Pollen Tube Tip Growth Depends on Plasma Membrane Polarization Mediated by Tobacco PLC3 Activity and Endocytic Membrane Recycling

Abstract

Phosphatidyl inositol 4,5-bisphosphate (PI 4,5-P2) accumulates in a Rac/Rop-dependent manner in the pollen tube tip plasma membrane, where it may control actin organization and membrane traffic. PI 4,5-P2 is hydrolyzed by phospholipase C (PLC) activity to the signaling molecules inositol 1,4,5-trisphosphate and diacyl glycerol (DAG). To investigate PLC activity during tip growth, we cloned Nt PLC3, specifically expressed in tobacco (Nicotiana tabacum) pollen tubes. Recombinant Nt PLC3 displayed Ca2+-dependent PI 4,5-P2–hydrolyzing activity sensitive to U-73122 and to mutations in the active site. Nt PLC3 overexpression, but not that of inactive mutants, inhibited pollen tube growth. Yellow fluorescent protein (YFP) fused to Nt PLC3, or to its EF and C2 domains, accumulated laterally at the pollen tube tip plasma membrane in a pattern complementary to the distribution of PI 4,5-P2. The DAG marker Cys1:YFP displayed a similar intracellular localization as PI 4,5-P2. Blocking endocytic membrane recycling affected the intracellular distribution of DAG but not of PI 4,5-P2. U-73122 at low micromolar concentrations inhibited and partially depolarized pollen tube growth, caused PI 4,5-P2 spreading at the apex, and abolished DAG membrane accumulation. We show that Nt PLC3 is targeted by its EF and C2 domains to the plasma membrane laterally at the pollen tube tip and that it maintains, together with endocytic membrane recycling, an apical domain enriched in PI 4,5-P2 and DAG required for polar cell growth.

Ca2+-Dependent Protein Kinase11 and 24 Modulate the Activity of the Inward Rectifying K+ Channels inArabidopsisPollen Tubes

“…A number of Ca 2+ -regulated cellular activities, such as cytoskeleton dynamics, exocytosis, and endocytosis, are directly or indirectly involved in the regulation of pollen tube growth (Roy et al, 1999;Hwang et al, 2005;Helling et al, 2006;Konrad et al, 2011). Based on the observation that Ca 2+ is known to regulate the activity of ion channels in many other physiological processes, Ca 2+ and its downstream components are assumed to play crucial roles in modulating ion channel activity during pollen tube growth.…”

Plant phosphoinositide-specific phospholipase C

“…However, there is a report on tobacco PI-PLC (NtPLC3), interacting with G-protein NtRac5. 23 Furthermore, in Lilium davidii, PI-PLC activity was stimulated by the addition of G-protein activator, cholera toxin. 24 In another study, overexpression of G-protein-coupled receptor was linked to the activation of PI-PLC.…”

“…25 The regulation of plant PI-PLC by G-protein-coupled receptor is by and large obscure but activation by calcium is well established. 10,16,22,23 There are two levels of regulation by calcium: (1) its requirement for the catalytic activity of XY domain and (2) its requirement for the membrane targeting activity of C2 domain. 13 We attempted to explain the mechanism of membrane targeting of PI-PLC based on the ability of C2 domain to expose hydrophobic surfaces upon stimulation by calcium.…”

“…Using yellow fluorescent protein (YFP) tag, it was demonstrated that EF-YFP-C2 domain was capable of binding to membranes, whereas C2-YFP construct did not show a similar binding. 23 In vitro lipid binding assays with N-terminal deletion mutant corresponding to EF-hand in A. thaliana PI-PLC displayed membrane binding capacity but lacked catalytic activity. 17 In our earlier study, using sedimentation based lipid binding assay, fluorescence spectroscopy, and surface plasmon resonance spectroscopy (SPR), we demonstrated that C2 domain could bind membranes independent of EF-hand or XY catalytic domain.…”

Deciphering the Possible Mechanism of GABA in Tobacco Pollen Tube Growth and Guidance

“…25 Interestingly, the roles of all these molecular components have been studied in pollen tubes. [26][27][28][29] It is important to investigate whether these signal components are regulated by GABA via GPCRs in plants, as they are in animals. 30 In our preliminary study, we found that exogenous GABA could regulate the level of Ca 2+ in tobacco pollen tubes.…”