Characterisation of detergent-insoluble membranes in pollen tubes of Nicotiana tabacum (L.)

Abstract

Pollen tubes are the vehicle for sperm cell delivery to the embryo sac during fertilisation of Angiosperms. They provide an intriguing model for unravelling mechanisms of growing to extremes. The asymmetric distribution of lipids and proteins in the pollen tube plasma membrane modulates ion fluxes and actin dynamics and is maintained by a delicate equilibrium between exocytosis and endocytosis. The structural constraints regulating polarised secretion and asymmetric protein distribution on the plasma membrane are mostly unknown. To address this problem, we investigated whether ordered membrane microdomains, namely membrane rafts, might contribute to sperm cell delivery. Detergent insoluble membranes, rich in sterols and sphingolipids, were isolated from tobacco pollen tubes. MALDI TOF/MS analysis revealed that actin, prohibitins and proteins involved in methylation reactions and in phosphoinositide pattern regulation are specifically present in pollen tube detergent insoluble membranes. Tubulins, voltage-dependent anion channels and proteins involved in membrane trafficking and signalling were also present. This paper reports the first evidence of membrane rafts in Angiosperm pollen tubes, opening new perspectives on the coordination of signal transduction, cytoskeleton dynamics and polarised secretion.

Retarded germination of Nicotiana tabacum seeds following insertion of exogenous DNA mimics the seed persistent behavior

“…Protein identification was performed by peptide mass fingerprinting [ 33 , 34 ] using an Ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA). Bands and spots of interest were manually excised, destained, as previously described [ 34 , 35 ], and acetonitrile dehydrated. Before protein digestion, 1D gel-resolved proteins were reduced and alkylated as previously reported [ 35 ].…”

“…Bands and spots of interest were manually excised, destained, as previously described [ 34 , 35 ], and acetonitrile dehydrated. Before protein digestion, 1D gel-resolved proteins were reduced and alkylated as previously reported [ 35 ]. 1D and 2D-gel resolved proteins were rehydrated in trypsin solution (Sigma Aldrich, Italy), and in-gel protein digestion was performed by an overnight incubation at 37°C.…”

Mapping of Membrane Lipid Order in Root Apex Zones of Arabidopsis thaliana

“…In consideration of the relative high cytotoxity of filipin to plant cells ( Ovečka et al, 2010 ; Boutté et al, 2011 ), we have used di-4-ANEPPDHQ as an optimal probe to visualize and quantify the lipid order in membranes of living plant cells ( Zhao et al, 2015 ). Moreover, di-4-ANEPPDHQ was used for imaging pf polarly growing pollen tubes as well, and it was clearly reported that the apical regions of Nicotiana tabacum, Picea meyeri have significant higher GP values than other regions ( Liu et al, 2009 ; Moscatelli et al, 2015 ).…”

Emerging roles for microtubules in angiosperm pollen tube growth highlight new research cues

“…It has also been proposed that sterols in DRMs provide a primer for cellulose biosynthesis ( Peng et al, 2002 ) or a scaffold to ensure the proper structural conformation of CESA and to stabilize CSCs ( Schrick et al, 2012 ). In pollen tubes, DRMs containing actin and tubulin, together with proteins involved in membrane trafficking, have been identified ( Moscatelli et al, 2015 , paper in press). Further functional studies are necessary to elucidate the role of membrane microdomains in exo-endocytosis and in cytoskeleton dynamics.…”

“…The presence of CalS in the pollen tube tip ( Cai et al, 2011 ) where callose is absent intriguingly suggests that CalS activity could be controlled by the retrieval of the enzyme from the apical PM to be repositioned in the shank and that MTs could be involved in this process. Even if CalS has not been identified in tobacco pollen tube membrane microdomains ( Moscatelli et al, 2015 ), further studies could better characterize DRM-proteins and investigate the role of MTs in functional relationships between membrane rafts, membrane trafficking and cell wall composition in polarized growth.…”