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.

• Klíčová slova
• Genetically encoded biosensors, lipid manipulation, membrane lipid imaging, microscopy, peripheral membrane proteins, protein–lipid interactions,
• MeSH
• buněčná membrána * metabolismus   MeSH
• membránové lipidy metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• rostlinné proteiny * metabolismus   MeSH
• rostliny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• membránové lipidy MeSH
• membránové proteiny MeSH
• rostlinné proteiny * MeSH

• Publikační typ
• zprávy 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.

• Klíčová slova
• autophagy, biologically active substance, diacylglycerol kinase, phosphatidic acid, phospholipase, phospholipid, signal transduction, targets,
• MeSH
• fosfolipasa D * metabolismus   MeSH
• hormony metabolismus   MeSH
• kyseliny fosfatidové * metabolismus   MeSH
• proteiny metabolismus   MeSH
• rostlinné proteiny genetika   MeSH
• rostliny metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• vývoj rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• fosfolipasa D * MeSH
• hormony MeSH
• kyseliny fosfatidové * MeSH
• proteiny MeSH
• rostlinné proteiny MeSH

Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.

• Klíčová slova
• auxin carriers, correlative microscopy, nanodomains, plasma membrane,
• MeSH
• Arabidopsis genetika   růst a vývoj   MeSH
• buněčná membrána genetika   metabolismus   ultrastruktura   MeSH
• konfokální mikroskopie MeSH
• kovové nanočástice chemie   MeSH
• kyseliny indoloctové metabolismus   MeSH
• mikroskopie elektronová rastrovací * MeSH
• počítačové zpracování obrazu MeSH
• protoplasty metabolismus   ultrastruktura   MeSH
• regulátory růstu rostlin genetika   metabolismus   MeSH
• tabák genetika   metabolismus   ultrastruktura   MeSH
• zlato chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• regulátory růstu rostlin MeSH
• zlato 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.

• Klíčová slova
• Arabidopsis, cell polarity, lateral diffusion, plant development, polar auxin transport, positive feedback, protein phosphorylation,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• biologický transport MeSH
• kořeny rostlin metabolismus   MeSH
• kyseliny indoloctové MeSH
• membránové transportní proteiny genetika   MeSH
• polarita buněk MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné buňky metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• proteiny huseníčku * MeSH

Plant roots are very plastic and can adjust their tissue organization and cell appearance during abiotic stress responses. Previous studies showed that direct root illumination and sugar supplementation mask root growth phenotypes and traits. Sugar and light signaling where further connected to changes in auxin biosynthesis and distribution along the root. Auxin signaling underpins almost all processes involved in the establishment of root traits, including total root length, gravitropic growth, root hair initiation and elongation. Root hair plasticity allows maximized nutrient uptake and therefore plant productivity, and root hair priming and elongation require proper auxin availability. In the presence of sucrose in the growth medium, root hair emergence is partially rescued, but the full potential of root hair elongation is lost. With our work we describe a combinatory study showing to which extent light and sucrose are antagonistically influencing root length, but additively affecting root hair emergence and elongation. Furthermore, we investigated the impact of the loss of PIN-FORMED2, an auxin efflux carrier mediating shootward auxin transporter, on the establishment of root traits in combination with all growth conditions.

• Klíčová slova
• PIN-FORMED2, dark grown roots, gravitropic index, light grown roots, root growth, root hair, root hair elongation, shootward auxin transport, sucrose, sugar, total root length,
• Publikační typ
• časopisecké články MeSH