A High-throughput Method for the Quantitative Analysis of Auxins

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2010 Oct 2

PMID 20885372

Citations 29

Authors

Lana S Barkawi

Yuen-Yee Tam

Julie A Tillman

Jennifer Normanly

Jerry D Cohen

Affiliations

Soon will be listed here.

Abstract

Auxin measurements in plants are critical to understanding both auxin signaling and metabolic homeostasis. The most abundant natural auxin is indole-3-acetic acid (IAA). This protocol is for the precise, high-throughput determination of free IAA in plant tissue by isotope dilution analysis using gas chromatography-mass spectrometry (GC-MS). The steps described are as follows: harvesting of plant material; amino and polymethylmethacrylate solid-phase purification followed by derivatization with diazomethane (either manual or robotic); GC-MS analysis; and data analysis. [¹³C₆]IAA is the standard used. The amount of tissue required is relatively small (25 mg of fresh weight) and one can process more than 500 samples per week using an automated system. To extract eight samples, this procedure takes ∼3 h, whether performed manually or robotically. For processing more than eight samples, robotic extraction becomes substantially more time efficient, saving at least 0.5 h per additional batch of eight samples.

Citing Articles

Analytical methods for stable isotope labeling to elucidate rapid auxin kinetics in Arabidopsis thaliana.

Tang Q, Tillmann M, Cohen J PLoS One. 2024; 19(5):e0303992.

PMID: 38776314 PMC: 11111016. DOI: 10.1371/journal.pone.0303992.

Can plant hormonomics be built on simple analysis? A review.

Vrobel O, Tarkowski P Plant Methods. 2023; 19(1):107.

PMID: 37833752 PMC: 10576392. DOI: 10.1186/s13007-023-01090-2.

Effect of ion source polarity and dopants on the detection of auxin plant hormones by ion mobility-mass spectrometry.

Ilbeigi V, Valadbeigi Y, Moravsky L, Matejcik S Anal Bioanal Chem. 2022; 414(20):6259-6269.

PMID: 35794348 DOI: 10.1007/s00216-022-04198-x.

Biphasic control of cell expansion by auxin coordinates etiolated seedling development.

Du M, Bou Daher F, Liu Y, Steward A, Tillmann M, Zhang X Sci Adv. 2022; 8(2):eabj1570.

PMID: 35020423 PMC: 8754305. DOI: 10.1126/sciadv.abj1570.

Protocol: analytical methods for visualizing the indolic precursor network leading to auxin biosynthesis.

Tillmann M, Tang Q, Cohen J Plant Methods. 2021; 17(1):63.

PMID: 34158074 PMC: 8220744. DOI: 10.1186/s13007-021-00763-0.

References

Barkawi L, Tam Y, Tillman J, Pederson B, Calio J, Al-Amier H . A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue. Anal Biochem. 2007; 372(2):177-88. DOI: 10.1016/j.ab.2007.08.009. View

Ribnicky D, Cooke T, Cohen J . A microtechnique for the analysis of free and conjugated indole-3-acetic acid in milligram amounts of plant tissue using a benchtop gas chromatograph-mass spectrometer. Planta. 1998; 204(1):1-7. DOI: 10.1007/s004250050223. View

Rao R, Hunter A, Kashpur O, Normanly J . Aberrant synthesis of indole-3-acetic acid in Saccharomyces cerevisiae triggers morphogenic transition, a virulence trait of pathogenic fungi. Genetics. 2010; 185(1):211-20. PMC: 2870956. DOI: 10.1534/genetics.109.112854. View

Hamilton R . Isolation of indole-3-acetic acid from corn kernels & etiolated corn seedlings. Plant Physiol. 1961; 36(3):354-9. PMC: 406147. DOI: 10.1104/pp.36.3.354. View

Ljung K, Bhalerao R, Sandberg G . Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth. Plant J. 2001; 28(4):465-74. DOI: 10.1046/j.1365-313x.2001.01173.x. View

Turian G, Hamilton R . Chemical detection of 3-indolylacetic acid in Ustilago zeae tumors. Biochim Biophys Acta. 1960; 41:148-50. DOI: 10.1016/0006-3002(60)90381-4. View

Tam Y, Normanly J . Overexpression of a bacterial indole-3-acetyl-l-aspartic acid hydrolase in Arabidopsis thaliana. Physiol Plant. 2002; 115(4):513-522. DOI: 10.1034/j.1399-3054.2002.1150405.x. View

Quint M, Barkawi L, Fan K, Cohen J, Gray W . Arabidopsis IAR4 modulates auxin response by regulating auxin homeostasis. Plant Physiol. 2009; 150(2):748-58. PMC: 2689969. DOI: 10.1104/pp.109.136671. View

Ljung K, Hull A, Celenza J, Yamada M, Estelle M, Normanly J . Sites and regulation of auxin biosynthesis in Arabidopsis roots. Plant Cell. 2005; 17(4):1090-104. PMC: 1087988. DOI: 10.1105/tpc.104.029272. View

10.

Chen K, Miller A, Patterson G, Cohen J . A Rapid and Simple Procedure for Purification of Indole-3-Acetic Acid Prior to GC-SIM-MS Analysis. Plant Physiol. 1988; 86(3):822-5. PMC: 1054577. DOI: 10.1104/pp.86.3.822. View

11.

Mertens R, Eberle J, Arnscheidt A, Ledebur A, Weiler E . Monoclonal antibodies to plant growth regulators. II. Indole-3-acetic acid. Planta. 2013; 166(3):389-93. DOI: 10.1007/BF00401177. View

12.

Cohen J, Baldi B, Slovin J . C(6)-[benzene ring]-indole-3-acetic Acid: a new internal standard for quantitative mass spectral analysis of indole-3-acetic Acid in plants. Plant Physiol. 1986; 80(1):14-9. PMC: 1075048. DOI: 10.1104/pp.80.1.14. View

13.

Armstrong J, Yuan S, Dale J, Tanner V, Theologis A . Identification of inhibitors of auxin transcriptional activation by means of chemical genetics in Arabidopsis. Proc Natl Acad Sci U S A. 2004; 101(41):14978-83. PMC: 522024. DOI: 10.1073/pnas.0404312101. View

14.

Barkawi L, Cohen J . A method for concurrent diazomethane synthesis and substrate methylation in a 96-sample format. Nat Protoc. 2010; 5(10):1619-26. DOI: 10.1038/nprot.2010.119. View

15.

Sun J, Xu Y, Ye S, Jiang H, Chen Q, Liu F . Arabidopsis ASA1 is important for jasmonate-mediated regulation of auxin biosynthesis and transport during lateral root formation. Plant Cell. 2009; 21(5):1495-511. PMC: 2700526. DOI: 10.1105/tpc.108.064303. View

16.

Edlund A, Eklof S, Sundberg B, Moritz T, Sandberg G . A Microscale Technique for Gas Chromatography-Mass Spectrometry Measurements of Picogram Amounts of Indole-3-Acetic Acid in Plant Tissues. Plant Physiol. 1995; 108(3):1043-1047. PMC: 157455. DOI: 10.1104/pp.108.3.1043. View

17.

Bandurski R, Schulze A, Cohen J . Photo-regulation of the ratio of ester to free indole-3-acetic acid. Biochem Biophys Res Commun. 1977; 79(4):1219-23. DOI: 10.1016/0006-291x(77)91136-6. View

18.

Cohen J, Bausher M, Bialek K, Buta J, Gocal G, Janzen L . Comparison of a commercial ELISA assay for indole-3-acetic Acid at several stages of purification and analysis by gas chromatography-selected ion monitoring-mass spectrometry using a c(6)-labeled internal standard. Plant Physiol. 1987; 84(4):982-6. PMC: 1056712. DOI: 10.1104/pp.84.4.982. View

19.

Ilic N, Habus I, Barkawi L, Park S, Stefanic Z, Kojic-Prodic B . Aminoethyl-substituted indole-3-acetic acids for the preparation of tagged and carrier-linked auxin. Bioorg Med Chem. 2005; 13(9):3229-40. DOI: 10.1016/j.bmc.2005.02.046. View

20.

Negi S, Sukumar P, Liu X, Cohen J, Muday G . Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato. Plant J. 2009; 61(1):3-15. DOI: 10.1111/j.1365-313X.2009.04027.x. View