Biology subjects

Pejchar, P.

Publications and source records attributed to Pejchar, P..

11 recordsLinked to original sources

bioRxiv2026

A plasmodesmata-specific exocyst complex regulates symplastic connectivity by affecting callose turnover

Plasmodesmata are intercellular channels that mediate symplastic communication between plant cells. Molecular transport through these channels is critically regulated by dynamic callose deposition and degradation, yet the secretory mechanisms that deliver regulatory components to plasmodesmata remain poorly understood. Here, we identify and characterize a non-canonical plasmodesmata-associated module of the exocyst, an evolutionarily conserved protein complex involved in secretory vesicle tethering and exocytosis. Exocyst subunits EXO70G1, SEC15A, EXO84C, and SEC10A specifically accumulate at plasmodesmata, whereas the canonical exocyst subunits EXO70A1 and SEC8 do not. Genetic and interaction analyses show that EXO70G1 acts as a landmark for recruiting SEC15A and EXO84C to plasmodesmata, revealing a distinct mode of exocyst targeting at these membrane domains. EXO70G1-dependent exocyst targeting to plasmodesmata depends on phosphoinositides and sphingolipids, consistent with the specialized lipid environment of plasmodesmal membranes. Loss of EXO70G1 results in increased callose accumulation and reduced symplastic transport, and strongly enhances developmental defects of a callose-overproducing mutant. In addition, exo70G1 mutants display enhanced resistance to bacterial pathogen Pseudomonas syringae, linking reduced plasmodesmal permeability to anti-bacterial defense. Cross-species analysis further indicates that plasmodesmata association is a derived feature of the EXO70G clade, present in angiosperms but absent from non-angiosperm EXO70 homologs. Together, our findings show that exocyst diversification in plants has generated a specialized trafficking module - plasmodesmata-associated exocyst - that links vesicle delivery to callose homeostasis at plasmodesmata, thereby regulating intercellular communication, development, and immunity. TeaserA specialized secretion module of the exocyst complex regulates plant cell-to-cell connectivity by controlling callose turnover at plasmodesmata

plant biology↗

bioRxiv2026

Loss of PR1 function enhances Arabidopsis resistance to Botrytis cinerea

PATHOGENESIS-RELATED 1 (PR1) is one of the most widely used markers of salicylic acid (SA)-dependent plant immunity, yet its direct functional contribution to pathogen defence remains poorly understood. Here, we investigated the role of PR1 in Arabidopsis thaliana by analyzing a pr1 loss-of-function mutant challenged with bacterial and fungal pathogens and fumonisin B1 (FB1)-induced cell death. Notably, loss of PR1 led to markedly different responses to distinct pathogens; while it moderately increased susceptibility to the pathogenic bacterium Pseudomonas syringae, it substantially enhanced resistance to the necrotrophic fungus Botrytis cinerea, and the responses to the necrotroph Sclerotinia sclerotiorum remained unaltered. The pr1 mutant also displayed reduced spread of FB1-induced cell death, linking PR1 function to the promotion of stress-associated cell death. In line with the susceptibility changes, we observed the strongest PR1 accumulation and cell wall enrichment during B. cinerea infection using mCherry-tagged PR1 expressed under its endogenous promoter. Complementation with full-length PR1 and with a C-terminally truncated PR1 variant lacking the CAPE peptide restored wild-type susceptibility, whereas a non-cleavable PR1 variant did not. These results indicate that proteolytic processing at the CAPE cleavage motif, rather than the CAPE peptide itself, is required for PR1 function. Our data thus strongly suggest that PR1 may act as a susceptibility factor for necrotrophic pathogens by promoting host cell death.

Plant Biology↗

bioRxiv2026

Unstructured regions differentially modulate the activation of RBOHD and RBOHH

Reactive oxygen species (ROS) produced by plant NADPH oxidases (RBOHs) must be precisely controlled in their concentration and spatial distribution to support diverse developmental and stress responses. RBOHs are activated by Ca2+ binding and phosphorylation, yet how internal regulatory domains within RBOHs have evolved to translate these inputs into precise levels of ROS production remains unclear. To address this, we performed phylogenetic analyses to define RBOH subfamilies and identify protein regions underlying functional diversification. This analysis revealed that the most variable regions across land-plant RBOHs are two unstructured regions in the N-terminus, UR1 and UR2, which flank the EF-hand Ca2+-binding domain (EFD). We dissected the roles of these regions in Arabidopsis RBOHD, which is central to plant immunity, and in RBOHH, which drives pollen tube elongation and exhibits high Ca2+-induced ROS production. Our analyses revealed that UR1 plays opposing roles in these RBOHs: in RBOHD, UR1 functions as an autoinhibitory module that restrains Ca2+-mediated activation, whereas in RBOHH, UR1 is essential for Ca2+-dependent activation and has coevolved with the EFD to maximize Ca2+-induced ROS production. We further uncovered divergent regulatory roles for UR2. In RBOHD, but not in RBOHH, phosphorylation of UR2 stabilizes an -helical conformation that promotes interaction with the catalytic domain required for enzymatic activation. Furthermore, unlike in RBOHH, the EFD of RBOHD has coevolved with UR2 to maximize phosphorylation-induced activity. Together, our results show how evolution of unstructured regulatory regions adapts a conserved enzymatic core to distinct demands of immune signaling and polarized growth.

plant biology↗

bioRxiv2026

Evolutionary analysis of the exocyst complex in streptophytes links early EXO70 diversification with dominance over SEC3 in membrane targeting

The exocyst is a conserved octameric vesicle-tethering complex essential for targeted secretion. It is organized into two modules (I: SEC3, SEC5, SEC6, SEC8; II: SEC10, SEC15, EXO70, EXO84). In plants, the module II subunits SEC15, EXO84, and especially EXO70 have diversified into multiple subfamilies, yet the evolutionary origins and functional consequences of this diversification remain unclear. Here we reconstruct exocyst evolution across streptophytes using phylogenomic, functional complementation, and structural modeling analyses. We show that the three major EXO70 subfamilies originated in anydrophytes--the common ancestor of Zygnematophyceae and land plants--indicating that EXO70 diversification had begun at the dawn of plant terrestrialization. Complementation of Arabidopsis exo70 mutants with EXO70 paralogs from the liverwort Marchantia polymorpha and the streptophyte alga Klebsormidium nitens demonstrates that the ancestral canonical function is retained in the EXO70.1 lineage, whereas other subfamilies have undergone substantial functional specialization. We further uncover an evolutionary shift in exocyst membrane targeting: while Klebsormidium SEC3 retains autonomous membrane-recruitment capacity, land-plant SEC3 subunits have lost this ability, rendering exocyst targeting increasingly dependent on EXO70. Together, these findings suggest that early EXO70 diversification, combined with the redistribution of membrane-targeting functions within the exocyst, enabled paralog-specific exocyst recruitment and facilitated the emergence of specialized secretion pathways during plant terrestrialization. Significance StatementThe exocyst is a conserved protein complex that targets secretory vesicles to the plasma membrane across eukaryotes. In land plants, the EXO70 subunit diversified extensively, but the origins and consequences of this diversification remained unclear. We show that three major EXO70 subfamilies arose in the common ancestor of land plants and their closest algal relatives, at the dawn of plant terrestrialization. Cross-species complementation reveals that the EXO70.1 lineage retains the ancestral canonical function, whereas other subfamilies specialized. We further uncover an evolutionary shift in the mechanism of exocyst membrane targeting, from SEC3 toward EXO70, that could have enabled distinct EXO70 paralogs to direct secretion to different cellular sites during the colonization of land.

plant biology↗

bioRxiv2025

Independent roles of Arp2/3 complex and RIC4 protein in the control of epidermal cell shape

The aim of this study was to determine whether RIC4, a CRIB domain-containing protein that functions as an effector of ROP GTPases, and the Arp2/3 complex, an actin nucleator, functionally cooperate in controlling the shape of Arabidopsis cotyledon epidermal cells. The combination of KO mutants demonstrated that loss of RIC4 and loss of the Arp2/3 complex result in completely opposite epidermal cell shape phenotypes. The double KO mutation is phenotypically similar to the Arp2/3 mutation, and the effect of RIC4 loss is completely eliminated. Analysis of overexpression revealed that excess RIC4 significantly suppresses the formation of pavement cell lobes. However, RIC4 does not require an active Arp2/3 complex for this effect. Our data further show that overexpression of RIC4 has a specific actin stabilization effect in cotyledon epidermal cells. However, we could not demonstrate that actin stabilization is directly related to the cell shape changes that RIC4 overexpression induces. In conclusion, RIC4 and the Arp2/3 complex do not share the same signaling pathway in their function in the control of cotyledon epidermal cell shape.

plant biology↗

bioRxiv2025

Non-classical Nuclear Signal PY-NLS Mediates Karyopherinβ2-Dependent Nuclear Import and Condensate Formation in Plants

Nuclear localization signals (NLSs) direct proteins to the nucleus, but whether the non-classical proline-tyrosine NLS (PY-NLS), characterized mainly in animals and fungi, functions in plants is unknown. Here we identify a basic PY-NLS pathway in tobacco and Arabidopsis and show that TRANSPORTIN 1 (TRN1) is its receptor. TRN1 binds PY-NLS cargoes in vitro, whereas loss of TRN1 causes their cytoplasmic accumulation. Mutational, structural and quantitative localization analyses of motifs from the jasmonate signaling repressor JAZ1 and a PLA2-like protein reveal an extended linker between binding epitopes and a short -helix preceding the conserved PY dipeptide as features that distinguish plant PY-NLSs from established eukaryotic models. A validated prediction pipeline identifies 179 Arabidopsis proteins containing candidate PY-NLSs. Together, our findings establish TRN1-mediated PY-NLS recognition as a previously unrecognized plant nuclear import pathway and uncover a plant-specific signal architecture with broad implications for the nuclear control of gene expression, RNA metabolism and development.

plant biology↗

bioRxiv2022

AtEH/Pan1 proteins drive phase separation of the TPLATE complex and clathrin polymerisation during plant endocytosis

Clathrin-mediated endocytosis (CME) is an essential cellular internalisation pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. In plants, the evolutionarily ancient TSET/TPLATE complex (TPC) plays an essential, but not well-defined role in CME. 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, early-, and late-stage endocytic accessory proteins. Importantly, clathrin forms ordered assemblies within the condensate environment. Biomolecular condensation therefore acts to promote dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, the disordered region sequence properties of AtEH1 regulate the material properties of the endocytic condensates in vivo and alteration of these material properties influences endocytosis dynamics, and consequently plant adaptive growth. HighlightsO_LIAtEH subunits are endocytic scaffolds which drive condensation of the TPC C_LIO_LIAtEH1 condensates nucleate on the plasma membrane via lipid interactions C_LIO_LICondensation of AtEH1/TPC facilitates clathrin re-arrangement and assembly C_LIO_LIAtEH1 IDR1 composition controls condensate properties to regulate endocytosis C_LI

cell biology↗

bioRxiv2019

EXO70A2 is critical for the exocyst complex function in Arabidopsis pollen

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 fertilize ovules. Pollen grains undergo two major developmental switches: dehydration characterized by metabolic quiescent state, and rehydration upon pollination that leads to extraordinary metabolic and membrane trafficking activity, resulting in germination and rapid tip growth of pollen tubes. To sustain these processes, many plant housekeeping genes evolved their pollen-specific paralogs. 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 describe that EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in Arabidopsis pollen, which governs the conventional secretory function of the exocyst, analogically 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 similarly to other exocyst subunits to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for the EXO70A1 function in the sporophyte, indicating functional redundancy of these two closely related isoforms. Phylogenetic analysis revealed that the ancient duplication of EXO70A to two (or more) paralogs, one of which is highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in the Arabidopsis pollen required for efficient plant sexual reproduction.

plant biology↗

bioRxiv2019

Functional analysis of phospholipase Dδ family in tobacco pollen tubes

Phosphatidic acid (PA), important signalling and metabolic phospholipid, is predominantly localized in the subapical plasma membrane (PM) of growing pollen tubes. PA can be produced from structural phospholipids by phospholipase D (PLD) but the isoforms responsible for production of plasma membrane PA were not identified yet and their functional roles remain unknown. Following genome-wide bioinformatic analysis of PLD family in tobacco, we focused on the pollen-overrepresented PLD{delta} class. Combining live-cell imaging, gene overexpression or knock-down, lipid-binding and structural bioinformatics, we characterized 5 NtPLD{delta} isoforms. Distinct PLD{delta} isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane-bound PLD{delta}s are tightly bound to PM, primarily via the central catalytic domain. Knock-down, overexpression and in vivo PA level analyses revealed isofom PLD{delta}3 as the most important member of the PLD{delta} subfamily active in pollen tubes. PA promotes binding of PLD{delta}3 to the PM, thus creating a positive feedback loop, where PA accumulation leads to the formation of massive PM invaginations. Tightly controlled production of PA generated by PLD{delta}3 at the PM is important for maintaining the balance between various membrane trafficking processes, that are crucial for plant cell tip growth.

plant biology↗

bioRxiv2018

Arabidopsis class I formin FH1 relocates between membrane compartments during root cell ontogeny and associates with plasmodesmata

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 behaviour 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 an ancestral role at cell-cell junctions.

plant biology↗

bioRxiv2018

A combinatorial lipid code shapes the electrostatic landscape of plant endomembranes

Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In all eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidic acidic (PA), phosphatidylserine (PS) and phosphatidylinositol-4-phosphate (PI4P) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of cellular organelles not only defines the plant electrostatic territory but also distinguishes different compartments within this territory by specifying their varying surface charges.

plant biology↗