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.
Highlights
Pollen tubes are tip-growing cells with a key function in plant sexual reproduction [1]
It has previously been reported that the distribution of PtdIns(4,5)P2 in pollen tubes, as indicated by fluorescent biosensors such as RedStar-PLCδ1-PH, closely matches the distribution of PI4P 5-kinases [12, 13]; a similar pattern was reported for fungal hyphae [23]
The dynamic control of PtdIns(4,5)P2 production in the subapical plasma membrane of pollen tubes is critical for polar cell expansion
Summary
Pollen tubes are tip-growing cells with a key function in plant sexual reproduction [1]. A loss of apical cell polarity upon overproduction of PtdIns(4,5)P2 gives rise to substantial changes in pollen tube cell shape, including apical tip swelling [13,19] or in more complex cases, branched cell morphologies [12,14,16,19] From these observations, it is clear that the extent of the subapical PtdIns(4,5)P2 domain must be tightly controlled to enable "normal" pollen tube growth. It was shown that a fluorescence-tagged variant of the tobacco (Nicotiana tabacum) PLC, NtPLC3, displays a subapical plasma membrane distribution in tobacco pollen tubes [20], which at first approximation appears similar to that observed for PI4P 5-kinases. Together with a plasma membrane distribution in a discontinuous pattern omitting punctate areas determined by spinning disc (SD) analysis, our data suggest that NtPLC3 may contribute to the spatial restriction of PtdIns(4,5)P2 in the plasma membrane of pollen tubes
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