Although angiosperm plants generally react to immunity elicitors like chitin or chitosan by the cell wall callose deposition, this response in particular cell types, especially upon chitosan treatment, is not fully understood. Here we show that the growing root hairs (RHs) of Arabidopsis can respond to a mild (0.001%) chitosan treatment by the callose deposition and by a deceleration of the RH growth. We demonstrate that the glucan synthase-like 5/PMR4 is vital for chitosan-induced callose deposition but not for RH growth inhibition. Upon the higher chitosan concentration (0.01%) treatment, RHs do not deposit callose, while growth inhibition is prominent. To understand the molecular and cellular mechanisms underpinning the responses to two chitosan treatments, we analysed early Ca2+ and defence-related signalling, gene expression, cell wall and RH cellular endomembrane modifications. Chitosan-induced callose deposition is also present in the several other plant species, including functionally analogous and evolutionarily only distantly related RH-like structures such as rhizoids of bryophytes. Our results point to the RH callose deposition as a conserved strategy of soil-anchoring plant cells to cope with mild biotic stress. However, high chitosan concentration prominently disturbs RH intracellular dynamics, tip-localised endomembrane compartments, growth and viability, precluding callose deposition.

• Keywords
• arabidopsis, cell wall, defence, gene expression, signalling,
• MeSH
• Arabidopsis * growth & development   drug effects   metabolism   physiology   MeSH
• Cell Membrane metabolism   MeSH
• Cell Wall * metabolism   MeSH
• Chitosan * pharmacology   MeSH
• Glucans * metabolism   MeSH
• Glucosyltransferases metabolism   MeSH
• Plant Roots * growth & development   metabolism   drug effects   MeSH
• Arabidopsis Proteins * metabolism   genetics   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Calcium metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• callose MeSH Browser
• Chitosan * MeSH
• Glucans * MeSH
• Glucosyltransferases MeSH
• Arabidopsis Proteins * MeSH
• Calcium MeSH

Once regarded as mere membrane building blocks, lipids are now recognized as diverse and intricate players that mold the functions, identities, and responses of cellular membranes. Although the interactions of lipids with integral and peripheral membrane proteins are crucial for their localization, activity, and function, how proteins bind lipids is still far from being thoroughly explored. Describing and characterizing these dynamic protein-lipid interactions is thus essential to understanding the membrane-associated processes. Here we review the current range of experimental techniques employed to study plant protein-lipid interactions, integrating various methods. We summarize the principles, advantages, and limitations of classical in vitro biochemical approaches, including protein-lipid overlays and various liposome binding assays, and complement them with in vivo microscopic techniques centered around the use of genetically encoded lipid sensors and pharmacological or genetic membrane lipid manipulation tools. We also highlight several emerging techniques still awaiting their advancement into plant membrane research and emphasize the need to use complementary experimental strategies as key for elucidating the mechanistic roles of protein-lipid interactions in plant cell biology.

• Keywords
• Genetically encoded biosensors, lipid manipulation, membrane lipid imaging, microscopy, peripheral membrane proteins, protein–lipid interactions,
• MeSH
• Cell Membrane * metabolism   MeSH
• Membrane Lipids metabolism   MeSH
• Membrane Proteins metabolism   MeSH
• Plant Proteins * metabolism   MeSH
• Plants metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Membrane Lipids MeSH
• Membrane Proteins MeSH
• Plant Proteins * MeSH

Exocyst component of 70-kDa (EXO70) proteins are constituents of the exocyst complex implicated in vesicle tethering during exocytosis. MILDEW RESISTANCE LOCUS O (MLO) proteins are plant-specific calcium channels and some MLO isoforms enable fungal powdery mildew pathogenesis. We here detected an unexpected phenotypic overlap of Arabidopsis thaliana exo70H4 and mlo2 mlo6 mlo12 triple mutant plants regarding the biogenesis of leaf trichome secondary cell walls. Biochemical and Fourier transform infrared spectroscopic analyses corroborated deficiencies in the composition of trichome cell walls in these mutants. Transgenic lines expressing fluorophore-tagged EXO70H4 and MLO exhibited extensive colocalization of these proteins. Furthermore, mCherry-EXO70H4 mislocalized in trichomes of the mlo triple mutant and, vice versa, MLO6-GFP mislocalized in trichomes of the exo70H4 mutant. Expression of GFP-marked PMR4 callose synthase, a known cargo of EXO70H4-dependent exocytosis, revealed reduced cell wall delivery of GFP-PMR4 in trichomes of mlo triple mutant plants. In vivo protein-protein interaction assays in plant and yeast cells uncovered isoform-preferential interactions between EXO70.2 subfamily members and MLO proteins. Finally, exo70H4 and mlo6 mutants, when combined, showed synergistically enhanced resistance to powdery mildew attack. Taken together, our data point to an isoform-specific interplay of EXO70 and MLO proteins in the modulation of trichome cell wall biogenesis and powdery mildew susceptibility.

• MeSH
• Arabidopsis * metabolism   MeSH
• Cell Wall metabolism   MeSH
• Plant Diseases microbiology   MeSH
• Disease Resistance genetics   MeSH
• Protein Isoforms genetics   metabolism   MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Plant Proteins metabolism   MeSH
• Trichomes genetics   metabolism   MeSH
• Vesicular Transport Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• EXO70H4 protein, Arabidopsis MeSH Browser
• Protein Isoforms MeSH
• Arabidopsis Proteins * MeSH
• Plant Proteins MeSH
• Vesicular Transport Proteins MeSH

Flagellin perception is a keystone of pattern-triggered immunity in plants. The recognition of this protein by a plasma membrane (PM) receptor complex is the beginning of a signaling cascade that includes protein phosphorylation and the production of reactive oxygen species (ROS). In both Arabidopsis (Arabidopsis thaliana) seedlings and suspension cells, we found that treatment with flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, caused a rapid and transient decrease in the level of phosphatidylinositol (PI) 4,5-bisphosphate along with a parallel increase in phosphatidic acid (PA). In suspension cells, inhibitors of either phosphoinositide-dependent phospholipases C (PLC) or diacylglycerol kinases (DGKs) inhibited flg22-triggered PA production and the oxidative burst. In response to flg22, receptor-like kinase-deficient fls2, bak1, and bik1 mutants (FLAGELLIN SENSITIVE 2, BRASSINOSTEROID INSENSITIVE 1-associated kinase 1, and BOTRYTIS-INDUCED KINASE 1, respectively) produced less PA than wild-type (WT) plants, whereas this response did not differ in NADPH oxidase-deficient rbohD (RESPIRATORY BURST OXIDASE HOMOLOG D) plants. Among the DGK-deficient lines tested, the dgk5.1 mutant produced less PA and less ROS after flg22 treatment compared with WT seedlings. In response to flg22, dgk5.1 plants showed lower callose accumulation and impaired resistance to Pseudomonas syringae pv. tomato DC3000 hrcC-. Transcriptomics revealed that the basal expression of defense-related genes was altered in dgk5.1 seedlings compared with the WT. A GFP-DGK5 fusion protein localized to the PM, where RBOHD and PLC2 (proteins involved in plant immunity) are also located. The role of DGK5 and its enzymatic activity in flagellin signaling and fine-tuning of early immune responses in plant-microbe interactions is discussed.

• MeSH
• Arabidopsis * metabolism   MeSH
• Diacylglycerol Kinase genetics   metabolism   MeSH
• Flagellin pharmacology   genetics   MeSH
• Plant Immunity MeSH
• Protein Serine-Threonine Kinases MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Pseudomonas syringae physiology   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BIK1 protein, Arabidopsis MeSH Browser
• Diacylglycerol Kinase MeSH
• Flagellin MeSH
• Protein Serine-Threonine Kinases MeSH
• Arabidopsis Proteins * MeSH
• Reactive Oxygen Species MeSH

Extracellular vesicles are small membrane particles (30-1000 nm) released by Bacteria, Eukaryotes and Archaea. They have been shown to play an important role in intracellular and intercellular communication, within and between kingdoms via transport of bioactive molecules. Thus, they can be involved in altering gene expression and regulation of physiological and pathological processes of the recipient. Their unique properties make extracellular vesicles a perfect candidate vector for targeted drug delivery or a biomarker. For a long time, animal and mainly mammal extracellular vesicles have been used in research. But for plants, there had been speculations about the existence of nanovesicles due to the presence of a cell wall. Today, awareness of plant extracellular vesicles is on the rise and their research has proved they have various functions, such as protein secretion, transport of bioactive molecules or defense against pathogens. Further potential of plant extracellular vesicles is stressed in this review.

• MeSH
• Bacteria MeSH
• Biomarkers metabolism   MeSH
• Extracellular Vesicles * metabolism   MeSH
• Drug Delivery Systems MeSH
• Humans MeSH
• Cell Communication MeSH
• Mammals MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Biomarkers MeSH

Cells sense a variety of extracellular signals balancing their metabolism and physiology according to changing growth conditions. Plasma membranes are the outermost informational barriers that render cells sensitive to regulatory inputs. Membranes are composed of different types of lipids that play not only structural but also informational roles. Hormones and other regulators are sensed by specific receptors leading to the activation of lipid metabolizing enzymes. These enzymes generate lipid second messengers. Among them, phosphatidic acid (PA) is a well-known intracellular messenger that regulates various cellular processes. This lipid affects the functional properties of cell membranes and binds to specific target proteins leading to either genomic (affecting transcriptome) or non-genomic responses. The subsequent biochemical, cellular and physiological reactions regulate plant growth, development and stress tolerance. In the present review, we focus on primary (genome-independent) signaling events triggered by rapid PA accumulation in plant cells and describe the functional role of PA in mediating response to hormones and hormone-like regulators. The contributions of individual lipid signaling enzymes to the formation of PA by specific stimuli are also discussed. We provide an overview of the current state of knowledge and future perspectives needed to decipher the mode of action of PA in the regulation of cell functions.

• Keywords
• autophagy, biologically active substance, diacylglycerol kinase, phosphatidic acid, phospholipase, phospholipid, signal transduction, targets,
• MeSH
• Phospholipase D * metabolism   MeSH
• Hormones metabolism   MeSH
• Phosphatidic Acids * metabolism   MeSH
• Proteins metabolism   MeSH
• Plant Proteins genetics   MeSH
• Plants metabolism   MeSH
• Signal Transduction physiology   MeSH
• Plant Development MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Phospholipase D * MeSH
• Hormones MeSH
• Phosphatidic Acids * MeSH
• Proteins MeSH
• Plant Proteins 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.

• Keywords
• EXO70A1, cell polarity, exocyst, phospholipids, plasma membrane,
• MeSH
• Arabidopsis metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Cytoplasm metabolism   MeSH
• Exocytosis MeSH
• Phospholipids metabolism   MeSH
• Cell Polarity MeSH
• Arabidopsis Proteins metabolism   MeSH
• Proteomics methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• EXO70A1 protein, Arabidopsis MeSH Browser
• Phospholipids MeSH
• Arabidopsis Proteins MeSH

Polar subcellular localization of the PIN exporters of the phytohormone auxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into how these factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. We thus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.

• Keywords
• Arabidopsis, cell polarity, lateral diffusion, plant development, polar auxin transport, positive feedback, protein phosphorylation,
• MeSH
• Arabidopsis * genetics   metabolism   MeSH
• Biological Transport MeSH
• Plant Roots metabolism   MeSH
• Indoleacetic Acids MeSH
• Membrane Transport Proteins genetics   MeSH
• Cell Polarity MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Cells metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Indoleacetic Acids MeSH
• Membrane Transport Proteins MeSH
• Arabidopsis Proteins * MeSH

Pollen development, pollen grain germination, and pollen tube elongation are crucial biological processes in angiosperm plants that need precise regulation to deliver sperm cells to ovules for fertilization. Highly polarized secretion at a growing pollen tube tip requires the exocyst tethering complex responsible for specific targeting of secretory vesicles to the plasma membrane. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in the male gametophyte, governing the conventional secretory function of the exocyst, analogous to EXO70A1 (At5g03540) in the sporophyte. Our analysis of a CRISPR-generated exo70a2 mutant revealed that EXO70A2 is essential for efficient pollen maturation, pollen grain germination, and pollen tube growth. GFP:EXO70A2 was localized to the nucleus and cytoplasm in developing pollen grains and later to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for EXO70A1 function in the sporophyte, but not vice versa, indicating partial functional redundancy of these two closely related isoforms and higher specificity of EXO70A2 for pollen development-related processes. Phylogenetic analysis revealed that the ancient duplication of EXO70A, one of which is always highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in Arabidopsis pollen that is required for efficient plant sexual reproduction.

• MeSH
• Arabidopsis genetics   growth & development   MeSH
• Exocytosis genetics   physiology   MeSH
• Phylogeny MeSH
• Genetic Variation MeSH
• Genotype MeSH
• Pollen Tube genetics   growth & development   MeSH
• Gene Expression Regulation, Plant MeSH
• Genes, Plant MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

Exocyst is a heterooctameric protein complex crucial for the tethering of secretory vesicles to the plasma membrane during exocytosis. Compared to other eukaryotes, exocyst subunit EXO70 is represented by many isoforms in land plants whose cell biological and biological roles, as well as modes of regulation remain largely unknown. Here, we present data on the phospho-regulation of exocyst isoform EXO70C2, which we previously identified as a putative negative regulator of exocyst function in pollen tube growth. A comprehensive phosphoproteomic analysis revealed phosphorylation of EXO70C2 at multiple sites. We have now performed localization and functional studies of phospho-dead and phospho-mimetic variants of Arabidopsis EXO70C2 in transiently transformed tobacco pollen tubes and stably transformed Arabidopsis wild type and exo70C2 mutant plants. Our data reveal a dose-dependent effect of AtEXO70C2 overexpression on pollen tube growth rate and cellular architecture. We show that changes of the AtEXO70C2 phosphorylation status lead to distinct outcomes in wild type and exo70c2 mutant cells, suggesting a complex regulatory pattern. On the other side, phosphorylation does not affect the cytoplasmic localization of AtEXO70C2 or its interaction with putative secretion inhibitor ROH1 in the yeast two-hybrid system.

• Keywords
• Exo70, exocyst, membrane trafficking, phosphorylation, pollen tube, secretion inhibitor, tip-growth,
• Publication type
• Journal Article MeSH