Coordinated Localization and Antagonistic Function of NtPLC3 and PI4P 5-Kinases in the Subapical Plasma Membrane of Tobacco Pollen Tubes

Abstract

Polar tip growth of pollen tubes is regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂), which localizes in a well-defined region of the subapical plasma membrane. How the PtdIns(4,5)P₂ region is maintained is currently unclear. In principle, the formation of PtdIns(4,5)P₂ by PI4P 5-kinases can be counteracted by phospholipase C (PLC), which hydrolyzes PtdIns(4,5)P₂. Here, we show that fluorescence-tagged tobacco NtPLC3 displays a subapical plasma membrane distribution which frames that of fluorescence-tagged PI4P 5-kinases, suggesting that NtPLC3 may modulate PtdIns(4,5)P₂-mediated processes in pollen tubes. The expression of a dominant negative NtPLC3 variant resulted in pollen tube tip swelling, consistent with a delimiting effect on PtdIns(4,5)P₂ production. When pollen tube morphologies were assessed as a quantitative read-out for PtdIns(4,5)P₂ function, NtPLC3 reverted the effects of a coexpressed PI4P 5-kinase, demonstrating that NtPLC3-mediated breakdown of PtdIns(4,5)P₂ antagonizes the effects of PtdIns(4,5)P₂ overproduction in vivo. When analyzed by spinning disc microscopy, fluorescence-tagged NtPLC3 displayed discontinuous membrane distribution omitting punctate areas of the membrane, suggesting that NtPLC3 is involved in the spatial restriction of plasma membrane domains also at the nanodomain scale. Together, the data indicate that NtPLC3 may contribute to the spatial restriction of PtdIns(4,5)P₂ in the subapical plasma membrane of pollen tubes.

Keywords: PI4P 5-kinase; Phosphatidylinositol 4,5-bisphosphate; PtdIns(4,5)P2; phospholipase C; pollen tube tip swelling.

Figure 1

NtPLC3 localizes to a subapical plasma membrane domain encompassing domains occupied by PIP5K2 or PIP5K11. The localization of NtPLC3 was monitored by confocal imaging upon transient expression in tobacco pollen tubes. For these localization studies, expression levels were kept low to minimize morphological changes of the pollen tube cells. (A) Coexpression of EYFP-PIP5K11 with RedStar-PLCδ1-PH, a fluorescent biosensor for PtdIns(4,5)P₂, as indicated. B-E, Association of fluorescence-tagged NtPLC3 with an extended subapical plasma membrane domain, which spans the region occupied by coexpressed PIP5K2-mCherry or EYFP-PIP5K11. (B) Coexpression of EYFP-NtPLC3 with PIP5K2-mCherry. (C) Coexpression of NtPLC3-RFP with EYFP-PIP5K11, monitored in a time lapse experiment. The time series shown was recorded for 600 s at a frame rate of 2.14 frames min^-1. Three selected frames are shown, as indicated. (D) Kymograph analysis of median confocal LSM sections of a growing pollen tube from (C), indicating the dynamic relative movements of PIP5K2-EYFP (green) and NtPLC3-RFP (red). Right panel: merged images (overlap indicated by yellow). (E) Intensity and distribution of the EYFP-PIP5K11 (green) and NtPLC3-RFP (red) signals were further analyzed by the machine learning program Ilastik to provide nonbiased results. Note that the relative distances from the pollen tube tip are roughly constant, and that the region occupied by NtPLC3-RFP continuously spans the region occupied by PIP5K2-EYFP. Data are representative for five independent experiments. Scale bars = 10 µm.

Figure 2

A catalytically inactive NtPLC3 variant, NtPLC3 H124A. A catalytically inactive variant of NtPLC3 to study dominant negative effects in vivo was generated by substituting histidine 124 for alanine. The effect of this substitution on enzyme function was tested using protein recombinantly produced in E. coli as a fusion to an N-terminal maltose-binding protein (MBP)-tag. (A) Immuno detection of recombinant MBP control, MBP-NtPLC3 and the dominant negative MBP-NtPLC3 H124A using an anti-MBP antibody, as indicated. (B) Catalytic activity of recombinant MBP, MBP-NtPLC3 and MBP-Nt-PLC3 H124A against a 2[³H]PtdIns(4,5)P₂ substrate in vitro. Data are the mean ± SD from three experiments.

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.

Figure 4

NtPLC3-RFP counteracts PIP5K2-EYFP-induced PtdIns(4,5)P₂-dependent tip swelling. The effects of NtPLC3 on cell morphologies mediated by the overexpression of PIP5K2 were tested upon transient co-overexpression in tobacco pollen tubes. The expression of all constructs was driven by the strong pollen-specific Lat52 promoter. (A) Incidence of normal, branched or swollen cell morphologies upon transient overexpression of AtPIP5K2-EYFP alone, or co-overexpressed either with NtPLC3-RFP or with NtPLC3-RFP H124A in tobacco pollen tubes. Data are from three independent experiments, and > 50 cells were analyzed for each transformation. Asterisks indicate a significant difference to the AtPIP5K2-EYFP control according to a Student’s t-test (*, p ≤ 0.05; ***, p ≤ 0.01). (B) Fluorescence intensities recorded during the co-overexpression of PIP5K2-EYFP with NtPLC3-RFP or with NtPLC3-RFP H124A. Scale bars = 10 µm.

Figure 5

NtPLC3-EYFP localizes in a discontinuous pattern, omitting circular plasma membrane nanodomains. The fluorescence distribution of overexpressed NtPLC3-EYFP in the plasma membrane was assessed by SD microscopy. Only cells displaying low fluorescence intensities were imaged; they exhibited no morphological changes. (A) Association of NtPLC3-EYFP with the pollen tube plasma membrane in a discontinuous pattern omitting circular nanodomains, determined by SD microscopy. The pattern is representative for data from three independent experiments, and 15 cells were analyzed. Scale bar = 5 µm. (B) Upper panel: Enlargement of the area indicated by the dashed box in (A). Dashed line, trace recorded for intensity analysis. Lower panel: Intensity profile for NtPLC3-EYFP fluorescence along the line. (C) Diameter of NtPLC3-EYFP-excluded domains, analyzed at different distances from the pollen tube tip, as indicated. Data are from three independent experiments, and 25 nanodomains were analyzed per distance; (D) Area density of NtPLC3-EYFP- excluded domains, analyzed at different distances from the pollen tube tip, as indicated. Data are from three independent experiments and 12 areas were analyzed per distance. (E) Association of PIP5K5-EYFP with the pollen tube plasma membrane in a continuous pattern of the plasma membrane, determined by SD microscopy as a control. Left panel, medial confocal section; right panel, peripheral section at the plasma membrane surface. The pattern is representative for data from three independent experiments, and nine cells were analyzed. Scale bar = 5 µm. (F) Upper panel: Enlargement of the area indicated by the dashed box in (A). Dashed line, trace recorded for intensity analysis. Lower panel: Intensity profile for PIP5K5-EYFP fluorescence along the line. Asterisks indicate a significant difference from the values at distance zero according to a Student’s t-test (*, p ≤ 0.05).

Figure 6

A simplified model of NtPLC3 antagonizing PtdIns(4,5)P₂-formation. A possible role for NtPLC3 is in restricting PtdIns(4,5)P₂ production in some areas of the plasma membrane while enabling it in other areas. The punctate plasma membrane areas not occupied by NtPLC3-EYFP fluorescence might be sites of enhanced PtdIns(4,5)P₂ formation and/or residence of PI4P 5-kinases. PtdIns(4,5)P₂ abundance might thus be controlled and restricted to nanodomains by the action of degrading enzymes, such as PLC. Other explanations are possible.