Pollen tubes exhibit regular periodic membrane trafficking events in the absence of apical extension

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Abstract
The growing pollen tube provides an excellent single cell model system in which to study the mechanisms determining growth regulation, polarity and periodic behaviour. Previously, using FM4-64, we identified periodic movements within the apical vesicle accumulation that were related to the period of oscillatory growth. This suggested a more complex interdependence between membrane traffic, apical extension and periodicity than previously thought. To investigate this a comparison was made between normally growing and Brefeldin-A-treated, non-growing, tubes. Brefeldin-A treatment established an intriguing, stable yet dynamic system of membrane aggregations in the pollen tube tip that exhibited regular movements of material with a 5-7 second period compared with the normal ∼30 second periodicity observed in growing tubes. Heat treatment was found to reduce period length in both cases. After BFA treatment membrane was demonstrated to flow from the extreme pollen tube apex back through a distinct subapical Brefeldin-A-induced membrane accumulation. The effects of Brefeldin-A on the distribution of ER- and Golgi-targeted fluorescent proteins revealed that ER did not contribute directly to the system of membrane aggregations while only certain compartments of the Golgi might be involved. The involvement of membrane derived from the apical vesicle accumulation was strongly implicated. Calcium measurements revealed that Brefeldin-A abolished the typical tip-focused calcium gradient associated with growth and there were no obvious periodic fluctuations in apical calcium associated with the continued periodic Brefeldin-A membrane aggregation associated movements. Our experiments reveal an underlying periodicity in the pollen tube that is independent of secretion, apical extension and the oscillating tip-focused calcium gradient normally associated with growth, but requires an active actin cytoskeleton.