Current models of plasma membrane (PM) postulate its organization in various nano-and micro-domains with distinct protein and lipid composition. While metazoan PM nanodomains usually display high lateral mobility, the dynamics of plant nanodomains is often highly spatially restricted. Here we have focused on the determination of the PM distribution in nanodomains for Arabidopsis thaliana flotillin (AtFLOT) and hypersensitive induced reaction proteins (AtHIR), previously shown to be involved in response to extracellular stimuli. Using in vivo laser scanning and spinning disc confocal microscopy in Arabidopsis thaliana we present here their nanodomain localization in various epidermal cell types. Fluorescence recovery after photobleaching (FRAP) and kymographic analysis revealed that PM-associated AtFLOTs contain significantly higher immobile fraction than AtHIRs. In addition, much lower immobile fractions have been found in tonoplast pool of AtHIR3. Although members of both groups of proteins were spatially restricted in their PM distribution by corrals co-aligning with microtubules (MTs), pharmacological treatments showed no or very low role of actin and microtubular cytoskeleton for clustering of AtFLOT and AtHIR into nanodomains. Finally, pharmacological alteration of cell wall (CW) synthesis and structure resulted in changes in lateral mobility of AtFLOT2 and AtHIR1. Accordingly, partial enzymatic CW removal increased the overall dynamics as well as individual nanodomain mobility of these two proteins. Such structural links to CW could play an important role in their correct positioning during PM communication with extracellular environment.

The dynamic microtubule cytoskeleton plays fundamental roles in the growth and development of plants including regulation of their responses to environmental stress. Plants exposed to hyper-osmotic stress commonly acclimate, acquiring tolerance to variable stress levels. The underlying cellular mechanisms are largely unknown. Here, we show, for the first time, by in vivo imaging approach that linear patterns of phospholipase Dδ match the localization of microtubules in various biological systems, validating previously predicted connection between phospholipase Dδ and microtubules. Both the microtubule and linear phospholipase Dδ structures were disintegrated in a few minutes after treatment with oryzalin or salt. Moreover, by using immunofluorescence confocal microscopy of the cells in the root elongation zone of Arabidopsis, we have shown that the cortical microtubules rapidly depolymerized within 30 min of treatment with 150 or 200 mM NaCl. Within 5 h of treatment, the density of microtubule arrays was partially restored. A T-DNA insertional mutant lacking phospholipase Dδ showed poor recovery of microtubule arrays following salt exposition. The restoration of microtubules was significantly retarded as well as the rate of root growth, but roots of overexpressor GFP-PLDδ prepared in our lab, have grown slightly better compared to wild-type plants. Our results indicate that phospholipase Dδ is involved in salt stress tolerance, possibly by direct anchoring and stabilization of de novo emerging microtubules to the plasma membrane, providing novel insight into common molecular mechanism during various stress events.

The superior properties of silver nanoparticles (AgNPs) has resulted in their broad utilization worldwide, but also the risk of irreversible environment infestation. The plant cuticle and cell wall can trap a large part of the nanoparticles and thus protect the internal cell structures, where the cytoskeleton, for example, reacts very quickly to the threat, and defense signaling is subsequently triggered. We therefore used not only wild-type Arabidopsis seedlings, but also the glabra 1 mutant, which has a different composition of the cuticle. Both lines had GFP-labeled microtubules (MTs), allowing us to observe their arrangement. To quantify MT dynamics, we developed a new microscopic method based on the FRAP technique. The number and growth rate of MTs decreased significantly after AgNPs, similarly in both lines. However, the layer above the plasma membrane thickened significantly in wild-type plants. The levels of three major stress phytohormone derivatives—jasmonic, abscisic, and salicylic acids—after AgNP (with concomitant Ag+) treatment increased significantly (particularly in mutant plants) and to some extent resembled the plant response after mechanical stress. The profile of phytohormones helped us to estimate the mechanism of response to AgNPs and also to understand the broader physiological context of the observed changes in MT structure and dynamics.

Phosphatidylinositol 4-kinases (PI4Ks) are the first enzymes that commit phosphatidylinositol into the phosphoinositide pathway. Here, we show that Arabidopsis thaliana seedlings deficient in PI4Kβ1 and β2 have several developmental defects including shorter roots and unfinished cytokinesis. The pi4kβ1β2 double mutant was insensitive to exogenous auxin concerning inhibition of root length and cell elongation; it also responded more slowly to gravistimulation. The pi4kß1ß2 root transcriptome displayed some similarities to a wild type plant response to auxin. Yet, not all the genes displayed such a constitutive auxin-like response. Besides, most assessed genes did not respond to exogenous auxin. This is consistent with data with the transcriptional reporter DR5-GUS. The content of bioactive auxin in the pi4kß1ß2 roots was similar to that in wild-type ones. Yet, an enhanced auxin-conjugating activity was detected and the auxin level reporter DII-VENUS did not respond to exogenous auxin in pi4kß1ß2 mutant. The mutant exhibited altered subcellular trafficking behavior including the trapping of PIN-FORMED 2 protein in rapidly moving vesicles. Bigger and less fragmented vacuoles were observed in pi4kß1ß2 roots when compared to the wild type. Furthermore, the actin filament web of the pi4kß1ß2 double mutant was less dense than in wild-type seedling roots, and less prone to rebuilding after treatment with latrunculin B. A mechanistic model is proposed in which an altered PI4K activity leads to actin filament disorganization, changes in vesicle trafficking, and altered auxin homeostasis and response resulting in a pleiotropic root phenotypes.