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

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.

Rab11 GTPase-Regulated Membrane Trafficking Is Crucial for Tip-Focused Pollen Tube Growth in Tobacco

“…(A) Epifluorescence image of a wild-type pollen tube treated with FM4-64 (3.2 mM, 10 min). The accumulation of labeled transport vesicles in the apical clear zone was as previously reported (Camacho and Malho, 2003;Parton et al, 2003). (B) Epifluorescence images of a GFP-Rab11b-expressing pollen tube treated with FM4-64 (3.2 mM, 10 min) taken in the green and red channels (top and middle panels).…”

“…The A confocal image from the medial section of a pollen tube expressing GFP-HDEL, revealing the endoplasmic reticulum system. The endoplasmic reticulum membranes are extensive and present throughout the entire pollen tube except in the extreme apex (see also Parton et al, 2003). The densest network can be seen at the subapical region.…”

“…FM dyes are taken up efficiently from the growth medium by elongating pollen tubes (O'Driscoll et al, 1993;Camacho and Malho, 2003;Parton et al, 2003). The temporal pattern of FM4-64 labeling suggests an initial internalization by endocytosis followed by membrane recycling from the endosomal system to the apex, resulting in the accumulation of FM4-64-labeled transport vesicles in the clear zone ( Figure 8A; Camacho and Malho, 2003;Parton et al, 2003).…”

“…The temporal pattern of FM4-64 labeling suggests an initial internalization by endocytosis followed by membrane recycling from the endosomal system to the apex, resulting in the accumulation of FM4-64-labeled transport vesicles in the clear zone ( Figure 8A; Camacho and Malho, 2003;Parton et al, 2003). When GFP-Rab11b-expressing pollen tubes were treated with FM4-64, the localization of the fusion protein in the apical clear zone and in the retrograde cytoplasmic stream overlapped with that of the fluorescent dye ( Figures 8B and 8C), consistent with exocytic vesicles and vesicles derived from endocytic membranes co-occupying the apical clear zone.…”

The Maize aberrant pollen transmission 1 Gene Is a SABRE/KIP Homolog Required for Pollen Tube Growth

“…The importance of an intact Golgi network for pollen tube elongation was first demonstrated with the organelle movement inhibitor brefeldin A. Treatment of pollen tubes with this drug alters cytoplasmic streaming, causes the disappearance of Golgi bodies, and arrests pollen tube growth (Rutten and Knuiman 1993;Cheung et al 2002;Parton et al 2003). Recent genetic data confirm the importance of the Golgi for pollen tube growth.…”