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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: 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.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.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.

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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Enhanced PIN2-eGFP vacuolar accumulation in dark-grown det3-1 mutant.PIN2-eGFP was slightly enhanced in diffuse and punctate cytoplasmic structures in light-grown det3-1 mutant, compared to the eir1-1 control plant (A, C). In dark-grown det3-1 mutant, a high level of PIN2-eGFP was detected both at the PM and in vacuolar compartments, compared to the dark-grown control plant, where PIN2-eGFP was greatly reduced from the PM and a greatly reduced level accumulated in vacuolar compartments (B, D). Shown were root epidermal cells imaged under identical confocal settings. Scale bars, 50 µm (left), 10 µm (right).
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pone-0001510-g004: Enhanced PIN2-eGFP vacuolar accumulation in dark-grown det3-1 mutant.PIN2-eGFP was slightly enhanced in diffuse and punctate cytoplasmic structures in light-grown det3-1 mutant, compared to the eir1-1 control plant (A, C). In dark-grown det3-1 mutant, a high level of PIN2-eGFP was detected both at the PM and in vacuolar compartments, compared to the dark-grown control plant, where PIN2-eGFP was greatly reduced from the PM and a greatly reduced level accumulated in vacuolar compartments (B, D). Shown were root epidermal cells imaged under identical confocal settings. Scale bars, 50 µm (left), 10 µm (right).

Mentions: By contrast, in 5-day-old seedlings grown in dark, PIN2-eGFP was greatly reduced from the PM and a detectable level of PIN2-eGFP was accumulated in intracellular compartments resembling vacuoles in both epidermal and cortical cells (Figs. 1F, G; 2A, B; 4A, B). To confirm that PIN2-eGFP intracellular compartments were vacuoles, we carried out differential interference contrast (DIC) microscopic analysis of root epidermis cells (Figs. 2A, B; middle panels) and labeling experiments with lysotracker red, a fluorescence dye that specifically marks acidic endomembrane compartments (Figs. 2C, D; middle panels). These data collectively confirmed that the intracellular compartments where PIN2-eGFP accumulated in dark-grown, but not in light-grown plants, were vacuolar compartments. Immuno-fluorescence labeling of the endogenous PIN2 proteins, using affinity-purified anti-PIN2 antibodies [50], confirms that the PIN2-eGFP fluorescence patterns in light- and dark-grown seedlings represent the patterns of the endogenous protein (Figs. 2E, F).


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)

Enhanced PIN2-eGFP vacuolar accumulation in dark-grown det3-1 mutant.PIN2-eGFP was slightly enhanced in diffuse and punctate cytoplasmic structures in light-grown det3-1 mutant, compared to the eir1-1 control plant (A, C). In dark-grown det3-1 mutant, a high level of PIN2-eGFP was detected both at the PM and in vacuolar compartments, compared to the dark-grown control plant, where PIN2-eGFP was greatly reduced from the PM and a greatly reduced level accumulated in vacuolar compartments (B, D). Shown were root epidermal cells imaged under identical confocal settings. Scale bars, 50 µm (left), 10 µm (right).
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Related In: Results  -  Collection

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

pone-0001510-g004: Enhanced PIN2-eGFP vacuolar accumulation in dark-grown det3-1 mutant.PIN2-eGFP was slightly enhanced in diffuse and punctate cytoplasmic structures in light-grown det3-1 mutant, compared to the eir1-1 control plant (A, C). In dark-grown det3-1 mutant, a high level of PIN2-eGFP was detected both at the PM and in vacuolar compartments, compared to the dark-grown control plant, where PIN2-eGFP was greatly reduced from the PM and a greatly reduced level accumulated in vacuolar compartments (B, D). Shown were root epidermal cells imaged under identical confocal settings. Scale bars, 50 µm (left), 10 µm (right).
Mentions: By contrast, in 5-day-old seedlings grown in dark, PIN2-eGFP was greatly reduced from the PM and a detectable level of PIN2-eGFP was accumulated in intracellular compartments resembling vacuoles in both epidermal and cortical cells (Figs. 1F, G; 2A, B; 4A, B). To confirm that PIN2-eGFP intracellular compartments were vacuoles, we carried out differential interference contrast (DIC) microscopic analysis of root epidermis cells (Figs. 2A, B; middle panels) and labeling experiments with lysotracker red, a fluorescence dye that specifically marks acidic endomembrane compartments (Figs. 2C, D; middle panels). These data collectively confirmed that the intracellular compartments where PIN2-eGFP accumulated in dark-grown, but not in light-grown plants, were vacuolar compartments. Immuno-fluorescence labeling of the endogenous PIN2 proteins, using affinity-purified anti-PIN2 antibodies [50], confirms that the PIN2-eGFP fluorescence patterns in light- and dark-grown seedlings represent the patterns of the endogenous protein (Figs. 2E, F).

Bottom Line: 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.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.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.

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