Abstract
During the last 10 years, the analytical techniques used in different areas of “life science” have improved tremendously. Mass spectrometry (MS) has become the most versatile and sensitive technique available for identifying and quantifying organic molecules, and liquid chromatography-mass spectrometry is the modern analytical tool of choice for analyzing samples of plant, animal and human origin. Both the sensitivity and the selectivity of the available techniques have increased immensely; modern instruments are much smaller, more user-friendly and more versatile than before, and the overall cost of the method has been greatly reduced. However, the required equipment is not available to most plant research laboratories, and most researchers in biology have limited experience with MS techniques. In this chapter, we aim to explain the advantages and limitations of these techniques, and how they can be used in plant research today. More specifically, we demonstrate how different MS techniques can be used for auxin metabolite identification, quantification and profiling. Efficient sample extraction and purification is essential for highly sensitive and selective analyses. We therefore describe selected novel approaches that have been developed to increase the sensitivity of these analyses and make them applicable at the tissue and cellular levels. We also discuss how these techniques can be combined with isotope labelling and mutant analyses to get a better understanding of the metabolic pathways involved in auxin biosynthesis and degradation. Finally, we examine the future prospects for the use of MS and other analytical techniques in auxin research as well as the potential for combining these techniques to obtain more information from single samples, and perhaps even from single cells.
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References
Baldi BG, Maher BR, Cohen JD (1989) Hydrolysis of indole-3-acetic acid esters exposed to mild alkaline conditions. Plant Physiol 91:9–12
Barkawi LS, Tam YY, Tillman JA et al (2008) A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue. Anal Biochem 372:177–188
Barkawi LS, Tam YY, Tillman JA et al (2010) A high-throughput method for the quantitative analysis of auxins. Nat Protoc 5:1609–1618
Chen CB, Chen YJ, Zhou J et al (2006) A 9-vinyladenine-based molecularly imprinted polymeric membrane for the efficient recognition of plant hormone 1H-indole-3-acetic acid. Anal Chim Acta 569:58–65
Chen ML, Huang YQ, Liu JQ et al (2011) Highly sensitive profiling assay of acidic plant hormones using a novel mass probe by capillary electrophoresis-time of flight-mass spectrometry. J Chromatogr B 879:938–944
Chiwocha SD, Abrams SR, Ambrose SJ et al (2003) A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds. Plant J 35:405–417
Cooney TP, Nonhebel HM (1991) Biosynthesis of indole-3-acetic acid in tomato shoots: measurement, mass spectral identification and incorporation of 2H from 2H2O into indole-3-acetic acid, D- and L-tryptophan, indole-3-pyruvate and tryptamine. Planta 184:368–376
Dobrev PI, Kamínek M (2002) Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J Chromatogr A 950:21–29
Dobrev PI, Havlíček L, Vágner M et al (2005) Purification and determination of plant hormones auxin and abscisic acid using solid phase extraction and two-dimensional high performance liquid chromatography. J Chromatogr A 1075:159–166
Du F, Ruan G, Liang S et al (2012a) Monolithic molecularly imprinted solid-phase extraction for the selective determination of trace cytokinins in plant samples with liquid chromatography-electrospray tandem mass spectrometry. Anal Bioanal Chem 404:489–501
Du F, Ruan G, Liu H (2012b) Analytical methods for tracing plant hormones. Anal Bioanal Chem 403:55–74
Durgbanshi A, Arbona V, Pozo O et al (2005) Simultaneous determination of multiple phytohormones in plant extracts by liquid chromatography-electrospray tandem mass spectrometry. J Agric Food Chem 53:8437–8442
Edlund A, Eklöf S, Sundberg B et al (1995) A microscale technique for gas chromatography-mass spectrometry measurements of picogram amounts of indole-3-acetic acid in plant tissues. Plant Physiol 108:1043–1047
Eklund DM, Thelander M, Landberg K et al (2010) Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens. Development 137:1275–1284
Ernstsen A, Sandberg G, Crozier A (1986) Effects of sodium diethyldithiocarbamate, solvent, temperature and plant extracts on the stability of indoles. Physiol Plant 68:519–522
Farrow SC, Emery RN (2012) Concurrent profiling of indole-3-acetic acid, abscisic acid, and cytokinins and structurally related purines by high-performance-liquid-chromatography tandem electrospray mass spectrometry. Plant Methods 8:42
Flores MI, Romero-González R, Frenich AG et al (2011) QuEChERS-based extraction procedure for multifamily analysis of phytohormones in vegetables by UHPLC-MS/MS. J Sep Sci 34:1517–1524
Holčapek M, Jirásko R, Lísa M (2012) Recent developments in liquid chromatography-mass spectrometry and related techniques. J Chromatogr A 1259:3–15
Hu Y, Li Y, Zhang Y et al (2011) Development of sample preparation method for auxin analysis in plants by vacuum microwave-assisted extraction combined with molecularly imprinted clean-up procedure. Anal Bioanal Chem 399:3367–3374
Huang MT, Ho CT, Lee CY (eds) (1992) Phenolic compounds in food and their effects on health I and II. American Chemical Society, Washington, DC
Izumi Y, Okazawa A, Bamba T et al (2009) Development of a method for comprehensive and quantitative analysis of plant hormones by highly sensitive nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry. Anal Chim Acta 648:215–225
Jensen PJ, Bandurski RS (1995) Incorporation of deuterium into indole-3-acetic acid and tryptophan in Zea mays seedlings grown on 30 % deuterium oxide. Plant Physiol 147:697–702
Kai K, Horita J, Wakasa K et al (2007a) Three oxidative metabolites of indole-3-acetic acid from Arabidopsis thaliana. Phytochemistry 68:1651–1663
Kai K, Wakasa K, Miyagawa H (2007b) Metabolism of indole-3-acetic acid in rice: identification and characterization of N-beta-D-glucopyranosyl indole-3-acetic acid and its conjugates. Phytochemistry 68:2512–2522
Kaufmann A (2012) The current role of high-resolution mass spectrometry in food analysis. Anal Bioanal Chem 403:1233–1249
Kirwan GM, Johansson E, Kleemann R et al (2012) Building multivariate systems biology models. Anal Chem 84:7064–7071
Kojima M, Sakakibara H (2012) Highly sensitive high-throughput profiling of six phytohormones using MS-probe modification and liquid chromatography-tandem mass spectrometry. Methods Mol Biol 918:151–164
Kojima M, Kamada-Nobusada T, Komatsu H et al (2009) Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: an application for hormone profiling in Oryza sativa. Plant Cell Physiol 50:1201–1214
Kowalczyk M, Sandberg G (2001) Quantitative analysis of indole-3-acetic acid metabolites in Arabidopsis. Plant Physiol 127:1845–1853
Kugimiya A, Takeuchi T (1999a) Application of indoleacetic acid-imprinted polymer to solid phase extraction. Anal Chim Acta 395:251–255
Kugimiya A, Takeuchi T (1999b) Effects of 2-hydroxyethyl methacrylate on polymer network and interaction in hydrophilic molecularly imprinted polymers. Anal Sci 15:29–33
Liu BF, Zhong XH, Lu YT (2002) Analysis of plant hormones in tobacco flowers by micellar electrokinetic capillary chromatography coupled with on-line large volume sample stacking. J Chromatogr A 945:257–265
Liu X, Hegeman AD, Gardner G et al (2012) Protocol: high-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples. Plant Methods 8:31
Ljung K, Östin A, Lioussanne L et al (2001a) Developmental regulation of indole-3-acetic acid turnover in Scots pine seedlings. Plant Physiol 125:464–475
Ljung K, Bhalerao RP, Sandberg G (2001b) Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth. Plant J 28:465–474
Ljung K, Hull AK, Celenza J et al (2005) Sites and regulation of auxin biosynthesis in Arabidopsis roots. Plant Cell 17:1090–1094
Lu QM, Chen LH, Lu MH et al (2010) Extraction and analysis of auxins in plants using dispersive liquid-liquid microextraction followed by high-performance liquid chromatography with fluorescence detection. J Agric Food Chem 58:2763–2770
Mashiguchi K, Tanaka K, Sakai T et al (2011) The main auxin biosynthesis pathway in Arabidopsis. Proc Natl Acad Sci USA 108:18512–18517
Müller M, Munné-Bosch S (2011) Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Plant Methods 7:37
Müller A, Düchting P, Weiler EW (2002) A multiplex GC-MS/MS technique for the sensitive and quantitative single-run analysis of acidic phytohormones and related compounds, and its application to Arabidopsis thaliana. Planta 216:44–56
Normanly J (2010) Approaching cellular and molecular resolution of auxin biosynthesis and metabolism. Cold Spring Harb Perspect Biol 2:a001594
Novák O, Hényková E, Sairanen I et al (2012) Tissue specific profiling of the Arabidopsis thaliana auxin metabolome. Plant J 72:523–536
Nováková L, Vlčková H (2009) A review of current trends and advances in modern bio-analytical methods: chromatography and sample preparation. Anal Chim Acta 656:8–35
Núñez O, Gallart-Ayala H, Martins CP et al (2012) New trends in fast liquid chromatography for food and environmental analysis. J Chromatogr A 1228:298–323
Oikawa A, Saito K (2012) Metabolite analyses of single cells. Plant J 70:30–38
Östin A, Catalá C, Chamarro J et al (1995) Identification of glucopyranosyl-β-1,4- glucopyranosyl-β-1-N-oxindole-3-acetyl-N-aspartic acid, a new IAA catabolite, by liquid chromatography/tandem mass spectrometry. J Mass Spectrom 30:1007–1017
Östin A, Kowalyczk M, Bhalerao RP et al (1998) Metabolism of indole-3-acetic acid in Arabidopsis. Plant Physiol 118:285–296
Pan X, Wang W (2009) Profiling of plant hormones by mass spectrometry. J Chromatogr B 877:2806–2813
Pan X, Welti R, Wang X (2008) Simultaneous quantification of major phytohormones and related compounds in crude plant extracts by liquid chromatography-electrospray tandem mass spectrometry. Phytochemistry 69:1773–1781
Pěnčík A, Rolčík J, Novák O et al (2009) Isolation of novel indole-3-acetic acid conjugates by immunoaffinity extraction. Talanta 80:651–655
Pengelly WL, Bandurski RS (1983) Analysis of indole-3-acetic acid metabolism in Zea mays using deuterium oxide as a tracer. Plant Physiol 73:445–449
Petersson SV, Johansson AI, Kowalczyk M et al (2009) An auxin gradient and maximum in the Arabidopsis root apex show by high-resolution cell-specific analysis of IAA distribution and synthesis. Plant Cell 21:1659–1668
Prinsen E, Van Dongen W, Esmans EL et al (1998) Micro and capillary liquid chromatography tandem mass spectrometry: a new dimension in phytohormone research. J Chromatogr A 826:25–37
Quittenden LJ, Davies NW, Smith JA et al (2009) Auxin biosynthesis in pea: characterization of the tryptamine pathway. Plant Physiol 151:1130–1138
Rapparini F, Tam YY, Cohen JD et al (2002) Indole-3-acetic acid metabolism in Lemna gibba undergoes dynamic changes in response to growth temperature. Plant Physiol 128:1410–1416
Reinecke DM, Bandurski RS (1983) Oxindole-3-acetic acid, an indole-3-acetic acid catabolite in Zea mays. Plant Physiol 71:211–213
Rittenberg D, Foster L (1940) A new procedure for quantitative analysis by isotope dilution, with application to the determination of amino acids and fatty acids. J Biol Chem 133:727–744
Sairanen I, Novák O, Pěnčík A et al (2012) Soluble carbohydrates regulate auxin biosynthesis via PIF proteins in Arabidopsis. Plant Cell 24:4907–4916
Schmelz EA, Engelberth J, Alborn HT et al (2003) Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants. Proc Natl Acad Sci USA 100:10552–10557
Shi X, ** F, Huang Y et al (2012) Simultaneous determination of five plant growth regulators in fruits by modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction and liquid chromatography-tandem mass spectrometry. Agric Food Chem 60:60–65
Sugawara S, Hishiyama S, Jikumaru Y et al (2009) Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis. Proc Natl Acad Sci USA 106:5430–5435
Sundberg B (1990) Influence of extraction solvent (buffer, methanol, acetone) and time on the quantification of indole-3-acetic acid in plants. Physiol Plant 78:293–297
Sundberg B, Sandberg G, Crozier A (1986) Purification of indole-3-acetic acid in plant extracts by immunoaffinity chromatography. Phytochemistry 25:295–298
Svačinová J, Novák O, Plačková L et al (2012) A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction. Plant Methods 8:17
Tam YY, Normanly J (1998) Determination of indole-3-pyruvic acid levels in Arabidopsis thaliana by gas chromatography-selected ion monitoring-mass spectrometry. J Chromatogr A 800:101–108
Tam YY, Epstein E, Normanly J (2000) Characterization of auxin conjugates in Arabidopsis. Low steady-state levels of indole-3-acetyl-aspartate, indole-3-acetyl-glutamate, and indole-3-acetyl-glucose. Plant Physiol 123:589–596
Taylor PJ (2005) Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry. Clin Biochem 38:328–334
Tivendale ND, Davies NW, Molesworth PP et al (2010) Reassessing the role of N-hydroxytryptamine in auxin biosynthesis. Plant Physiol 154:1957–1965
Van Meulebroek L, Vanden Bussche J, Steppe K et al (2012) Ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry for metabolomic profiling of the endogenous phytohormonal status of the tomato plant. J Chromatogr A 1260:67–80
Wiklund S, Johansson E, Sjöström L et al (2008) Visualization of GC/TOF-MS-based metabolomics data for identification of biochemically interesting compounds using OPLS class models. Anal Chem 80:115–122
Wu YL, Hu B (2009) Simultaneous determination of several phytohormones in natural coconut juice by hollow fiber-based liquid-liquid-liquid microextraction-high performance liquid chromatography. J Chromatogr A 1216:7657–7663
Zhang Y, Li Y, Hu Y, Li G et al (2010) Preparation of magnetic indole-3-acetic acid imprinted polymer beads with 4-vinylpyridine and β-cyclodextrin as binary monomer via microwave heating initiated polymerization and their application to trace analysis of auxins in plant tissues. J Chromatogr A 1217:7337–7344
Zhao YD, Christensen SK, Fankhauser C et al (2001) A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science 291:306–309
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Novák, O., Pěnčík, A., Ljung, K. (2014). Identification and Profiling of Auxin and Auxin Metabolites. In: Zažímalová, E., Petrášek, J., Benková, E. (eds) Auxin and Its Role in Plant Development. Springer, Vienna. https://doi.org/10.1007/978-3-7091-1526-8_3
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DOI: https://doi.org/10.1007/978-3-7091-1526-8_3
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