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)P 2 ), which localizes in a well-defined region of the subapical plasma membrane. How the PtdIns(4,5)P 2 region is maintained is currently unclear. In principle, the formation of PtdIns(4,5)P 2 by PI4P 5-kinases can be counteracted by phospholipase C (PLC), which hydrolyzes PtdIns(4,5)P 2 . 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)P 2 -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)P 2 production. When pollen tube morphologies were assessed as a quantitative read-out for PtdIns(4,5)P 2 function, NtPLC3 reverted the effects of a coexpressed PI4P 5-kinase, demonstrating that NtPLC3-mediated breakdown of PtdIns(4,5)P 2 antagonizes the effects of PtdIns(4,5)P 2 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)P 2 in the subapical plasma membrane of pollen tubes.

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

“…Petunia PiPLC1 and tobacco NtPLC3 both localise to the plasma membrane in growing pollen tubes, in overlapping regions with their substrate PI(4,5)P 2 , although the protein localisation tends to elongate further towards the shank [ 185 , 239 ]. A similar co-localisation was observed for NtPLC3 and the Arabidopsis PI4P5Ks AtPI4P5K2 and 11 after transient expression in tobacco pollen tubes [ 240 ]. For petunia PiPLC1, image time series indicated that the PLC localisation extends towards the extreme apex in phases of reduced growth and is less apical in phases of rapid growth [ 185 ].…”

“…In addition, so far, no Arabidopsis PI-PLC mutant was reported to be impaired in pollen tube growth. However, studies in other model systems highlighted PI-PLC importance to counterbalance PI4P5K-activity [ 185 , 239 , 240 ]. Petunia PiPLC1 and tobacco NtPLC3 both localise to the plasma membrane in growing pollen tubes, in overlapping regions with their substrate PI(4,5)P 2 , although the protein localisation tends to elongate further towards the shank [ 185 , 239 ].…”

“…For petunia PiPLC1, image time series indicated that the PLC localisation extends towards the extreme apex in phases of reduced growth and is less apical in phases of rapid growth [ 185 ]. Dominant negative expression of PLCs mutated in their active site or chemical inhibition of NtPLC3 caused a depolarisation of tip growth and swollen tube tips [ 185 , 239 , 240 ]. Additionally, PI(4,5)P 2 visualised by a lipid sensor was observed to spread further towards the shank upon PLC-inhibition [ 239 ].…”

“…Additionally, PI(4,5)P 2 visualised by a lipid sensor was observed to spread further towards the shank upon PLC-inhibition [ 239 ]. The pollen tube tip phenotype of swollen tips in tobacco pollen tubes was observed upon overexpression of inactive NtPLC3 and AtPI4P5K2; however, AtPI4P5K2-induced tip swelling could be reversed by simultaneous overexpression of active NtPLC3 [ 240 ]. Taken together, PLCs seem to be important in pollen tube tip growth as the main antagonist of PI4P5Ks, regulating PI(4,5)P 2 abundance and location.…”

A phosphoinositide 5‐phosphatase from Solanum tuberosum is activated by PAMP‐treatment and may antagonize phosphatidylinositol 4,5‐bisphosphate at Phytophthora infestans infection sites

“…To further test whether the expression of StIPP would interfere with PtdIns(4,5)P 2 -dependent aspects of pollen tube growth in vivo, we assessed the impact of StIPP-YFP expression on pollen tube cell morphologies arising from the modulation of PtdIns (4,5)P 2 . Pollen tubes display characteristic cell morphologies upon overproduction of PtdIns(4,5)P 2 , which can easily be scored, including pollen tubes with branched or stunted tips (Ischebeck et al, 2008(Ischebeck et al, , 2010Stenzel et al, 2012;Hempel et al, 2017) or displaying tip swelling (Ischebeck et al, 2011;Stenzel et al, 2012Stenzel et al, , 2020. The range of pollen tube morphologies observed is represented in Fig 4(c), as indicated.…”