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Open Access Methodology article

Single-cell-based system to monitor carrier driven cellular auxin homeostasis

Elke Barbez12, Martina Laňková3, Markéta Pařezová3, Alexis Maizel4, Eva Zažímalová3, Jan Petrášek3, Jiří Friml15 and Jürgen Kleine-Vehn12*

Author Affiliations

1 Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, 9052, Gent, Belgium

2 Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190, Vienna, Austria

3 Institute of Experimental Botany, The Academy of Sciences of the Czech Republic, 16502, Praha 6, Czech Republic

4 Department of Stem Cell Biology, Center for Organismal Studies, University of Heidelberg, 69120, Heidelberg, Germany

5 Department of Functional Genomics and Proteomics, Faculty of Science, and CEITEC, Masaryk University, Kamenice 5, CZ-62500, Brno, Czech Republic

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BMC Plant Biology 2013, 13:20  doi:10.1186/1471-2229-13-20

Published: 4 February 2013

Abstract

Background

Abundance and distribution of the plant hormone auxin play important roles in plant development. Besides other metabolic processes, various auxin carriers control the cellular level of active auxin and, hence, are major regulators of cellular auxin homeostasis. Despite the developmental importance of auxin transporters, a simple medium-to-high throughput approach to assess carrier activities is still missing. Here we show that carrier driven depletion of cellular auxin correlates with reduced nuclear auxin signaling in tobacco Bright Yellow-2 (BY-2) cell cultures.

Results

We developed an easy to use transient single-cell-based system to detect carrier activity. We use the relative changes in signaling output of the auxin responsive promoter element DR5 to indirectly visualize auxin carrier activity. The feasibility of the transient approach was demonstrated by pharmacological and genetic interference with auxin signaling and transport. As a proof of concept, we provide visual evidence that the prominent auxin transport proteins PIN-FORMED (PIN)2 and PIN5 regulate cellular auxin homeostasis at the plasma membrane and endoplasmic reticulum (ER), respectively. Our data suggest that PIN2 and PIN5 have different sensitivities to the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Also the putative PIN-LIKES (PILS) auxin carrier activity at the ER is insensitive to NPA in our system, indicating that NPA blocks intercellular, but not intracellular auxin transport.

Conclusions

This single-cell-based system is a useful tool by which the activity of putative auxin carriers, such as PINs, PILS and WALLS ARE THIN1 (WAT1), can be indirectly visualized in a medium-to-high throughput manner. Moreover, our single cell system might be useful to investigate also other hormonal signaling pathways, such as cytokinin.

Keywords:
Auxin homeostasis; DR5; Auxin carrier; Auxin transport