single-cell-based system to monitor carrier driven cellular auxin homeostasissingle-cell-based系统监测载体驱动细胞生长素内稳态.pdfVIP

Barbez et al. BMC Plant Biology 2013, 13:20 /1471-2229/13/20 METHODOLOGY ARTICLE Open Access Single-cell-based system to monitor carrier driven cellular auxin homeostasis 1,2 3 3 4 3 3 1,5 Elke Barbez , Martina Laňková , Markéta Pařezová , Alexis Maizel , Eva Zažímalová , Jan Petrášek , Jiří Friml and Jürgen Kleine-Vehn1,2* 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 t