Functional analysis of phospholipase Dδ family in tobacco pollen tubes

Abstract

Phosphatidic acid (PA), important signalling and metabolic phospholipid, is predominantly localized in the subapical plasma membrane (PM) of growing pollen tubes. PA can be produced from structural phospholipids by phospholipase D (PLD) but the isoforms responsible for production of plasma membrane PA were not identified yet and their functional roles remain unknown. Following genome-wide bioinformatic analysis of PLD family in tobacco, we focused on the pollen-overrepresented PLDδ class. Combining live-cell imaging, gene overexpression or knock-down, lipid-binding and structural bioinformatics, we characterized 5 NtPLDδ isoforms. Distinct PLDδ isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane-bound PLDδs are tightly bound to PM, primarily via the central catalytic domain. Knock-down, overexpression and in vivo PA level analyses revealed isofom PLDδ3 as the most important member of the PLDδ subfamily active in pollen tubes. PA promotes binding of PLDδ3 to the PM, thus creating a positive feedback loop, where PA accumulation leads to the formation of massive PM invaginations. Tightly controlled production of PA generated by PLDδ3 at the PM is important for maintaining the balance between various membrane trafficking processes, that are crucial for plant cell tip growth.

Phosphatidic Acid in Plant Hormonal Signaling: From Target Proteins to Membrane Conformations

“…PA has a specific pattern of intracellular localization. Under basal conditions, PA has been detected in the plasma membrane (PM) in various plant objects: growing tobacco pollen tubes [ 41 , 42 ], mature trichomes [ 43 ], epidermal and meristematic root cells and shoot tissues [ 44 ]. However, PA has also been detected in cell membranes other than PM.…”

“…A recent study on the functions of PLDδ-family phospholipase in tobacco showed that pollen tubes support a high level of expression of PLDδ genes during growth. For some of the PLDδ isoforms, the unique localization was detected, and the sequences important for binding to the PM were determined [ 42 ]. It has been noted that the increased level of PA, formed by the overexpression of Nt PLDδ3 isoform, causes specific morphological phenotypes indicating a violation of the balance of vesicular traffic and membrane recirculation [ 42 ].…”

Signalling Pinpointed to the Tip: The Complex Regulatory Network That Allows Pollen Tube Growth

“…Interestingly, ROS-production by NtNOX itself is in part regulated by PLD-produced PA [62]. Recently, bioinformatic analysis of tobacco PLDδs classified five different NTPLDδs, NtPLDδ1 to 5 [276]. Overexpression of these PLDs in tobacco pollen tubes showed preferential cytosolic localisation for NtPLDδ1 and 2, plasma membrane localisation for NtPLDδ4 and 5, and an equilibrium between both compartments for NtPLDδ3.…”

“…Tobacco pollen tubes overexpressing NtPLDδ3 showed the most severe phenotypes, including apical membrane invaginations. Due to the observed phenotypes, a role for NtPLDδ3 in membrane trafficking was proposed, highlighting the importance of lipid-mediated signalling for the maintenance of secretion in physiological tip growth [276].…”

Mechanical stress effects on transcriptional regulation of genes encoding microtubule- and actin-associated proteins

“…Among good candidates for mechanotransduction is plant-specific phospholipase D delta (PLDδ) (Ho et al 2009 ; Cvrčkova 2013 ; Pleskot et al 2013 ; Pejchar et al 2020 ), a possible mediator in the cell wall–PM-cytoskeleton continuum (Marc et al 1996 ; Gardiner et al 2003 ) and also able to regulate the activity of both MTs and AFs (Petrasek and Schwarzerova 2009 ). In particular, PLDδ binds to plant flotillin homolog (Ho et al 2009 ), a lipid microdomain marker (Martin et al 2005 ), constituting the sites where multimolecular signaling complexes containing G-proteins or kinases, flotillin, PLDδ, MTs, actin filaments, Hsp70 and others are assembled and take part in cell signaling and vesicle trafficking (Martin et al 2005 ; Ho et al 2009 ; Tapken and Murphy 2015 ).…”