Figure 3.

Overexpression of dominant negative RFP-NtPLC3 H124A induces pollen tube tip swelling, similar to expressed PI4P 5-kinases. The effects of NtPLC3 or NtPLC3 H124A on cell morphologies were tested upon transient overexpression in tobacco pollen tubes. The expression of all constructs was driven by the strong pollen-specific Lat52 promoter. (A) The extent of pollen tube tip swelling was assessed upon overexpression of NtPLC3, NtPLC3 H124A, PIP5K2, PIP5K11, or PIP5K5. Negative controls included untransformed pollen tubes, stained with the membrane dye, FM 4-64, or pollen tubes expressing EYFP, as indicated. (B) Pollen tube tip widths as determined for the respective expressed constructs. Asterisks indicate a significant difference to the YFP control according to a Student’s t-test (***, p ≤ 0.01); n.s., not significant. Data are from at least five indepen dent experiments and >50 cells were analyzed for each transformation. (C) Based on the same data set, the incidence of normal, branched or swollen cell morphologies was determined upon transient expression of RFP, NtPLC3-RFP or NtPLC3-RFP H124A in tobacco pollen tubes. Note that overex pression of the dominant negative NtPLC3-RFP H124A mediated pollen tube tip swelling, not tip branching. Asterisks indicate a significant difference to the RFP control according to a Student’s t-test (***, p ≤ 0.01). Data are from at least five independent experiments, and >50 cells were analyzed for each transformation. (D) Fluorescence intensities recorded during the expression of RFP, NtPLC3-RFP or NtPLC3-RFP H124A. Scale bars, 10 µm.