Abstract
BackgroundThe continuous polarized vesicle secretion in pollen tubes is essential for tip growth but the location of endo- and exocytic sub-domains remains however controversial. In this report we aimed to show that Arabidopsis thaliana syntaxins are involved in this process and contribute to spatially define exocytosis and membrane recycling.ResultsUsing GFP-fusion constructs, we imaged the distribution of pollen-specific (AtSYP124) and non-pollen syntaxins (AtSYP121 and AtSYP122) in transiently transformed Nicotiana tabacum pollen tubes. All three proteins associate with the plasma membrane and with apical vesicles indicating a conserved action mechanism for all SYPs. However, the GFP tagged SYP124 showed a specific distribution with a higher labelling at the plasma membrane flanks, 10-25 μm behind the apex. This distribution is affected by Ca2+ fluxes as revealed by treatment with Gd3+ (an inhibitor of extracellular Ca2+ influx) and TMB-8 (an inhibitor of intracellular Ca2+ release). Both inhibitors decreased growth rate but the distribution of SYP124 at the plasma membrane was more strongly affected by Gd3+. Competition with a related dominant negative mutant affected the specific distribution of SYP124 but not tip growth. In contrast, co-expression of the phosphatidylinositol-4-monophosphate 5-kinase 4 (PIP5K4) or of the small GTPase Rab11 perturbed polarity and the normal distribution of GFP-SYP but did not inhibit the accumulation in vesicles or at the plasma membrane.ConclusionsThe results presented suggest that in normal growing pollen tubes, a net exocytic flow occurs in the flanks of the tube apex mediated by SYP124. The specific distribution of SYP124 at the plasma membrane is affected by changes in Ca2+ levels in agreement with the importance of this ion for exocytosis. Apical growth and the specific localization of SYP124 were affected by regulators of membrane secretion (Ca2+, PIP5K4 and Rab11) but competition with a dominant negative mutant affected only SYP distribution. These data thus suggest that syntaxins alone do not provide the level of specificity that is required for apical growth and that additional signalling and functional mechanisms are required.
Highlights
The continuous polarized vesicle secretion in pollen tubes is essential for tip growth but the location of endo- and exocytic sub-domains remains controversial
To study the distribution of syntaxins during pollen tube growth, pollen of Nicotiana tabacum was transiently transfected with a GFP-AtSYP124 fusion construct with the chimera cloned downstream of the Lat52 promoter [25]
AtSYP124 is a pollen-specific gene [Microarray Database Genevestigator, GeneChip; [17,28]]) from a highly conserved family. To investigate if this specificity is translated into a specific distribution or if syntaxin distribution follows the cell’s secretory requirements, we transformed pollen with GFP fusion constructs of AtSYP121 and AtSYP122, non-pollen syntaxins that have been previously characterized in protoplasts by us [23] and with dominant-negative (DN) versions of all three proteins
Summary
The continuous polarized vesicle secretion in pollen tubes is essential for tip growth but the location of endo- and exocytic sub-domains remains controversial. The membrane material provided by complex reported at the extreme tip [9]; and (d) the absence of clathrin-coated vesicles from the extreme apex in pollen tubes preserved by freeze-fixation [10]. Some of these data only indirectly concerned secretion or were obtained with methods that precluded a proper analysis of vesicle dynamics. It allowed us to generate other working hypothesis namely the existence of a rapid endocytosis (or kiss-and-run) mechanism at the extreme apex coupled to a “conventional” mechanism with clathrin-mediated endocytosis occurring at sub-apical regions [12,13]
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