Asymmetric localization of Arabidopsis SYP124 syntaxin at the pollen tube apical and sub-apical zones is involved in tip growth

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.

Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth

“…Conversely, based on colabeling with FM1-43 and FM4-64, it was concluded that exocytosis takes place in a subapical collar located in the transition zone between the tip and the shank, as well as at the shank, but not at the tip (Bove et al, 2008;Zonia and Munnik, 2008). In agreement, the pollen tube-specific syntaxin GFP-SYP124 was observed in the inverted cone, 10 to 25 mm away from the tip (Silva et al, 2010), and fluorescence recovery after photobleaching experiments with FM dyes also have indicated that exocytosis takes place at the subapical region (Bove et al, 2008;Moscatelli et al, 2012;Idilli et al, 2013). Yet, based on pollen tube reorientation experiments in a microfluidics device, it was concluded that growth takes place at the tip rather than at a subapical collar located in the transition zone between the apex and the shank (Sanati Nezhad et al, 2014).…”

The RING finger E3 ligase STRF1 is involved in membrane trafficking and modulates salt‐stress response in Arabidopsis thaliana

“…These membrane proteins include the SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins, which mediate intracellular vesicle trafficking, fusion and secretion in plant growth and development and are also involved in ABA signaling pathway and in abiotic stress response (Leyman et al, 1999;Zhu et al, 2002;Leshem et al, 2006;Zhang et al, 2007b;Bassham and Blatt, 2008;Ebine et al, 2008;Grefen and Blatt, 2008;Sokolovski et al, 2008;Zouhar et al, 2009;Shirakawa et al, 2010;Silva et al, 2010). Another membrane trafficking protein, Rab7, a member of the Rab family of small GTPases which in yeast and mammals controls intracellular vesicle trafficking from the late endosome to the vacuole (Rutherford and Moore, 2002;Woollard and Moore, 2008), has been reported to be involved in salt and osmotic stress response in plants (Mazel et al, 2004;Agarwal et al, 2008;George and Parida, 2010).…”

Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: Engineering attempts using a modified SNARE protein

“…The functional specificity is characteristic of several SNAREs belonging to multigenic families (Silva et al, 2010) and is generally related to regulated processes. The interesting distribution pattern of the deletion mutant GFP:At51H3, labelling small vacuolar compartments well separated from the central vacuole, indicates the possibility to delay the fusion process.…”