Figure 4: Cytoplasmic acidification inhibits CME.

(a) Confocal images of Arabidopsis root cells stained with Lyso Tracker Red DND 99 (30 min) followed by an additional 30 min in the presence of DMSO (Ø), ES9, antimycin A (AA), carbonyl cyanide m-chlorophenyl hydrazine (CCCP), tyrphostinA23 (TyrA23), and TyrA51. The presence of ES9, CCCP and TyrA23 relocalized the dye from the tonoplast to the cytoplasm (b) Boxplot representation of cytoplasmic pH measurements using pH-GFP in the presence of 10 μM ES9, 50 μM TyrA23, and 1 μM CCCP, showing cytoplasmic acidification after treatment. n=59, 60, 54, and 61 seedlings respectively. Individual data points are represented. (c) Confocal images comparing FM4-64 uptake (30 min) at pH 5.5, pH 7, or pH 5.5 and 100 mM KCl in the presence of mock (DMSO, Ø), 1 μM CCCP, 50 μM TyrA23, and 10 μM ES9. (d) Quantification of the FM4-64 signal intensity under the different conditions in c. Values represent the ratio of cytoplasmic/plasma membrane signal intensity. Stars indicate significance compared to the corresponding treatment at pH 5.5. **P<0.01 and ***P<0.001 with a Kruskal-Wallis analysis of variance (ANOVA) on Ranks. The boxplot centre lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. For each condition five to seven seedlings were measured. Scale bars, 5 μm.