Figure 2. Time-lapse analysis of the depletion of PI(4,5)P₂ in plant cells using iDePP.

(a) Dual color time-course analysis during dex induction, monitoring mCIT-2xPH^PLC PI(4,5)P₂ biosensor subcellular localization and MAP-mCH-dOCRL appearance at the root tip in epidermal cells every five minutes for 6h30. White and orange arrowheads indicate mCIT-PH^PLC partial- and full release from the plasma membrane into the cytosol, respectively. Images were colored in green fire blue, where yellow showed the max intensity and dark blue the low level of fluorescence. Scale bars: 10 μm. (b) Dissociation indexes of mCIT-2xPH^PLC over-time. 30 cells from the displayed root, during the induction of the expression of MAP-mCH-dOCRL, were analyzed. (c-e) Time-course analysis of ³²P-PIP₂ incorporation (which is related to changed levels of the lipids) in iDePP seedlings ± dex in MAP-mCH-dOCRL line. Seedlings of MAP-mCH-dOCRL line were labelled for 20 hrs with ³²Pi and co-incubated with or without dex for the times indicated (0-20 hrs). Each sample contained the lipid extract of three seedlings of which 1/5^th was analyzed by TLC (d) and quantified by phosphoimaging, where ³²P-PIP₂ was calculated as percentage of total ³²P-lipids (d) or as fold-change compared to incubation without dex (e). In the plots, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values. All statistical tests were two-sided.