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.

• MeSH
• Arabidopsis * metabolismus   MeSH
• fosfatidylinositolfosfáty MeSH
• fosfatidylinositoly metabolismus   MeSH
• kořeny rostlin genetika   metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidylinositolfosfáty MeSH
• fosfatidylinositoly MeSH
• kyseliny indoloctové MeSH
• phosphatidylinositol 4-phosphate MeSH Prohlížeč
• proteiny huseníčku * MeSH

Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid-protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3-SEC5-SEC6-SEC8 and SEC10-SEC15-EXO70-EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3-EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1-phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein-lipid code for the recruitment of peripheral membrane proteins.

• Klíčová slova
• EXO70A1, cell polarity, exocyst, phospholipids, plasma membrane,
• MeSH
• Arabidopsis metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• exocytóza MeSH
• fosfolipidy metabolismus   MeSH
• polarita buněk MeSH
• proteiny huseníčku metabolismus   MeSH
• proteomika metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EXO70A1 protein, Arabidopsis MeSH Prohlížeč
• fosfolipidy MeSH
• proteiny huseníčku MeSH

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.

• Klíčová slova
• Arabidopsis, cytoskeleton, mitochondrial protein import, phospholipase D alpha1, proteomics, quality control, vesicular transport,
• MeSH
• Arabidopsis metabolismus   MeSH
• endocytóza MeSH
• fosfolipasa D genetika   metabolismus   MeSH
• genová ontologie MeSH
• glukosinoláty biosyntéza   MeSH
• kořeny rostlin metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteom metabolismus   MeSH
• proteomika * MeSH
• synaptotagmin I metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• transport proteinů MeSH
• uncoupling protein 1 metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfolipasa D MeSH
• glukosinoláty MeSH
• mitochondriální proteiny MeSH
• PLDA1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• proteom MeSH
• synaptotagmin I MeSH
• SYT1 protein, Arabidopsis MeSH Prohlížeč
• uncoupling protein 1 MeSH

Phospholipase D alpha 1 (PLDα1, At3g15730) and its product phosphatidic acid (PA) are involved in a variety of cellular and physiological processes, such as cytoskeletal remodeling, regulation of stomatal closure and opening, as well as biotic and abiotic stress signaling. Here we aimed to study developmental expression patterns and subcellular localization of PLDα1 in Arabidopsis using advanced microscopy methods such as light-sheet fluorescence microscopy (LSFM) and structured illumination microscopy (SIM). We complemented two knockout pldα1 mutants with a YFP-tagged PLDα1 expressed under the PLDα1 native promoter in order to study developmental expression pattern and subcellular localization of PLDα1 in Arabidopsis thaliana under natural conditions. Imaging of tissue-specific and developmentally-regulated localization of YFP-tagged PLDα1 by LSFM in roots of growing seedlings showed accumulation of PLDα1-YFP in the root cap and the rhizodermis. Expression of PLDα1-YFP in the rhizodermis was considerably higher in trichoblasts before and during root hair formation and growth. Thus, PLDα1-YFP accumulated in emerging root hairs and in the tips of growing root hairs. PLDα1-YFP showed cytoplasmic subcellular localization in root cap cells and in cells of the root transition zone. In aerial parts of plants PLDα1-YFP was also localized in the cytoplasm showing enhanced accumulation in the cortical cytoplasmic layer of epidermal non-dividing cells of hypocotyls, leaves, and leaf petioles. However, in dividing cells of root apical meristem and leaf petiole epidermis PLDα1-YFP was enriched in mitotic spindles and phragmoplasts, as revealed by co-visualization with microtubules. Finally, super-resolution SIM imaging revealed association of PLDα1-YFP with both microtubules and clathrin-coated vesicles (CCVs) and pits (CCPs). In conclusion, this study shows the developmentally-controlled expression and subcellular localization of PLDα1 in dividing and non-dividing Arabidopsis cells.

• Klíčová slova
• Arabidopsis thaliana, At3g15730, development, light-sheet fluorescence microscopy, localization, microtubules, phospholipase D,
• Publikační typ
• časopisecké články MeSH

Microtubules (MTs) are involved in key processes in plant cells, including cell division, growth and development. MT-interacting proteins modulate MT dynamics and organization, mediating functional and structural interaction of MTs with other cell structures. In addition to conventional microtubule-associated proteins (MAPs) in plants, there are many other MT-binding proteins whose primary function is not related to the regulation of MTs. This review focuses on enzymes, chaperones, or proteins primarily involved in other processes that also bind to MTs. The MT-binding activity of these multifunctional MAPs is often performed only under specific environmental or physiological conditions, or they bind to MTs only as components of a larger MT-binding protein complex. The involvement of multifunctional MAPs in these interactions may underlie physiological and morphogenetic events, e.g., under specific environmental or developmental conditions. Uncovering MT-binding activity of these proteins, although challenging, may contribute to understanding of the novel functions of the MT cytoskeleton in plant biological processes.

• Klíčová slova
• MAP, microtubules, multifunctional MAP, plants, tubulin,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Phytohormone salicylic acid (SA) is a crucial component of plant-induced defense against biotrophic pathogens. Although the key players of the SA pathway are known, there are still gaps in the understanding of the molecular mechanism and the regulation of particular steps. In our previous research, we showed in Arabidopsis suspension cells that n-butanol, which specifically modulates phospholipase D activity, significantly suppresses the transcription of the pathogenesis related (PR-1) gene, which is generally accepted as the SA pathway marker. In the presented study, we have investigated the site of n-butanol action in the SA pathway. We were able to show in Arabidopsis plants treated with SA that n-butanol inhibits the transcription of defense genes (PR-1, WRKY38). Fluorescence microscopy of Arabidopsis thaliana mutants expressing 35S::NPR1-GFP (nonexpressor pathogenesis related 1) revealed significantly decreased nuclear localization of NPR1 in the presence of n-butanol. On the other hand, n-butanol did not decrease the nuclear localization of NPR1 in 35S::npr1C82A-GFP and 35S::npr1C216A-GFP mutants constitutively expressing NPR1 monomers. Mass spectrometric analysis of plant extracts showed that n-butanol significantly changes the metabolic fingerprinting while t-butanol had no effect. We found groups of the plant metabolites, influenced differently by SA and n-butanol treatment. Thus, we proposed several metabolites as markers for n-butanol action.

• Klíčová slova
• NPR1, PR-1, metabolome, n-butanol, phospholipase D, salicylic acid, signaling,
• Publikační typ
• časopisecké články MeSH