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Quantitative evaluation of IAA conjugate pools in Arabidopsis thaliana

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Abstract

Main conclusion

This work has demonstrated that the major method of estimating the amount of unknown IAA conjugates—base hydrolysis—can be significantly complicated by chemical artifacts such as glucobrassicin or protein degradation.

The concept of ‘bound auxin’ traces its origin back to more than 80 years ago and has driven research on the sources and forms of these plant hormones since. Indeed, analytical studies have demonstrated that the majority of cellular auxin is conjugated to simple sugars, cyclitols, glycans, amino acids, and other biomolecules. A number of studies have confirmed the enzymatic systems responsible for the synthesis and hydrolysis of a number of such conjugates in Arabidopsis thaliana and some of these compounds have been identified in situ. However, the amount of indole-3-acetic acid (IAA) released upon treating Arabidopsis tissue extracts with base, a commonly employed technique for estimating the amount of IAA conjugates, greatly exceeded the summation of all the IAA conjugates known individually to be present in Arabidopsis. This discrepancy has remained as an unsolved question. In this study, however, we found that a significant portion of the IAA found after base treatment could be attributed to chemical conversions other than conjugate hydrolysis. Specifically, we showed that glucobrassicin conversion, previously thought to occur at insignificant levels, actually accounted for the majority of solvent soluble IAA released and that proteinaceous tryptophan degradation accounted for a large portion of solvent insoluble IAA. These studies clearly demonstrated the limits associated with using a harsh technique like base hydrolysis in determining IAA conjugates and support using more direct approaches such as mass spectrometry-based strategies for unambiguous characterizations of the total complement of IAA conjugates in new plant materials under study.

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Abbreviations

4-Cl-IAA:

4-Chloro-indole-3-acetic acid

CV:

Column volumes

DTT:

Dithiothreitol

HEPES:

2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

IAA:

Indole-3-acetic acid

IAN:

Indole-3-acetonitrile

IA-Ala:

Indole-3-acetyl alanine

IA-Asp:

Indole-3-acetyl aspartate

IA-Glu:

Indole-3-acetyl glutamate

IA-Leu:

Indole-3-acetyl leucine

IA-Trp:

Indole-3-acetyl tryptophan

MES:

2-(N-Morpholino)ethanesulfonic acid

OxIAA:

Oxindole-3-acetic acid

TCA:

Trichloroacetic acid

TFA:

Trifluoroacetic acid

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Acknowledgments

We thank Dr. William Gray (University of Minnesota) for help with FPLC, Dr. Seijin Park (Colorado State University) for advice on IAA conjugates, Dr. Xing Liu (California Institute of Technology) for sharing her expertise in IAA analysis, Dr. LeeAnn Higgins and Mr. Todd Markowski (both from the University of Minnesota) for help with protein identification. P. Y. was partially supported by a University of Minnesota Monsanto Fellowship, a University of Minnesota Department of Horticultural Science Plant Development Scholarship and by funds from the National Science Foundation (IOS-1238812). The research was funded by the National Science Foundation Plant Genome Research Program (IOS-1238812), the Minnesota Agricultural Experiment Station, and the Gordon and Margaret Bailey Endowment for Environmental Horticulture.

Conflict of interest

The authors declare no conflict of interests.

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Author notes

  1. Peng Yu

    Present address: Emil-Erlenmeyer-Forum 5, Chair of Proteomics and Bioanalytics, Technische Universität München, 85354, Freising, Germany

Authors and Affiliations

  1. Department of Horticultural Science, Microbial and Plant Genomics Institute, University of Minnesota, 1970 Folwell Avenue, Saint Paul, MN, 55108, USA

    Peng Yu, Paula Lor, Adrian D. Hegeman & Jerry D. Cohen

  2. Plant Biological Sciences Graduate Program, University of Minnesota, Saint Paul, USA

    Peng Yu, Adrian D. Hegeman & Jerry D. Cohen

  3. Institut für Botanik, Technische Universität Dresden, 01062, Dresden, Germany

    Jutta Ludwig-Müller

  4. Department of Plant Biology, University of Minnesota, Saint Paul, USA

    Adrian D. Hegeman

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  1. Peng Yu
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Correspondence to Peng Yu or Jerry D. Cohen.

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Yu, P., Lor, P., Ludwig-Müller, J. et al. Quantitative evaluation of IAA conjugate pools in Arabidopsis thaliana . Planta 241, 539–548 (2015). https://doi.org/10.1007/s00425-014-2206-z

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