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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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Root growth, auxin transport and intracellular localization of PIN proteins in Arabidopsis plants grown in the presence and absence of light.(A) A 5-day-old seedling grown under light developed a long root, short hypocotyl and two fully-expanded cotyledons (left); by contrast, a dark-grown seedling developed a short root, long hypocotyl, two un-expanded cotyledons and an apical hook (right). Arrows marked hypocotyl-root junction. (B) Root elongation rate was 6.4±1 and 1.8±0.1 mm/day for light- and dark-grown plants, respectively (n = 10; repeated three times, p<0.05). (C) Root diameter was 148±12 and 90±5 µm for light- and dark-grown seedlings, respectively (n = 10; repeated three times, p<0.05). Normalized root basipetal auxin transport (D) and acropetal auxin transport (E) in dark-grown plants was 77% and 50% that of light-grown counterparts (n = 8; repeated three times, p<0.05). (F-K) Shown were median optical sections of root tips of plants grown in light (F, H, I) and dark (G, I, K), expressing PIN2-eGFP (F, G), PIN1-eGFP (H, I) and PIN7-eGFP (J, K), and counter stained for cell walls with propidium iodide (red). Error bars represent standard deviations. Scale bars, 2 mm (A); 50 µm (F-K; left panels); 10 µm (F-K; right panels).
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pone-0001510-g001: Root growth, auxin transport and intracellular localization of PIN proteins in Arabidopsis plants grown in the presence and absence of light.(A) A 5-day-old seedling grown under light developed a long root, short hypocotyl and two fully-expanded cotyledons (left); by contrast, a dark-grown seedling developed a short root, long hypocotyl, two un-expanded cotyledons and an apical hook (right). Arrows marked hypocotyl-root junction. (B) Root elongation rate was 6.4±1 and 1.8±0.1 mm/day for light- and dark-grown plants, respectively (n = 10; repeated three times, p<0.05). (C) Root diameter was 148±12 and 90±5 µm for light- and dark-grown seedlings, respectively (n = 10; repeated three times, p<0.05). Normalized root basipetal auxin transport (D) and acropetal auxin transport (E) in dark-grown plants was 77% and 50% that of light-grown counterparts (n = 8; repeated three times, p<0.05). (F-K) Shown were median optical sections of root tips of plants grown in light (F, H, I) and dark (G, I, K), expressing PIN2-eGFP (F, G), PIN1-eGFP (H, I) and PIN7-eGFP (J, K), and counter stained for cell walls with propidium iodide (red). Error bars represent standard deviations. Scale bars, 2 mm (A); 50 µm (F-K; left panels); 10 µm (F-K; right panels).

Mentions: Compared to that of plants grown in continuous light, root radial expansion and rate of root elongation of plants grown in dark were reduced by 38% (90±5 vs. 148±12 µm) and 72% (1.8±0.1 vs. 6.4±1 mm/day), respectively (Figs. 1A–C). In addition, root gravitropic response was greatly reduced in plants grown in dark compared to the light-grown counterparts (data not shown). The phenotypes of dark-grown plants mimics that of auxin transport mutants [36], suggesting that auxin transport may be affected in plants grown in dark. To test this, we measured auxin transport activities in roots of 5-day-old plants grown either in continuous light or in dark. Because radial expansion was significantly reduced in the root tip region of dark-grown plants, which may indirectly affect auxin transport measurements, auxin transport activities were normalized, taken into account the differences in root radial expansion. The normalized data show that root acropetal (base-to-tip) and basipetal (tip-to-base) auxin transport activities in dark-grown plants were significantly reduced to 50% and 77%, respectively, that of light-grown plants (Figs. 1D, E; n = 8, three replicates, t-test p<0.05). The reduced root basipetal auxin transport in dark-grown plants was similar to that of light-grown agr1–5 mutant (an allele of pin2 mutants; [50].


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)

Root growth, auxin transport and intracellular localization of PIN proteins in Arabidopsis plants grown in the presence and absence of light.(A) A 5-day-old seedling grown under light developed a long root, short hypocotyl and two fully-expanded cotyledons (left); by contrast, a dark-grown seedling developed a short root, long hypocotyl, two un-expanded cotyledons and an apical hook (right). Arrows marked hypocotyl-root junction. (B) Root elongation rate was 6.4±1 and 1.8±0.1 mm/day for light- and dark-grown plants, respectively (n = 10; repeated three times, p<0.05). (C) Root diameter was 148±12 and 90±5 µm for light- and dark-grown seedlings, respectively (n = 10; repeated three times, p<0.05). Normalized root basipetal auxin transport (D) and acropetal auxin transport (E) in dark-grown plants was 77% and 50% that of light-grown counterparts (n = 8; repeated three times, p<0.05). (F-K) Shown were median optical sections of root tips of plants grown in light (F, H, I) and dark (G, I, K), expressing PIN2-eGFP (F, G), PIN1-eGFP (H, I) and PIN7-eGFP (J, K), and counter stained for cell walls with propidium iodide (red). Error bars represent standard deviations. Scale bars, 2 mm (A); 50 µm (F-K; left panels); 10 µm (F-K; right panels).
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pone-0001510-g001: Root growth, auxin transport and intracellular localization of PIN proteins in Arabidopsis plants grown in the presence and absence of light.(A) A 5-day-old seedling grown under light developed a long root, short hypocotyl and two fully-expanded cotyledons (left); by contrast, a dark-grown seedling developed a short root, long hypocotyl, two un-expanded cotyledons and an apical hook (right). Arrows marked hypocotyl-root junction. (B) Root elongation rate was 6.4±1 and 1.8±0.1 mm/day for light- and dark-grown plants, respectively (n = 10; repeated three times, p<0.05). (C) Root diameter was 148±12 and 90±5 µm for light- and dark-grown seedlings, respectively (n = 10; repeated three times, p<0.05). Normalized root basipetal auxin transport (D) and acropetal auxin transport (E) in dark-grown plants was 77% and 50% that of light-grown counterparts (n = 8; repeated three times, p<0.05). (F-K) Shown were median optical sections of root tips of plants grown in light (F, H, I) and dark (G, I, K), expressing PIN2-eGFP (F, G), PIN1-eGFP (H, I) and PIN7-eGFP (J, K), and counter stained for cell walls with propidium iodide (red). Error bars represent standard deviations. Scale bars, 2 mm (A); 50 µm (F-K; left panels); 10 µm (F-K; right panels).
Mentions: Compared to that of plants grown in continuous light, root radial expansion and rate of root elongation of plants grown in dark were reduced by 38% (90±5 vs. 148±12 µm) and 72% (1.8±0.1 vs. 6.4±1 mm/day), respectively (Figs. 1A–C). In addition, root gravitropic response was greatly reduced in plants grown in dark compared to the light-grown counterparts (data not shown). The phenotypes of dark-grown plants mimics that of auxin transport mutants [36], suggesting that auxin transport may be affected in plants grown in dark. To test this, we measured auxin transport activities in roots of 5-day-old plants grown either in continuous light or in dark. Because radial expansion was significantly reduced in the root tip region of dark-grown plants, which may indirectly affect auxin transport measurements, auxin transport activities were normalized, taken into account the differences in root radial expansion. The normalized data show that root acropetal (base-to-tip) and basipetal (tip-to-base) auxin transport activities in dark-grown plants were significantly reduced to 50% and 77%, respectively, that of light-grown plants (Figs. 1D, E; n = 8, three replicates, t-test p<0.05). The reduced root basipetal auxin transport in dark-grown plants was similar to that of light-grown agr1–5 mutant (an allele of pin2 mutants; [50].

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