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

Abstract

BackgroundAbundance 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.ResultsWe 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.ConclusionsThis 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.

Functional Analysis of the Hydrophilic Loop in Intracellular Trafficking of Arabidopsis PIN-FORMED Proteins

“…Studies done with ectopically expressed PIN5 have shown that PIN5 localizes predominantly in the ER and is involved in intracellular auxin homeostasis by possibly transporting cytosolic auxin to the ER lumen (Mravec et al, 2009;Ganguly et al, 2010;Barbez et al, 2013). In this study, we observed that PIN5 can also localize to the PM and undergoes endocytosis in certain cell types, including its own expression domain.…”

Auxin transport sites are visualized in planta using fluorescent auxin analogs

“…S15), suggesting that the localization of NBDauxins was not due to the nonspecific binding of the NBD fluorophores to organelles or membranes. Several auxin transporters, such as PIN5, PIN8, and PILS, are subcellularly localized to the endoplasmic reticulum (ER) (34)(35)(36). NBD-auxin signals showed a similar localization pattern to the signals of PIN5-GFP and PIN8-GFP observed in BY-2 cells (Fig.…”

“…These results imply that NBD-auxins might aggregate locally at application sites; therefore, NBD-auxins are not versatile for the visualization of auxin transport streams in the shoot. Several ER-localized auxin transporters, such as PIN5, PIN8, and PILS, play a role in subcellular auxin homeostasis (34)(35)(36). Because of the general nature of small-molecule compounds, a small fraction of NBDauxins might be passively diffused and nonspecifically bound to organelle membranes.…”

“…Our fluorescent auxin analogs will provide insights into auxin biology in combination with multiple tools such as SCF TIR1 -based sensors (8,35), pharmacological auxin probes (26,27), and mathematical modeling (43).…”

PIN-Dependent Auxin Transport: Action, Regulation, and Evolution

“…The agravitropic pin2 phenotype along with the polarized localization of PIN2 in the root identified PIN2 as the PAT component of root gravitropic response. The auxin efflux capacity of PIN proteins has since been shown in a number of transport assays in different systems (Petrásek et al, 2006;Yang and Murphy, 2009;Barbez et al, 2013;Zourelidou et al, 2014). An important question concerned the relation between PIN polarity and directionality of auxin transport.…”