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

Pollen germination and subsequent pollen tube elongation are essential for successful land plant reproduction. These processes are achieved through well-documented activation of membrane trafficking and cell metabolism. Despite this, our knowledge of the dynamics of cellular phospholipids remains scarce. Here we present the turnover of the glycerolipid composition during the establishment of cell polarity and elongation processes in tobacco pollen and show the lipid composition of pollen plasma membrane-enriched fraction for the first time. To achieve this, we have combined several techniques, such as lipidomics, plasma membrane isolation, and live-cell microscopy, and performed a study with different time points during the pollen germination and pollen tube growth. Our results showed that tobacco pollen tubes undergo substantial changes in their whole-cell lipid composition during the pollen germination and growth, finding differences in most of the glycerolipids analyzed. Notably, while lysophospholipid levels decrease during germination and growth, phosphatidic acid increases significantly at cell polarity establishment and continues with similar abundance in cell elongation. We corroborated these findings by measuring several phospholipase activities in situ. We also observed that lysophospholipids and phosphatidic acid are more abundant in the plasma membrane-enriched fraction than that in the whole cell. Our results support the important role for the phosphatidic acid in the establishment and maintenance of cellular polarity in tobacco pollen tubes and indicate that plasma membrane lysophospholipids may be involved in pollen germination.

• Klíčová slova
• lipidomics, phosphatidic acid, phospholipid, plasma membrane, pollen, pollen tube, tip growth, tobacco,
• Publikační typ
• časopisecké články 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

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.

• Klíčová slova
• Exo70, exocyst, membrane trafficking, phosphorylation, pollen tube, secretion inhibitor, tip-growth,
• Publikační typ
• časopisecké články MeSH

Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall-radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.

• Klíčová slova
• EXO70, cell wall, exocyst complex, phosphatidic acid, phosphatidylinositol 4,5-bisphosphate, phospholipids, plasma membrane domains, polar exocytosis, trichome,
• MeSH
• Arabidopsis chemie   genetika   MeSH
• buněčná membrána chemie   genetika   MeSH
• exocytóza genetika   MeSH
• fosfatidylinositol-4,5-difosfát chemie   metabolismus   MeSH
• fosfatidylseriny chemie   genetika   MeSH
• membránové lipidy genetika   metabolismus   MeSH
• proteiny huseníčku chemie   genetika   MeSH
• trichomy chemie   genetika   MeSH
• vezikulární transportní proteiny chemie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• EXO70A1 protein, Arabidopsis MeSH Prohlížeč
• EXO70H4 protein, Arabidopsis MeSH Prohlížeč
• fosfatidylinositol-4,5-difosfát MeSH
• fosfatidylseriny MeSH
• membránové lipidy MeSH
• proteiny huseníčku MeSH
• vezikulární transportní proteiny MeSH

The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell.

• MeSH
• buněčná membrána metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• exocytóza genetika   MeSH
• fylogeneze MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• konfokální mikroskopie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteomika metody   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny klasifikace   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• tabák genetika   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein - isoformy MeSH
• rostlinné proteiny MeSH