Fig 1.
Endosidin2 influences the dynamics of newly synthesized and endocytosed PIN2 populations.
The root treated with 50 μM ES2 for one hour in the experiment without photoconversion is shown in A. A fluorescence signal is evident in cytoplasmic ES2As (arrows) and vacuoles (asterisks). Clear ES2As are present in meristematic cells (arrows in D) and cells of the root transition zone (arrows in C). Vacuoles are labeled in the cells of the transition zone (asterisks in C) and the elongation zone (asterisks in B). After three hours of ES2 treatment, vacuoles are strongly fluorescent over the entire root (asterisks in E). The experiment without photoconversion shows that ES2 reduces the abundance of PIN2 protein in the PM (F and I, arrows in F point to ES2As). The photoconversion experiment indicates that ES2 accelerates disappearing the old red PIN2 population from the PM and inhibits the recovery of the newly synthesized green PIN2 pool in the PM (G-I). In this experiment, roots were imaged in green and red channels (BC), converted, then again pictured (AC) and transferred onto medium without (G) or with 50 μM ES2 (H) and reimaged at different time points. G and H show representative images captured during the time-lapse experiment. The detailed image, shown at high magnification and detector sensitivity, confirms the appearance of both red internalized and green newly synthesized PIN2-Dendra2 pools in ES2As (arrows). Charts (I) show changes in signal intensity of the PM in a time-lapse experiment without photoconversion (blue lines) and changes in red (red lines) and green fluorescence intensities (green lines) of the PM in the photoconversion experiment. The means of the PM's green signal intensities at each time point in the trial were normalized to the mean of PM green signal intensities of the same root assessed before conversion. Time-points 0 represent the relative green signal intensities recorded immediately after conversion. The means of red signal intensities at each time point during the experiment were related to the mean red fluorescence intensity measured immediately after conversion (time-point 0). In parallel non-photoconversion experiments, the means of green signal intensities at each time point in the trial were normalized to the mean of green signal intensity of the PM of the same root before application of treatment. Values of the time-points 0 were set to 1. The two-way ANOVA test showed that both, effects of ES2 concentrations and duration of treatment were statistically highly significant (p ≤ 0.001, n = 15) in all events (green signal intensity in the experiment without photoconversion and green and red signal intensities in the photoconverting experiment). Bars = 5 μm.
Fig 2.
Newly synthesized and internalized PIN2 populations appear in ES2As and vacuoles.
In the photoconversion experiment, vacuoles (arrow) and ES2As (arrowhead in A) are detectable in both green and red channels. Note that the green and red signals were partly overlain in large ES2As (yellow color). B shows detailed images of the cytoplasm area with green and red separated patches. These merge into larger aggregates where the signals partly overlap (yellow color). In this experiment, roots were photoconverted, then seedlings were placed on the medium with 50 μM ES2 for 1.5 hours and imaged with a Leica TCS SP8 STED 3X microscope. The numbers in the merged images combining red and green channels indicate optical slices' order in the Z-stack. Bars = 1 μm.
Fig 3.
The red PIN2 population in ES2As is of the PM origin.
In the presence of 50 μM cycloheximide, intra-cytoplasmic aggregates contain only the red PM-derived PIN2 population (A). The B charts show the normalized fluorescence intensities of the PM and ES2As. In this experiment, seedlings were pre-treated with cycloheximide for 30 minutes, then photoconverted and placed on the medium with 50 μM cycloheximide and with or without 50 μM ES2. Roots were immediately imaged and then reimaged after 90 minutes and three hours. The means of the green signal intensities of the PM and ES2As at each time point were normalized to the mean of green signal intensities of the PM of the same root before photoconversion. Time-points 0 represent the relative green signal intensities recorded immediately after conversion. The means of red signal intensities at each time point during the experiment were related to the mean of red fluorescence intensities of the PM measured immediately after conversion (time-point 0). Values of the time-points 0 were set to 1. Note that ES2As are only detectable in the green channel. In the experiment shown in C and D, seedlings were photoconverted and kept on the standard cultivation medium for two hours and then placed on the medium with ES2, imaged and then reimaged after 1.5 and three hours. Note ES2A accumulation of both red and green PIN2 populations. The charts in C show fluorescence intensities of ES2As and the PM in green and red channels. D depicts the representative image of cells in the meristematic zone of the root taken after three hours of ES2 treatment. Bars = 5 μm.
Fig 4.
PIN2 populations assemble with FM4-64 endocytic tracer in large ES2As but not in smaller bodies.
After photoconversion and co-treatment of seedlings with 50 μM ES2 and 2 μM FM4-64 for 1.5 hours, small differently fluorescent patches and large aggregates were detectable in the cytoplasm (A). B shows separate and merged channels of optical slice No. 5; the outlined area is shown at higher magnification in C, the large ES2A marked with an arrow is displayed in D at higher magnification and in separate and merged channels. Note that the aggregate is visible in all three fluorescence channels; nevertheless, signals did not entirely overlay. The numbers in the images indicate the order of optical slices in the Z-stack. Bars = 5 μm in A and B, = 1 μm in C and D.
Fig 5.
ES2 affects the Golgi apparatus.
In control root epidermal cells, the Golgi apparatus consists of stacks of a few flattened, straight cisternae (A). ES2 causes accumulation of enlarged vesicles near the Golgi (B) and even in Golgi stacks (asterisk in C), Golgi stack expansion, bending, circulation (B-D), and separation of encircled Golgi cisternae (asterisks in D). Aberrant Golgi units congregate (D), and further degrade so that large compartments composed of a heterogeneous mixture of multi-lamellar structures and vesicles (asterisks in E) are visible in the cytoplasm (E). Arrows in A-D point to Golgi cis face and arrowheads to trans-face. Note the formation of multi-vesicular bodies in the vicinity of aberrant Golgi (B), their appearance in the vacuole (double arrowhead in D), and the release of vesicle complexes to vacuoles (arrow in E). M = mitochondria, MVB = multivesicular body, CW = cell wall, V = vacuole, ER = endoplasmic reticulum, P = plastid. Bars = 0.5 μm.
Fig 6.
PIN3 and PIN4 are sensitive to ES2 treatment.
When seedlings were treated with 50 μM ES2 for 1.5 hours, ES2As were visible in the endodermal cells of PIN3-Dendra2 seedling roots (arrows in A) and root meristem cells adjacent to the root cap of PIN4-Dendra2 plants (arrows in B). Roots were imaged and then reimaged after 1.5 hours of treatment with 50 μM ES2. The chart in C depicts relative fluorescence intensities for the PM. The mean of green signal intensities after 1.5 hours of treatment with ES2 was normalized to the mean of green signal intensity of the PM of the same root before ES2 application. T-test shows statistically highly significant differences in the PM signal intensity between ES2 treated and untreated samples (in both cases p ≤ 0.001, 14 roots were analyzed for PIN3, 12 roots for PIN4) Bars = 5 μm.
Fig 7.
Endosidin2 has no apparent effect on SYT1 dynamics.
In the experiment without photoconversion, roots were placed on the medium with different ES2 concentrations, immediately imaged and then reimaged after 1,5 and 3 hours. In the experiment with photoconversion, seedlings were placed on the medium with different concentrations of ES2, then roots were immediately imaged, photoconverted, imaged again, and reimaged after 1.5 and 3 hours. A figure shows a representative image taken during experiments without photoconversion, B shows an image captured during the experiment with photoconversion, C and D show magnified images of the rectangle areas in A and E, respectively. Note that the SYT1-Dendra2 fusion protein does not accumulate in vacuoles or aggregate in the cytoplasm. The B chart shows relative PM fluorescence intensities after 1.5 and 3 hours of treatment with 50 or 100 μM ES2. In the experiment without photoconversion, the means of the PM's signal intensities in roots were normalized to the mean of signal intensities of the PM of the same root before ES2 application. In the photoconversion experiment, the means of PM green signal intensities in the roots after the photoconversion were normalized to the mean of green signal intensities of the PM of the same root before photoconversion (BC in the figure). The means of red signal intensities were normalized to the mean red fluorescence intensity measured immediately after photoconversion (AC in the figure). The two-way ANOVA test showed statistically significant differences among time points (p ≤ 0.05, n = 15) but no significant effect of ES2 treatment on green and red signal intensities in the photoconverting experiment. No effects were found in the experiment without photoconversion. Bars = 5 μm.