Modulation of plant root growth by nitrogen source-defined regulation of polar auxin transport (2020)
Figure 4

Fig. 4 | PIN2 protein abundance, polarity and vacuolar trafficking in roots on ammonium or nitrate containing medium.

a. Pseudo-colored, optical longitudinal cross sections of 5 DAG roots expressing PIN2::PIN2-GFP, eir1-4 12 HAT to ammonium or nitrate supplemented media. "e" denotes epidermis and "c" cortex, respectively. Color code represents GFP intensity from low (blue) to high (white) values. Scale bar=20 µm. Box plots display the distribution of the cell membrane derived PIN2-GFP fluorescence intensity (FI) values (in arbitrary units, a.u.) on ammonium (grey, epidermis (ep) and red, cortex (co), n=15) and nitrate (blue, epidermis (ep) and green, cortex (co), n=10) grown roots. 5 cells per root were analyzed. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.

b. Higher magnification of pseudo-colored confocal images of 5 DAG old roots expressing PIN2-GFP 12 HAT to ammonium or nitrate supplemented media. "e" denotes epidermis and "c" cortex, respectively. Color code represents GFP intensity from low (blue) to high (white) values. Scale bar=12 µm. White stars mark PIN2-GFP protein localization on the lateral membranes. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes divided by the signal value at inner/outer membranes) of roots on ammonium (n=31 cells from 6 roots) or nitrate (n=24 cells from 6 roots) supplemented medium. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.

c. Pseudo-colored PIN2-GFP signal in epidermal cells of 5 DAG old roots 12 HAT to ammonium or nitrate containing media. White arrows point to PIN2-GFP containing intracellular vesicles. Box plots represent the ratio in fluorescent signal detected inside the cell vs on the membranes (FI[cyt]/FI[m]). n=6 roots per condition, 5 cells per root were analyzed. Scale bar=5µm. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.

d. Microscopic images showing PIN2-Dendra fluorescent signal five hours after photoconversion of PIN2-Dendra into its red form. Depletion of the red signal and recovery of the green signal over a 6 hours period was followed in parallel in 5 DAG old roots12 HAT to ammonium or nitrate supplemented media. Note the increase in the intensity of the green signal in roots transferred to nitrate. Graph represents the mean signal + SD (n=6 roots per condition, 20 cells per root analyzed). The experiment was repeated 3 times. Scale bar=20 µm.

e. FRAP analysis of PIN2 protein mobility in PIN2::PIN2-GFP expressing epidermal cells 12 HAT to ammonium or nitrate. The graph shows polynomial regression fit with 95% confidence band of the mean signal recovery in the bleached region of interest (ROI) after background subtraction and normalization to photobleaching. Data are derived from 3 biological replicates, each consisting of 5 membranes from 3 different roots. Scale bar=2 µm.

f. Representative 3D SIM microscopic images of 10 DAG old epidermal cells expressing PIN2-GFP 12 HAT to ammonium or nitrate containing media. Green dots represent PIN2-GFP on the lateral cell surface (polar domain) of epidermal cells in the transition zone. Graph represents the number of GFP positive spots along a 30 µm long region starting at the apical side of the cell (8 cells per 4 roots and 9 cells per 4 roots) were analyzed per treatment, experiment was done 3 times. Error bars represent mean + SE. Note the effect of ammonium versus nitrate on the distribution of the PIN2-GFP spots.