Figure 1.
Localization pattern of PIN2-Dendra2 fusion protein.
The roots of PIN2-Dendra2 were photoconverted before imaging. (A) PIN2-Dendra2 when driven by the endogenous promoter is expressed in the root tip epidermis (e) and cortex (c) identically to PIN2-EGFP that was described previously [48]. (B) In both PIN2-Dendra2 and PIN2-EGFP transgenic lines, the fusion proteins localized polarly in shootwards transversal membranes (arrows). (C) PIN2-Dendra2 similarly to PIN2-EGFP accumulates in BFA bodies (arrows). (D) Expression and localization pattern of PIN2-Dendra2 was confirmed by immunohistochemistry using an anti-Dendra2 antibody (green) on chemically fixed and sectioned roots. Sections were counterstained by propidium iodide (red).
Figure 2.
Representative spectra of Dendra2 before and after photoconversion.
Dendra2 was expressed under the control of the 35S promoter and data for guard cells and root tips are presented. PIN2-Dendra2 driven by the endogenous promoter was analyzed in the membranes of root epidermis. Spectra were measured with a Zeiss LSM-510 Meta microscope before (labeled as unconverted) and after photoconversion (labeled as converted) for regions of interest (ROI) drawn in the coded images (left pictures in panel). Intact Dendra2 was analyzed in the nuclei (ROI1 drawn in the red color in the coded images) and in the cytoplasm (ROI2 drawn in green color in the coded images) of the guard cells and the cells of the root cap. Red lines in graphs represent the spectra emitted by nuclei, green lines represent the cytoplasm. The lower row in the panel shows the spectra emitted by the membrane-localized PIN2-Dendra2 (region drawn in red color in the coded image). The 458 nm excitation was combined with the HFT 458 beam splitter, the 488 nm excitation with the HFT 488 beam splitter and the 543 nm excitation with the HFT UV/488/543/633 beam splitter.
Figure 3.
Time-dependent photoconversion of PIN2-Dendra2 fusion protein.
(A) The same root was analyzed for green and red fluorescence before (time 0) and after 3 s and 20 s conversion. (B) Efficiency of photoconversion depends on the time of illumination with blue-violet light. Green and red fluorescence signals were collected and the intensities of 150 transversal membranes within 5 roots were assessed with the ImageJ software. The interrupted line with open circles represents changes in red signal intensities, the solid line with closed circles represents decreasing in green signal intensities, bars represent SE. (C) Emission spectra of green and red signals of unconverted and photoconverted membranes were analyzed in three roots (represented by three lines in the graph). The HFT UV/488/543/633 was used as a beam splitter. Each point on the graph with SE bar represents the mean of 30 transversal membranes.
Figure 4.
Values of green and red signal intensities in transversal plasma membranes containing PIN2-Dendra2.
(A) Means of green and red membrane fluorescence intensities examined before and after photoconversion in arbitrary unites. The graph summarize the results obtained in the course of the whole study (with 330 roots 20 to 25 membranes per root were analyzed). (B) Values of green signal intensities emitted by the membranes before photoconversion were plotted against the values of red signal intensities emitted by the same membranes after 15 s’ photoconversion. The graph shows the results from a total of 2500 membranes from 100 roots used in the course of study.
Figure 5.
Turnover of PIN2 in the membranes of root tips under normal and anaerobic conditions.
(A) The cover slips were either removed after every imaging (labeled as uncovered) or roots were permanently covered by cover slips (anaerobic conditions). Converted red in the labeling means red signal intensity in transversal membranes after photoconversion; converted green in the labeling means green signal intensity after photoconversion; unconverted in the labeling means green signal in the membrane without photoconversion. (B) Images of the same roots taken in green and red channels characterizing the decrease of red signal intensity and increase of green signal intensity in membranes within 12 h after photoconversion.
Figure 6.
Western blot analysis of PIN2-Dendra2 expression.
For Western blot analysis seedlings were grown and treated as described for microscopy and roots were collected after 12 h’ treatment for protein extraction. The lane marked as ‘covered’ represents extracts from seedlings whose roots grew for 12 h under anaerobic conditions under the cover slip. Control (untreated) samples, samples of untransformed seedlings and samples treated with ABA and jasmonates (MeJA and JA) were kept uncovered during the experiment. The blot was probed by a Dendra2 specific antibody (upper row), stripped and re-probed with an actin specific antibody (middle row). The lower row shows the membrane after Ponceau S staining. Bands intensities were quantified using ImageJ. The values obtained for Dendra2 were divided by the values for actin for that sample, normalized to the control and graphed. Columns in the graph represent the means of three blots, bars represent SD.
Figure 7.
Cycloheximide (50 µM) and actinomycin A (50 µg ml−1) block PIN2 membrane delivery and accelerates PIN2 internalization.
The differences between control and samples treated by antibiotics were statistically highly significant (p≤0.001) when analyzed by the two-way ANOVA test in all events i.e. green and red signal intensities in photoconverting experiments and also in green signal intensity of unconverted samples. Bonferroni’s post test showed highly statistically significant differences (p≤0.001) between control and cycloheximide-treated seedlings at all time points for red signal intensities in photoconverting experiments and for green signal intensity in experiments with unconverted samples. Significant differences were observed in green signal intensity in photoconverting experiments at every time point from 2 to 12 h (for all p≤0.001). In the experiments with actinomycin A statistically significant differences between control and treated samples in red signal intensity were observed at every time point from 1 to 3 h (p≤0.001) and at time point 4 h (p≤0.05), in green signal intensity at time point 3 h (p≤0.01) and at every time point from 4 to 12 h (p≤0.001) and in green signal intensity of unconverted samples at all time points (p≤0.001). For others, the differences were statistically not significant.
Figure 8.
Darkness treatment diminishes PIN2 abundance in the membrane mostly by suppressing its delivery.
Relocation of seedlings from the light to darkness significantly affected green and red signal intensities in photoconverting experiments and also green signal intensity of unconverted samples (in all cases p≤0.001). In photoconverting experiments, differences in green signal were detected at time point 6 h (p≤0.05) and 12 h (p≤0.001), in red signal at time point 1.5 h (p≤0.001) and in experiments without photoconversion at every time point from 3 to 12 h (p≤0.001). For others, the differences were statistically not significant.
Figure 9.
Low temperature treatment inhibits the internalization and membrane delivery of PIN2.
Differences between samples kept at 21°C and 4°C were statistically highly significant in all cases (p≤0.001) i.e. red signal intensities and green signal intensities in photoconverting experiments and green signal intensities in non-photoconverting experiments. In photoconverting experiments differences in green signal intensity were observed at every time point from 2 to 12 h (p≤0.001), in red signal intensity at time point 1 h (p≤0.001) and at every time point from 2 to 12 h (p≤0.001) and in experiments without photoconversion at time point 4 h (p≤0.01) and at time points from 6 and 12 h (p≤0.001).
Figure 10.
DMSO affects PIN2 internalization and membrane pool recovery.
A statistically highly significant effect (p≤0.001) of 3% (v/v) DMSO applied in the medium was observed in all events i.e. green and red signal intensities in photoconverting experiments and also in green signal intensity in non-photoconverting experiments. Differences between control and DMSO-treated samples in experiments with unconverted samples were observed at time point 6 h (p≤0.001), in photoconverting experiments for green signal intensity at time point 4 h (p≤0.001) and 6 h (p≤0.001) and for red signal intensity at time point 3 (p≤0.01), 4 and 6 h (both p≤0.001).
Figure 11.
Jasmonates influence PIN2 plasma membrane dynamic.
Both JA and MeJA at the concentrations 5 µM and 50 µM affect green signal intensity in transversal plasma membranes of unconverted samples (p≤0.01 for 5 µM JA and 5 µM MeJA, p≤0.001 for 50 µM JA and 50 µM MeJA,). Differences were significant at time point 6 h (p≤0.05) and 12 h (p≤0.001) for 5 µM JA, at time point 6 h (p≤0.001) and 12 h (p≤0.001) for 50 µM JA, at time point 6 h (p≤0.05) and 12 h (p≤0.01) for 5 µM MeJA, and at time point 6 h (p≤0.05) and 12 h (p≤0.001) for 50 µM MeJA. For others differences were statistically not significant. Both jasmonates at the concentrations examined slowed-down the disappearance of red signals from the membrane (labeled as converted red; for each jasmonate and concentration p≤0.001) For both jasmonates used at a concentration of 5 µM differences were significant at time point 1.5 h (p≤0.01), 3 h (p≤0.001) and 6 h (p≤0.001), for 50 µM JA at time point 3 h (p≤0.05) and 6 h (p≤0.001), for 50 µM MeJA at time point 3 h (p≤0.05) and 6 h (p≤0.05). For others, the differences were statistically not significant. Both jasmonates at the concentrations examined delayed membrane green signal recovery (labeled as converted green; for each jasmonate and concentration p≤0.001)). Differences were significant at time point 6 h (p≤0.001) and 12 h (p≤0.001) for 5 µM JA, at time point 6 h (p≤0.01) and 12 h (p≤0.01) for 5 µM MeJA, at time point 6 h (p≤0.001) and 12 h (p≤0.001) for 50 µM JA and 50 µM MeJA. For others, the differences were statistically not significant.
Figure 12.
ABA inhibits PIN2 membrane delivery.
ABA (5 µM) exhibited no effect on red signal intensity in photoconverting experiments, and affected green signal intensity in both photoconverting and non-photoconverting experiments (for both p≤0.001). The differences between control and treated samples in non-photoconverting experiments were significant at every time point from 2 to 12 h (for each p≤0.001), the differences in photoconverting experiments were significant at time point 1 h (p≤0.05) and at every time point from 2 to 12 h (for all p≤0.001).