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Light plays an essential role in intracellular distribution of auxin efflux carrier PIN2 in Arabidopsis thaliana.

Laxmi A, Pan J, Morsy M, Chen R - PLoS ONE (2008)

Bottom Line: In light-grown plants after shift to dark or to continuous red or far-red light, PIN2 also accumulated in vacuolar compartments.We show that PIN2 vacuolar targeting was derived from the PM via endocytic trafficking and inhibited by HY5-dependent light signaling.In addition, the ubiquitin 26S proteasome is involved in the process, since its inhibition by mutations in COP9 and a proteasome inhibitor MG132 impaired the process.

View Article: PubMed Central - PubMed

Affiliation: Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA.

ABSTRACT

Background: Light plays a key role in multiple plant developmental processes. It has been shown that root development is modulated by shoot-localized light signaling and requires shoot-derived transport of the plant hormone, auxin. However, the mechanism by which light regulates root development is not largely understood. In plants, the endogenous auxin, indole-3-acetic acid, is directionally transported by plasma-membrane (PM)-localized auxin influx and efflux carriers in transporting cells. Remarkably, the auxin efflux carrier PIN proteins exhibit asymmetric PM localization, determining the polarity of auxin transport. Similar to PM-resident receptors and transporters in animal and yeast cells, PIN proteins undergo constitutive cycling between the PM and endosomal compartments. Auxin plays multiple roles in PIN protein intracellular trafficking, inhibiting PIN2 endocytosis at some concentrations and promoting PIN2 degradation at others. However, how PIN proteins are turned over in plant cells is yet to be addressed.

Methodology and principle findings: Using laser confocal scanning microscopy, and physiological and molecular genetic approaches, here, we show that in dark-grown seedlings, the PM localization of auxin efflux carrier PIN2 was largely reduced, and, in addition, PIN2 signal was detected in vacuolar compartments. This is in contrast to light-grown seedlings where PIN2 was predominantly PM-localized. In light-grown plants after shift to dark or to continuous red or far-red light, PIN2 also accumulated in vacuolar compartments. We show that PIN2 vacuolar targeting was derived from the PM via endocytic trafficking and inhibited by HY5-dependent light signaling. In addition, the ubiquitin 26S proteasome is involved in the process, since its inhibition by mutations in COP9 and a proteasome inhibitor MG132 impaired the process.

Conclusions and significance: Collectively, our data indicate that light plays an essential role in PIN2 intracellular trafficking, promoting PM-localization in the presence of light and, on the other hand, vacuolar targeting for protein degradation in the absence of light. Based on these results, we postulate that light regulation of root development is mediated at least in part by changes in the intracellular distribution of auxin efflux carriers, PIN proteins, in response to the light environment.

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Related in: MedlinePlus

Vacuolar accumulation of PIN2-eGFP did not require de novo protein synthesis.Five-day-old light-grown PIN2-eGFP seedlings were pulse-labeled with an endocytosis marker, FM4-64, and pre-treated on growth media with or without cycloheximide (CHX; 50 µM) for 30 min. The plants were then shifted to dark and incubated for 4 hrs. In the absence of cycloheximide, PIN2-eGFP (green) accumulated in vacuolar compartments marked by FM4-64 (red) (A). Similarly, in the presence of cycloheximide, PIN2-eGFP also accumulated in vacuolar compartments in plants after shift to dark (B). Shown were root epidermis cells. Scale bar, 10 µm.
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pone-0001510-g005: Vacuolar accumulation of PIN2-eGFP did not require de novo protein synthesis.Five-day-old light-grown PIN2-eGFP seedlings were pulse-labeled with an endocytosis marker, FM4-64, and pre-treated on growth media with or without cycloheximide (CHX; 50 µM) for 30 min. The plants were then shifted to dark and incubated for 4 hrs. In the absence of cycloheximide, PIN2-eGFP (green) accumulated in vacuolar compartments marked by FM4-64 (red) (A). Similarly, in the presence of cycloheximide, PIN2-eGFP also accumulated in vacuolar compartments in plants after shift to dark (B). Shown were root epidermis cells. Scale bar, 10 µm.

Mentions: It has been previously shown that PIN2 undergoes constitutive cycling between the PM and endosomal compartments [45], [46], [61], [62]. However, vacuolar targeting of PIN2 protein has not been described so far. To gain insights of the mechanism underlying PIN2 intracellular distribution, we tested whether PIN2 accumulates to vacuolar compartments in light-grown seedlings after a short-term shift to dark. We found that PIN2-eGFP accumulated in vacuolar compartments in light-grown plants after shift to dark for a short period time (Figs. 5A; 6A–D).


Light plays an essential role in intracellular distribution of auxin efflux carrier PIN2 in Arabidopsis thaliana.

Laxmi A, Pan J, Morsy M, Chen R - PLoS ONE (2008)

Vacuolar accumulation of PIN2-eGFP did not require de novo protein synthesis.Five-day-old light-grown PIN2-eGFP seedlings were pulse-labeled with an endocytosis marker, FM4-64, and pre-treated on growth media with or without cycloheximide (CHX; 50 µM) for 30 min. The plants were then shifted to dark and incubated for 4 hrs. In the absence of cycloheximide, PIN2-eGFP (green) accumulated in vacuolar compartments marked by FM4-64 (red) (A). Similarly, in the presence of cycloheximide, PIN2-eGFP also accumulated in vacuolar compartments in plants after shift to dark (B). Shown were root epidermis cells. Scale bar, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2200863&req=5

pone-0001510-g005: Vacuolar accumulation of PIN2-eGFP did not require de novo protein synthesis.Five-day-old light-grown PIN2-eGFP seedlings were pulse-labeled with an endocytosis marker, FM4-64, and pre-treated on growth media with or without cycloheximide (CHX; 50 µM) for 30 min. The plants were then shifted to dark and incubated for 4 hrs. In the absence of cycloheximide, PIN2-eGFP (green) accumulated in vacuolar compartments marked by FM4-64 (red) (A). Similarly, in the presence of cycloheximide, PIN2-eGFP also accumulated in vacuolar compartments in plants after shift to dark (B). Shown were root epidermis cells. Scale bar, 10 µm.
Mentions: It has been previously shown that PIN2 undergoes constitutive cycling between the PM and endosomal compartments [45], [46], [61], [62]. However, vacuolar targeting of PIN2 protein has not been described so far. To gain insights of the mechanism underlying PIN2 intracellular distribution, we tested whether PIN2 accumulates to vacuolar compartments in light-grown seedlings after a short-term shift to dark. We found that PIN2-eGFP accumulated in vacuolar compartments in light-grown plants after shift to dark for a short period time (Figs. 5A; 6A–D).

Bottom Line: In light-grown plants after shift to dark or to continuous red or far-red light, PIN2 also accumulated in vacuolar compartments.We show that PIN2 vacuolar targeting was derived from the PM via endocytic trafficking and inhibited by HY5-dependent light signaling.In addition, the ubiquitin 26S proteasome is involved in the process, since its inhibition by mutations in COP9 and a proteasome inhibitor MG132 impaired the process.

View Article: PubMed Central - PubMed

Affiliation: Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA.

ABSTRACT

Background: Light plays a key role in multiple plant developmental processes. It has been shown that root development is modulated by shoot-localized light signaling and requires shoot-derived transport of the plant hormone, auxin. However, the mechanism by which light regulates root development is not largely understood. In plants, the endogenous auxin, indole-3-acetic acid, is directionally transported by plasma-membrane (PM)-localized auxin influx and efflux carriers in transporting cells. Remarkably, the auxin efflux carrier PIN proteins exhibit asymmetric PM localization, determining the polarity of auxin transport. Similar to PM-resident receptors and transporters in animal and yeast cells, PIN proteins undergo constitutive cycling between the PM and endosomal compartments. Auxin plays multiple roles in PIN protein intracellular trafficking, inhibiting PIN2 endocytosis at some concentrations and promoting PIN2 degradation at others. However, how PIN proteins are turned over in plant cells is yet to be addressed.

Methodology and principle findings: Using laser confocal scanning microscopy, and physiological and molecular genetic approaches, here, we show that in dark-grown seedlings, the PM localization of auxin efflux carrier PIN2 was largely reduced, and, in addition, PIN2 signal was detected in vacuolar compartments. This is in contrast to light-grown seedlings where PIN2 was predominantly PM-localized. In light-grown plants after shift to dark or to continuous red or far-red light, PIN2 also accumulated in vacuolar compartments. We show that PIN2 vacuolar targeting was derived from the PM via endocytic trafficking and inhibited by HY5-dependent light signaling. In addition, the ubiquitin 26S proteasome is involved in the process, since its inhibition by mutations in COP9 and a proteasome inhibitor MG132 impaired the process.

Conclusions and significance: Collectively, our data indicate that light plays an essential role in PIN2 intracellular trafficking, promoting PM-localization in the presence of light and, on the other hand, vacuolar targeting for protein degradation in the absence of light. Based on these results, we postulate that light regulation of root development is mediated at least in part by changes in the intracellular distribution of auxin efflux carriers, PIN proteins, in response to the light environment.

Show MeSH
Related in: MedlinePlus