PMC

Four-day-old light grown PIN2-eGFP seedlings, after removal of cotyledons, were transferred to either light (A) or dark (B) for 10 hrs. PIN2-eGFP was localized to the PM in excised roots incubated in the presence of light (A). By contrast, PIN2-eGFP was accumulated in vacuolar compartments in excised roots incubated in the absence of light (B). Shown in right were close-up images. Scale bars, 50 µm (left); 10 µm (right).

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).

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.

AUX1-YFP was mainly localized to the basal PM of lateral root cap and central pre-vascular cells, and to the PM of root columella cells in both light-grown (A) and dark-grown (B) seedling roots. PIP2A-GFP was predominantly on the PM of all root cells, except that it was excluded from the quiescent center and surrounding initial cells, of light-grown plants (C). In dark-grown seedlings, a detectable level of PIP2A-GFP accumulated in vacuolar compartments (D). deltaTIP-GFP labeled both the PM and tonoplast membrane of root cells of light-grown (E) and dark-grown (F) plants. Shown in right and insets were close-up images. Scale bars, 50 µm (left), 25 µm (right).

(A) PIN2-eGFP (green) asymmetric localization at the apical plasmamembrane (PM) of root epidermis cells of 5-day-old light-grown plants (left; inset). No vacuolar accumulation of PIN2-eGFP was observed by DIC imaging (middle and right; insets). (B) In dark-grown seedlings, PIN2-eGFP PM-localization was greatly reduced (left; inset) and a detectable level of PIN2-eGFP accumulated in vacuolar compartments (middle and right; insets). (C, D) Lysotracker red (red) labeling of vacuolar compartments in root epidermis cells of light-grown (C; middle) and dark-grown (D; middle) plants. PIN2-eGFP only accumulated in vacuoles of dark-grown plants (C, D). Insets were close-up images. (E, F) Immuno-fluorescence labeling of the endogenous PIN2 protein in light-grown and dark-grown wild type plants. The endogenous PIN2 protein was localized to the apical end of root epidermal cells of a 5-day-old light-grown plant (E; red). In 5-day-old dark-grown plants, PIN2 was greatly reduced from the PM, and localized in vacuolar compartments (F; red). Scale bar, 50 µm (A, B), 10 µm (C–F).

(A) PIN2-eGFP was localized at the apical PM of root epidermal cells of plants grown in continuous white light. (B) PIN2-eGFP asymmetric location in root epidermis cells was maintained in light-grown plants shifted to continuous blue light (475 nm) for 24 hrs. When light-grown plants were shifted to continuous red light (660 nm) for 5 hrs (C) or 24 hrs (D), PIN2-eGFP accumulated in vacuolar compartments. When light-grown plants were shifted to continuous far red light (730 nm) for 5 hrs (E) or 24 hrs (F), PIN2-eGFP accumulated in vacuolar compartments. (G) PIN2-eGFP was greatly reduced from the PM of root epidermis cells of light-grown hy5-1 mutant. PIN2-eGFP was moderately enhanced at the PM in light-grown homozygous cop9-1 mutant (I), compared to that of the wild type plant (H). In dark-grown cop9-1 mutant, PIN2-eGFP PM localization was enhanced and vacuolar accumulation was reduced (K), compared to that of the dark-grown wild type plant (J). In the presence of MG132 (50 µM), PIN2-eGFP PM localization was enhanced, while vacuolar accumulation was reduced in the light-grown plant after shift to dark for 20 hrs (M), compared to the reduced PM localization and increased vacuolar accumulation in the wild type plant after shift to dark for 20 hrs (L). Shown in right (A–G) were close-up images. Scale bars, 50 µm (A–G, left); 10 µm (A–G, right; H–M).

(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).

Five-day-old light-grown PIN2-eGFP seedlings were pulse-labeled with an endocytosis marker, FM4-64, and then transferred to dark (A–D) or kept in light (E–H). Both PIN2-eGFP (green) and FM4-64 (red) were restricted to the plasma membrane (PM) at T = 0 (A, E). At T = 30 min after transfer to dark, FM4-64 internalized to early endosomes and marked PIN2-eGFP-labeled endosomes (B). At T = 4 hrs after transfer to dark, PIN2-eGFP accumulated in vacuolar compartments, whose membrane was now labeled with FM4-64 (C). At T = 8 hrs after transfer to dark, strong vacuolar accumulation of PIN2-eGFP and FM4-64 labeling of vacuolar membrane were visible (D). On the other hand, FM4-64 labeled endosomes at T = 30 min (F) and vacuolar compartments at T = 4 and 8 hrs in seedling kept in the light condition (E–H). But, PIN2-eGFP remained at the PM under light (E–H). (A–H) Shown were root epidermal cells. Left, PIN2-eGFP (green); middle, FM4-64 (red); right, merged images. Scale bars, 10 µm. (I) RT-PCR analysis of steady state PIN2 transcript levels in seedlings grown in continuous light (left) or after light-to-dark transition (right) for up to 24 hrs. The steady state transcript levels of an Actin gene were used as internal loading controls. (J) Real-time qRT-PCR analysis of steady state PIN2 levels, normalized against the level of the Actin gene. Shown were the average PIN2/Actin ratios of three independent experiments. (K) Fluorescence intensities of PIN2-eGFP at the PM of root epidermis cells of light-grown plants kept in light (blue line) or shifted to dark (red line) for 0, 4, 8 and 12 hrs. Significant differences were observed between light- and dark-shifted plants at T = 4, 8 and 12 hrs (n = 168–466; Student's t-test, p<0.0001).