Specific gene knockdown mediated by the antisense oligodeoxynucleotides (AODNs) strategy emerged as a rapid and effective tool for probing gene role in plant cells, particularly tip-growing pollen ...tubes. Here, we describe the protocol for the successful employment of AODN technique in growing tobacco pollen tubes, covering AODN design, application, and analysis of the results. We also discuss the advantages and drawbacks of this method.
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
, 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.
Non-specific phospholipases C (NPCs) were discovered as a novel type of plant phospholipid-cleaving enzyme homologous to bacterial phosphatidylcholine-specific phospholipases C and responsible for ...lipid conversion during phosphate-limiting conditions. The six-gene family was established in Arabidopsis, and growing evidence suggests the involvement of two articles NPCs in biotic and abiotic stress responses as well as phytohormone actions. In addition, the diacylglycerol produced via NPCs is postulated to participate in membrane remodelling, general lipid metabolism and cross-talk with other phospholipid signalling systems in plants. This review summarises information concerning this new plant protein family and focusses on its sequence analysis, biochemical properties, cellular and tissue distribution and physiological functions. Possible modes of action are also discussed.
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
mutant plants. Our data reveal a dose-dependent effect of At
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
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.
The pathogenesis‐related 1 (PR1) proteins are members of the cross‐kingdom conserved CAP superfamily (from Cysteine‐rich secretory protein, Antigen 5, and PR1 proteins). PR1 mRNA expression is ...frequently used for biotic stress monitoring in plants; however, the molecular mechanisms of its cellular processing, localization, and function are still unknown. To analyse the localization and immunity features of Arabidopsis thaliana PR1, we employed transient expression in Nicotiana benthamiana of the tagged full‐length PR1 construct, and also disrupted variants with C‐terminal truncations or mutations. We found that en route from the endoplasmic reticulum, the PR1 protein transits via the multivesicular body and undergoes partial proteolytic processing, dependent on an intact C‐terminal motif. Importantly, only nonmutated or processing‐mimicking variants of PR1 are secreted to the apoplast. The C‐terminal proteolytic cleavage releases a protein fragment that acts as a modulator of plant defence responses, including localized cell death control. However, other parts of PR1 also have immunity potential unrelated to cell death. The described modes of the PR1 contribution to immunity were found to be tissue‐localized and host plant ontogenesis dependent.
Transiently expressed AtPR1 progresses from the endoplasmic reticulum via ARA6/7‐positive multivesicular bodies toward the cell wall; its effect on immunity is dependent on its C‐terminus integrity and host plant developmental stage.
Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ...ancient TSET-TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis.
Reactive oxygen species (ROS) generated by NADPH oxidase (NOX) are crucial for tip growth of pollen tubes. However, the regulation of NOX activity in pollen tubes remains unknown. Using purified ...plasma membrane fractions from tobacco and olive pollen and tobacco BY-2 cells, we demonstrate that pollen NOX is activated by calcium ions and low abundant signaling phospholipids, such as phosphatidic acid and phosphatidylinositol 4,5-bisphosphate in vitro and in vivo. Our data also suggest possible synergism between Ca2+ and phospholipid-mediated NOX activation in pollen. Rac/Rop small GTPases are also necessary for normal pollen tube growth and have been proposed to regulate ROS production in root hairs. We show here elevated ROS formation in pollen tubes overexpressing wild-type NtRac5 and constitutively active NtRac5, while overexpression of dominant-negative NtRac5 led to a decrease of ROS in pollen tubes. We also show that PA formed by distinct phospholipases D (PLD) is involved in pathways both upstream and downstream of NOX-mediated ROS generation and identify NtPLDδ as a PLD isoform acting in the ROS response pathway.
The first indication of the aluminum (Al) toxicity in plants growing in acidic soils is the cessation of root growth, but the detailed mechanism of Al effect is unknown. Here we examined the impact ...of Al stress on the activity of non-specific phospholipase C (NPC) in the connection with the processes related to the plasma membrane using fluorescently labeled phosphatidylcholine. We observed a rapid and significant decrease of labeled diacylglycerol (DAG), product of NPC activity, in Arabidopsis seedlings treated with AlCl
3
. Interestingly, an application of the membrane fluidizer, benzyl alcohol, restored the level of DAG during Al treatment. Our observations suggest that the activity of NPC is affected by Al-induced changes in plasma membrane physical properties.
Formins are evolutionarily conserved eukaryotic proteins engaged in actin nucleation and other aspects of cytoskeletal organization. Angiosperms have two formin clades with multiple paralogs; typical ...plant Class I formins are integral membrane proteins that can anchor cytoskeletal structures to membranes. For the main Arabidopsis housekeeping Class I formin, FH1 (At3g25500), plasmalemma localization was documented in heterologous expression and overexpression studies. We previously showed that loss of FH1 function increases cotyledon epidermal pavement cell shape complexity via modification of actin and microtubule organization and dynamics. Here, we employ transgenic Arabidopsis expressing green fluorescent protein-tagged FH1 (FH1-GFP) from its native promoter to investigate in vivo behavior of this formin using advanced microscopy techniques. The fusion protein is functional, since its expression complements the fh1 loss-of-function mutant phenotype. Accidental overexpression of FH1-GFP results in a decrease in trichome branch number, while fh1 mutation has the opposite effect, indicating a general role of this formin in controlling cell shape complexity. Consistent with previous reports, FH1-GFP associates with membranes. However, the protein exhibits surprising actin- and secretory pathway-dependent dynamic localization and relocates between cellular endomembranes and the plasmalemma during cell division and differentiation in root tissues, with transient tonoplast localization at the transition/elongation zones border. FH1-GFP also accumulates in actin-rich regions of cortical cytoplasm and associates with plasmodesmata in both the cotyledon epidermis and root tissues. Together with previous reports from metazoan systems, this suggests that formins might have a shared (ancestral or convergent) role at cell-cell junctions.
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