase for auxin oxidation 1 catalyzes the oxidation of IAA amino acid conjugates. Plant Physiol. 2021;187:103–115. PubMed PMC

Ludwig-Muller J. Auxin conjugates: their role for plant development and in the evolution of land plants. J Exp Bot. 2011;62:1757–1773. PubMed

Cooke TJ, Poli D, Sztein AE, Cohen JD. Evolutionary patterns in auxin action. Plant Mol Biol. 2002;49:319–338. PubMed

Zhang J, Peer WA. Auxin homeostasis: the DAO of catabolism. J Exp Bot. 2017;68:3145–3154. PubMed

Brunoni F, Collani S, Casanova-Saez R, Simura J, Karady M, Schmid M, et al. Conifers exhibit a characteristic inactivation of auxin to maintain tissue homeostasis. New Phytol. 2020;226:1753–1765. PubMed

Wasternack C, Hause B. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. an update to the review in annals of botany. Ann Bot. 2007;2013(111):1021–1058. PubMed PMC

Fonseca S, Chini A, Hamberg M, Adie B, Porzel A, Kramell R, et al. (+)-7-iso-Jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate. Nat Chem Biol. 2009;5:344–350. PubMed

Wasternack C. Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann Bot. 2007;100:681–697. PubMed PMC

Han GZ. Evolution of jasmonate biosynthesis and signaling mechanisms. J Exp Bot. 2017;68:1323–1331. PubMed

Chen K, Li GJ, Bressan RA, Song CP, Zhu JK, Zhao Y. Abscisic acid dynamics, signaling, and functions in plants. J Integr Plant Biol. 2020;62:25–54. PubMed

Nambara E, Marion-Poll A. Abscisic acid biosynthesis and catabolism. Ann Rev Plant Biol. 2005;56:165–185. PubMed

Zhang YL, Li X. Salicylic acid: biosynthesis, perception, and contributions to plant immunity. Curr Opin Plant Biol. 2019;50:29–36. PubMed

Lefevere H, Bauters L, Gheysen G. Salicylic acid biosynthesis in plants. Front Plant Sci. 2020;11:338. PubMed PMC

Dempsey MA, Vlot AC, Wildermuth MC, Klessig DF. Salicylic acid biosynthesis and metabolism. Arabidopsis Book. 2011;9:e0156. PubMed PMC

Hedden P. The current status of research on gibberellin biosynthesis. Plant Cell Physiol. 2020;61:1832–1849. PubMed PMC

Deng T, Wu DP, Duan CF, Yan XH, Du Y, Zou J, et al. Spatial profiling of gibberellins in a single leaf based on microscale matrix solid-phase dispersion and precolumn derivatization coupled with ultraperformance liquid chromatography-tandem mass spectrometry. Anal Chem. 2017;89:9537–9543. PubMed

Hao YH, Zhang Z, Wang L, Liu C, Lei AW, Yuan BF, et al. Stable isotope labeling assisted liquid chromatography-electrospray tandem mass spectrometry for quantitative analysis of endogenous gibberellins. Talanta. 2015;144:341–348. PubMed

Urbanova T, Tarkowska D, Novak O, Hedden P, Strnad M. Analysis of gibberellins as free acids by ultra performance liquid chromatography-tandem mass spectrometry. Talanta. 2013;112:85–94. PubMed

Ljung K. Auxin metabolism and homeostasis during plant development. Development. 2013;140:943–950. PubMed

Wasternack C, Strnad M. Jasmonate signaling in plant stress responses and development—active and inactive compounds. New Biotechnol. 2016;33:604–613. PubMed

Maruri-Lopez I, Yaniri Aviles-Baltazar N, Buchala A, Serrano M. Intra and extracellular journey of the phytohormone salicylic acid. Front Plant Sci. 2019;10:423. PubMed PMC

Trapp MA, De Souza GD, Rodrigues E, Boland W, Mithofer A. Validated method for phytohormone quantification in plants. Front Plant Sci. 2014;5:417. PubMed PMC

Erland LAE, Shukla MR, Glover WB, Saxena PK. A simple and efficient method for analysis of plant growth regulators: a new tool in the chest to combat recalcitrance in plant tissue culture. Plant Cell Tissue Organ Cult. 2017;131:459–470.

Sheflin AM, Kirkwood JS, Wolfe LM, Jahn CE, Broeckling CD, Schachtman DP, et al. High-throughput quantitative analysis of phytohormones in sorghum leaf and root tissue by ultra-performance liquid chromatography-mass spectrometry. Anal Bioanal Chem. 2019;411:4839–4848. PubMed

Yonny ME, Ballesteros-Gomez A, Adamo MLT, Torresi AR, Nazareno MA, Rubio S. Supramolecular solvent-based high-throughput sample treatment for monitoring phytohormones in plant tissues. Talanta. 2020;219:121249. PubMed

Cai WJ, Yu L, Wang W, Sun MX, Feng YQ. Simultaneous determination of multiclass phytohormones in submilligram plant samples by one-pot multifunctional derivatization-assisted liquid chromatography-tandem mass spectrometry. Anal Chem. 2019;91:3492–3499. PubMed

Cao ZY, Sun LH, Mou RX, Zhang LP, Lin XY, Zhu ZW, 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. PubMed

Flokova K, Tarkowska D, Miersch O, Strnad M, Wasternack C, Novak O. UHPLC-MS/MS based target profiling of stress-induced phytohormones. Phytochemistry. 2014;105:147–157. PubMed

Simura J, Antoniadi I, Siroka J, Tarkowska D, Strnad M, Ljung K, et al. Plant hormonomics: multiple phytohormone profiling by targeted metabolomics. Plant Physiol. 2018;177:476–489. PubMed PMC

Balcke GU, Handrick V, Bergau N, Fichtner M, Henning A, Stellmach H, et al. An UPLC-MS/MS method for highly sensitive high-throughput analysis of phytohormones in plant tissues. Plant Methods. 2012;8:47. PubMed PMC

Miggiels P, Wouters B, van Westen GJP, Dubbelman AC, Hankemeier T. Novel technologies for metabolomics: more for less. Trends Anal Chem. 2019;120:115323.

Burato JSD, Medina DAV, de Toffoli AL, Maciel EVS, Lancas FM. Recent advances and trends in miniaturized sample preparation techniques. J Sep Sci. 2020;43:202–225. PubMed

Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using stagetips. Nat Protoc. 2007;2:1896–1906. PubMed

Svacinova J, Novak O, Plackova L, Lenobel R, Holik 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;8:17. PubMed PMC

Pencik A, Casanova-Saez R, Pilarova V, Zukauskaite A, Pinto R, Micol JL, et al. Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis. J Exp Bot. 2018;69:2569–2579. PubMed PMC

Miersch O, Neumerkel J, Dippe M, Stenzel I, Wasternack C. Hydroxylated jasmonates are commonly occurring metabolites of jasmonic acid and contribute to a partial switch-off in jasmonate signaling. New Phytol. 2008;177:114–127. PubMed

Glauser G, Grata E, Dubugnon L, Rudaz S, Farmer EE, Wolfender JL. Spatial and temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response to wounding. J Biol Chem. 2008;283:16400–16407. PubMed

Novak O, Henykova E, Sairanen I, Kowalczyk M, Pospisil T, Ljung K. Tissue-specific profiling of the Arabidopsis thaliana auxin metabolome. Plant J. 2012;72:523–536. PubMed

Tureckova V, Novak O, Strnad M. Profiling ABA metabolites in Nicotiana tabacum L. leaves by ultra-performance liquid chromatography-electrospray tandem mass spectrometry. Talanta. 2009;80:390–9. PubMed

Stelmach BA, Muller A, Hennig P, Gebhardt S, Schubert-Zsilavecz M, Weiler EW. A novel class of oxylipins, sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride, from Arabidopsis thaliana. J Biol Chem. 2001;276:12832–12838. PubMed

Genva M, Akong FO, Andersson MX, Deleu M, Lins L, Fauconnier ML. New insights into the biosynthesis of esterified oxylipins and their involvement in plant defense and developmental mechanisms. Phytochem Rev. 2019;18:343–358.

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;1119:20–28. PubMed

Jang G, Shim JS, Jung C, Song JT, Lee HY, Chung PJ, et al. Volatile methyl jasmonate is a transmissible form of jasmonate and its biosynthesis is involved in systemic jasmonate response in wounding. Plant Biotechnol Rep. 2014;8:409–419.

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;75:3019–3030. PubMed

Stirk WA, Ordog V, Novak O, Rolcik J, Strnad M, Balint P, et al. Auxin and cytokinin relationships in 24 microalgal strains. J Phycol. 2013;49:459–467. PubMed

Zaveska Drabkova L, Dobrev PI, Motyka V. Phytohormone profiling across the bryophytes. PLoS ONE. 2015;10:e0125411. PubMed PMC

Kosakivska IV, Voytenko LV, Likhnyovskiy RV, Ustinova AY. Effect of temperature on accumulation of abscisic acid and indole-3-acetic acid in Triticum aestivum L seedlings. Genet Plant Physiol. 2014;4(3–4):201–8.

Pavlovic I, Petrik I, Tarkowska D, Lepedus H, Vujcic Bok V, Radic Brkanac S, et al. Correlations between phytohormones and drought tolerance in selected Brassica crops: Chinese cabbage, white cabbage and kale. Int J Mol Sci. 2018;19:2866. PubMed PMC

Junghans U, Polle A, Duchting P, Weiler E, Kuhlman B, Gruber F, et al. Adaptation to high salinity in poplar involves changes in xylem anatomy and auxin physiology. Plant Cell Environ. 2006;29:1519–1531. PubMed

De Zio E, Trupiano D, Karady M, Antoniadi I, Montagnoli A, Terzaghi M, et al. Tissue-specific hormone profiles from woody poplar roots under bending stress. Physiol Plant. 2019;165:101–113. PubMed

Stumpe M, Gobel C, Faltin B, Beike AK, Hause B, Himmelsbach K, et al. The moss Physcomitrella patens contains cyclopentenones but no jasmonates: mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology. New Phytol. 2010;188:740–749. PubMed

De Leon IP, Schmelz EA, Gaggero C, Castro A, Alvarez A, Montesano M. Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection. Mol Plant Pathol. 2012;13:960–974. PubMed PMC

Yamamoto Y, Ohshika J, Takahashi T, Ishizaki K, Kohchi T, Matusuura H, et al. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha. Phytochemistry. 2015;116:48–56. PubMed

Stintzi A, Weber H, Reymond P, Browse J, Farmer EE. Plant defense in the absence of jasmonic acid: The role of cyclopentenones. Proc Natl Acad Sci. 2001;98:12837–12842. PubMed PMC

Monte I, Kneeshaw S, Franco-Zorrilla JM, Chini A, Zamarreno AM, Garcia-Mina JM, et al. An ancient COI1-independent function for reactive electrophilic oxylipins in thermotolerance. Curr Biol. 2020;30:962–971. PubMed

Alallaq S, Ranjan A, Brunoni F, Novak O, Lakehal A, Bellini C. Red light controls adventitious root regeneration by modulating hormone homeostasis in Picea abies seedlings. Front Plant Sci. 2020;11:586140. PubMed PMC

Hartung W. The evolution of abscisic acid (ABA) and ABA function in lower plants, fungi and lichen. Funct Plant Biol. 2010;37:806–812.

Pashkovskiy PP, Vankova R, Zlobin IE, Dobrev P, Ivanov YV, Kartashov AV, et al. Comparative analysis of abscisic acid levels and expression of abscisic acid-related genes in scots pine and Norway spruce seedlings under water deficit. Plant Physiol Biochem. 2019;140:105–112. PubMed

Kramell R, Schmidt J, Schneider G, Sembdner G, Schreiber K. Synthesis Of N-(Jasmonoyl)amino acid conjugates. Tetrahedron. 1988;44:5791–5807.

Kowalczyk M, Sandberg G. Quantitative analysis of indole-3-acetic acid metabolites in Arabidopsis. Plant Physiol. 2001;127:1845–1853. PubMed PMC

Kai K, Horita J, Wakasa K, Miyagawa H. Three oxidative metabolites of indole-3-acetic acid from Arabidopsis thaliana. Phytochemistry. 2007;68:1651–1663. PubMed

Kai K, Nakamura S, Wakasa K, Miyagawa H. Facile preparation of deuterium-labeled standards of indole-3-acetic acid (IAA) and its metabolites to quantitatively analyze the disposition of exogenous IAA in Arabidopsis thaliana. Biosci Biotechnol Biochem. 2007;71:1946–1954. PubMed

Pavlovic I, Pencik A, Novak O, Vujcic V, Brkanac SR, Lepedus H, et al. Short-term salt stress in Brassica rapa seedlings causes alterations in auxin metabolism. Plant Physiol Biochem. 2018;125:74–84. PubMed

Hoagland DR, Arnon DI. The water-culture method for growing plants without soil. 2. California: California agricultural experiment station; 1950.

Bischoff HW, Bold HC. Phycological studies IV some soil algae from enchanted rock and related algal species. Texas: Univ Texas Publ; 1963.

Amide conjugates of the jasmonate precursor cis-(+)-12-oxo-phytodienoic acid regulate its homeostasis during plant stress responses

Cell wall integrity modulates HOOKLESS1 and PHYTOCHROME INTERACTING FACTOR4 expression controlling apical hook formation

Profiling of 1-aminocyclopropane-1-carboxylic acid and selected phytohormones in Arabidopsis using liquid chromatography-tandem mass spectrometry

In situ separation and visualization of isomeric auxin derivatives in Arabidopsis by ion mobility mass spectrometry imaging

Can plant hormonomics be built on simple analysis? A review

Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress

Metabolic profiles of 2-oxindole-3-acetyl-amino acid conjugates differ in various plant species