SH3P2 (At4g34660), an Arabidopsis thaliana SH3 and Bin/amphiphysin/Rvs (BAR) domain-containing protein, was reported to have a specific role in cell plate assembly, unlike its paralogs SH3P1 (At1g31440) and SH3P3 (At4g18060). SH3P family members were also predicted to interact with formins-evolutionarily conserved actin nucleators that participate in microtubule organization and in membrane-cytoskeleton interactions. To trace the origin of functional specialization of plant SH3Ps, we performed phylogenetic analysis of SH3P sequences from selected plant lineages. SH3Ps are present in charophytes, liverworts, mosses, lycophytes, gymnosperms, and angiosperms, but not in volvocal algae, suggesting association of these proteins with phragmoplast-, but not phycoplast-based cell division. Separation of three SH3P clades, represented by SH3P1, SH3P2, and SH3P3 of A. thaliana, appears to be a seed plant synapomorphy. In the yeast two hybrid system, Arabidopsis SH3P3, but not SH3P2, binds the FH1 and FH2 domains of the formin FH5 (At5g54650), known to participate in cytokinesis, while an opposite binding specificity was found for the dynamin homolog DRP1A (At5g42080), confirming earlier findings. This suggests that the cytokinetic role of SH3P2 is not due to its interaction with FH5. Possible determinants of interaction specificity of SH3P2 and SH3P3 were identified bioinformatically.
Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2 However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.
- MeSH
- Arabidopsis genetika růst a vývoj metabolismus MeSH
- buněčná membrána metabolismus MeSH
- časosběrné zobrazování metody MeSH
- exocytóza * MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- fosfatidylinositoly metabolismus MeSH
- fylogeneze MeSH
- geneticky modifikované rostliny MeSH
- konfokální mikroskopie MeSH
- mutace MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protein - isoformy genetika metabolismus MeSH
- proteiny huseníčku klasifikace genetika metabolismus MeSH
- pyl genetika růst a vývoj metabolismus MeSH
- pylová láčka genetika růst a vývoj metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční homologie nukleových kyselin MeSH
- simulace molekulární dynamiky MeSH
- stanovení celkové genové exprese metody MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- vezikulární transportní proteiny klasifikace genetika metabolismus MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
As sessile organisms, plants must sense environmental conditions and adjust their growth and development processes accordingly, through adaptive responses regulated by various internal factors, including hormones. A key environmental factor is temperature, but temperature-sensing mechanisms are not fully understood despite intense research. We investigated proteomic responses to temperature shocks (15 min cold or heat treatments) with and without exogenous applications of cytokinin in Arabidopsis. Image and mass spectrometric analysis of the two-dimensionally separated proteins detected 139 differentially regulated spots, in which 148 proteins were identified, most of which have not been previously linked to temperature perception. More than 70% of the temperature-shock response proteins were modulated by cytokinin, mostly in a similar manner as heat shock. Data mining of previous transcriptomic datasets supported extensive interactions between temperature and cytokinin signalling. The biological significance of this finding was tested by assaying an independent growth response of Arabidopsis seedlings to heat stress: hypocotyl elongation. This response was strongly inhibited in mutants with deficiencies in cytokinin signalling or endogenous cytokinin levels. Thus, cytokinins may directly participate in heat signalling in plants. Finally, large proportions of both temperature-shock and cytokinin responsive proteomes co-localize to the chloroplast, which might therefore host a substantial proportion of the temperature response machinery.
- MeSH
- Arabidopsis genetika růst a vývoj MeSH
- biologické modely MeSH
- cytokininy farmakologie MeSH
- fosforylace účinky léků MeSH
- geneticky modifikované rostliny MeSH
- hypokotyl účinky léků genetika růst a vývoj MeSH
- metabolismus sacharidů účinky léků genetika MeSH
- mutace MeSH
- proteiny huseníčku klasifikace genetika metabolismus MeSH
- proteom metabolismus MeSH
- reakce na tepelný šok genetika MeSH
- rostlinné geny MeSH
- teplota * MeSH
- transkriptom účinky léků genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
