Biology subjects

Kulich, I.

Publications and source records attributed to Kulich, I..

15 recordsLinked to original sources

bioRxiv2026

Hormone-dependent receptor docking controls calcium channel activity in plants

The phytohormone auxin is a central coordinator of plant growth and development. Besides its canonical effect on gene transcription1,2, auxin triggers an ultra-rapid calcium ion influx that initiates the root gravitropic response3. The nature of the so-called rapid auxin pathway connecting the AFB1 auxin receptor3,4 and plasma membrane calcium channels remained unknown. Here, we show that auxin induces the direct interaction of the AFB1 receptor with the CNGC14 calcium channel. As the AFB1 receptor is independent of the ubiquitin ligase complex5, the auxin-induced interaction translates into relocalization of the receptor to the plasma membrane. We identify the interaction interface and provide evidence that the docking of the receptor to the channel complex activates Ca2+ influx and triggers growth inhibition. These findings position a calcium channel as an unprecedented component of the AFB1 auxin receptor complex. The ligand-dependent localization shift of a TIR1/AFB family receptor represents a novel paradigm in signal transduction and opens the possibility of unforeseen branches of auxin signaling pathways.

plant biology↗

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↗

bioRxiv2025

Coordinated action of CRK2 and QSK1 regulate osmotic stress response in Arabidopsis

Precise control of intercellular communication is essential for normal growth and stress responses in all multicellular organisms. In Arabidopsis, two membrane-localized receptor like kinases (RLKs), the Cysteine-rich RLK CRK2 and the Leucine-rich repeat (LRR) RLK QSK1 relocalize from the general plasma membrane (PM) to plasmodesmata (PD) in response to osmotic stress. Both these RLKs regulate callose deposition thereby modulating PD permeability. However, unchecked callose deposition can block the PD and disrupt proper intercellular communication. Here, we show that under normal growth conditions, CRK2 phosphorylates and sequesters QSK1 at the general PM, preventing unnecessary callose deposition at PD. We show that osmotic stress-induced enrichment of QSK1 at PD requires functional CRK2 and establish that phosphorylation of QSK1 in its C-terminal region is inhibitory in this process. We propose that osmotic stress triggers dephosphorylation and release of QSK1 from the CRK2-QSK1 complex, enabling its relocalization from general PM to PD, where it promotes stress-induced callose deposition. Subsequently, CRK2 relocalizes to PD where it negatively influences callose deposition. Our work reveals a tightly coordinated distribution of QSK1 and CRK2 at PM, establishing a dynamic gating mechanism that balances growth and stress responsiveness.

plant biology↗

bioRxiv2025

Cytosolic Ca2+ as a universal signal for rapid root growth regulation

Roots must continuously adapt their growth and have evolved the ability to rapidly respond to diverse environmental and hormonal cues, including the phytohormone auxin. Auxin-induced cellular responses involve membrane depolarization, cytosolic calcium (Ca{superscript 2}) elevation, and extracellular pH increase, but how these processes lead to rapid growth regulation remains unclear. Here, we show that cytosolic Ca{superscript 2} acts as a universal signal integrating diverse cues for rapid regulation of root growth. Using live imaging, microfluidics, and optogenetics in Arabidopsis, we demonstrate that a swift rise in cytosolic Ca{superscript 2} is the primary target of auxin signaling affecting growth inhibition. Disruption of Ca{superscript 2} influx abolishes these responses, whereas light-gated Ca{superscript 2} influx from the apoplast or endoplasmic reticulum stores inhibits growth. Multiple unrelated stimuli--including auxin, extracellular ATP, RALF peptides, and hydrogen peroxide--converge on this Ca{superscript 2}-dependent mechanism. Cytosolic Ca{superscript 2} elevation thus represents a necessary and sufficient step for rapid growth inhibition, revealing a unifying principle of root signaling.

plant biology↗

bioRxiv2025

Pattern-triggered immunity in blue and white seed cultivars of Papaver somniferum

Papaver somniferum (poppy) is a traditional component of Central and Eastern European cuisine and an important oilseed crop in the region. The crucial thread for poppy stable yield is pathogen infection. Thus, we need to understand poppy defence mechanisms in detail. The first robust layer of plant immunity, which plays a crucial role in combat against pathogens, is pattern-triggered immunity (PTI). Here, we provide the first insights into PTI in poppy. We selected four poppy varieties used in the food industry. We investigated poppy response to various peptide elicitors acting as microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs). Flg22 induced the most robust reactive oxygen species (ROS) burst among all tested peptides. Flg22 also triggered putative mitogen-activated protein kinase (MAPK) phosphorylation and seedling growth inhibition in all tested cultivars. We identified PsWRKY22 and PsPR2 as candidate marker genes suitable for monitoring poppy PTI responses. The tested poppy cultivars have low levels of salicylic acid. Callose accumulation was triggered by wounding but not by flg22. For studying PTI in poppy, wounding is a challenge that needs to be considered as it can obscure potential PTI responses. Our findings highlight conserved aspects of poppy immunity and challenges in studying poppy PTI. The established pipeline facilitates improving our understanding of poppy immunity and has the potential for widespread application in poppy breeding and improving selection for broad-spectrum disease resistance provided by enhanced PTI. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/639761v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1680d52org.highwire.dtl.DTLVardef@42895eorg.highwire.dtl.DTLVardef@488ffeorg.highwire.dtl.DTLVardef@1e5a95d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract The establishment of the methods for studying pattern-triggered immunity (PTI) in Papaver somniferum L. (poppy) was inspired by the knowledge of the model plant Arabidopsis thaliana. The study showed a similarity between Arabidopsis and poppy in response to flg22 but also pointed out the obstacles for PTI analysis in poppy and the differences compared to the model plant. Created with BioRender.com. C_FIG

plant biology↗

bioRxiv2025

Calcium-triggered apoplastic ROS bursts balance gravity and mechanical signals to navigate soil

Reactive oxygen species (ROS) have been implicated repeatedly in multiple signaling processes in plants but the underlying mechanisms and roles remain enigmatic. Here, we developed live imaging of apoplastic ROS at the root surface. Different signals, including auxin, extracellular ATP and RALF1 peptide, all induce cytosolic calcium transients and apoplastic ROS bursts. Genetic and optogenetic manipulations identified calcium transients as necessary and sufficient for ROS bursts via activation of NADPH oxidases RBOHC and RBOHF. Apoplastic ROS bursts are not required but rather limit the gravity-induced root bending. Root bending is sensed by stretch-activated calcium channel MCA1 leading to NADPH oxidase activation at the stretched side. The resulting ROS production stiffens cell wall for better soil penetration. Apoplastic ROS thus provides a means to balance tissue flexibility and stiffness to efficiently navigate soil.

plant biology↗

bioRxiv2025

Armadillo Repeat Only Proteins Are Crucial for the Function of Plant CNGC Channels

The versatile Ca2+ signaling system governs plant responses to a wide array of environmental and developmental cues. CYCLIC NUCLEOTIDE-GATED CHANNELS (CNGCs) trigger cellular responses to diverse signals, including phytohormones, biotic and abiotic stresses; and as such their activity is tightly controlled. Unlike their animal paralogs, plant CNGCs does not seem to be gated by cyclic nucleotides, and the mechanism of their activation remains unresolved. Here we report ARMADILLO REPEAT ONLY (ARO) proteins as novel, plant-specific, and essential activators of plant CNGCs. Reciprocal proximity labeling revealed interactions between all sporophytic CNGCs and AROs. Loss-of-function aro mutants fail to induce Ca2+ transients in response to known CNGC-triggering stimuli. Structural modeling, mutational analysis, and electrophysiological data show that AROs assemble into a complex with CNGC tetramers by interaction with the conserved Calmodulin-binding IQ domain. AROs represent CNGC activators, competing with Calmodulins, showcasing an evolutionarily unique solution to regulation of calcium signaling in plants.

plant biology↗

bioRxiv2024

A genome-wide association screen for genes affecting leaf trichome development and epidermal metal accumulation in Arabidopsis

To identify novel genes engaged in plant epidermal development, we characterized the phenotypic variability of rosette leaf epidermis of 310 sequenced Arabidopsis thaliana accessions, focusing on trichome shape and distribution, compositional characteristics of the trichome cell wall, and histologically detectable metal ion distribution. Some of these traits correlated with climate parameters of ourq accessions locations of origin, suggesting environmental selection. A novel metal deposition pattern in stomatal guard cells was observed in some accessions. Subsequent GWAS analysis identified 1546 loci with protein sequence-altering SNPs associated with one or more traits, including 5 genes with previously reported relevant mutant phenotypes and 80 additional genes with known or predicted roles in relevant developmental and cellular processes. Some candidates, including GFS9/TT9, exhibited environmentally correlated allele distribution. Several large gene families, namely DUF674, DUF784, DUF1262, DUF1985, DUF3741, cytochrome P450, receptor-like kinases, Cys/His-rich C1 domain proteins and formins were overrepresented among the candidates for various traits, suggesting epidermal development-related functions. A possible participation of formins in guard cell metal deposition was supported by observations in available loss of function mutants. Screening of candidate gene lists against the STRING interactome database uncovered several predominantly nuclear protein interaction networks with possible novel roles in epidermal development.

plant biology↗

bioRxiv2023

Guanylate cyclase activity of TIR1/AFB auxin receptorsin rapid auxin responses

The plant hormone auxin is perceived by TIR1/AFB receptors, which mediate transcriptional reprogramming via a ubiquitination- and cAMP-dependent mechanism. Same receptors mediate also rapid, non-transcriptional growth responses that are mechanistically less well understood. Here, we demonstrate that TIR1/AFB receptors possess not only adenylate cyclase but also guanylate cyclase activity. Auxin perception independently activates cAMP and cGMP production by TIR1/AFBs in vitro and elevates both cAMP and cGMP levels in planta. Genetic and synthetic biology approaches revealed that AFB1 guanylate cyclase activity is required for the rapid cytosolic Ca2+ transients and root growth inhibition. This unprecedented combination of adenylate and guanylate cyclase activities within a single hormone receptor establishes a new paradigm for how one perception mechanism can mediate distinct downstream responses. One-Sentence SummaryTIR1/AFB auxin receptors possess both, adenylate and guanylate cyclase activities, mediating transcriptional reprogramming and rapid Ca2+-dependent growth responses, respectively.

plant biology↗

bioRxiv2023

Rapid redirection of auxin fluxes during root gravitropism by translocation of NGR proteins driving polarization of PIN-activating kinases

Root gravitropic bending represents a fundamental aspect of terrestrial plant physiology. Gravity is perceived by sedimentation of starch-rich plastids (statoliths) to the bottom of the central root cap cells. Following gravity perception, intercellular auxin transport is redirected downwards leading to an asymmetric auxin accumulation at the lower root side causing inhibition of cell expansion, ultimately resulting in downwards bending. How gravity-induced statoliths repositioning is translated into asymmetric auxin distribution remains unclear despite PIN auxin efflux carriers and the Negative Gravitropic Response of roots (NGR) proteins polarize along statolith sedimentation, thus providing a plausible mechanism for auxin flow redirection. In this study, using a functional NGR1-GFP construct, we visualized the NGR1 localization on the statolith surface and plasma membrane (PM) domains in close proximity to the statoliths, correlating with their movements. We determined that NGR1 binding to these PM domains is indispensable for NGR1 functionality and relies on cysteine acylation and adjacent polybasic regions as well as on lipid and sterol PM composition. Detailed timing of the early events following graviperception suggested that both NGR1 repolarization and initial auxin asymmetry precede the visible PIN3 polarization. This discrepancy motivated us to unveil a rapid, NGR-dependent translocation of PIN-activating AGCVIII kinase D6PK towards lower PMs of gravity-perceiving cells, thus providing an attractive model for rapid redirection of auxin fluxes following gravistimulation.

plant biology↗

bioRxiv2022

Interplay of EXO70 and MLO proteins modulates trichome cell wall composition and powdery mildew susceptibility

EXO70 proteins are essential constituents of the octameric exocyst complex implicated in vesicle tethering during exocytosis, while MLO proteins are plant-specific calcium channels of which some isoforms play a key role during fungal powdery mildew pathogenesis. We here detected by a variety of histochemical staining procedures an unexpected phenotypic overlap of A. 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 of isolated trichomes corroborated deficiencies in the composition of trichome cell walls in exo70H4 and mlo2 mlo6 mlo12 mutants. Transgenic lines expressing fluorophore- tagged EXO70H4 and MLO variants exhibited extensive co-localization of these proteins at the trichome plasma membrane and cell wall. Furthermore, mCherry- EXO70H4 mislocalized in trichomes of the mlo triple mutant and, vice versa, MLO6- GFP exhibited aberrant subcellular localization in trichomes of the exo70H4 mutant. Transgenic expression of GFP-marked PMR4 callose synthase, a previously identified cargo of EXO70H4 dependent exocytosis, revealed reduced cell wall delivery of GFP- PMR4 in mlo triple mutant plants. In vivo protein-protein interaction assays uncovered isoform-preferential physical interaction between EXO70 and MLO proteins. Finally, exo70H4 and mlo 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, possibly by (co-)regulating focal secretion of cell wall-related cargo.

plant biology↗

bioRxiv2022

Cell surface auxin signalling directly targets PIN-mediated auxin fluxes for adaptiveplant development

Phytohormone auxin and its directional transport mediate much of the remarkably plastic development of higher plants. Positive feedback between auxin signaling and transport is a key prerequisite for (i) self-organizing processes including vascular tissue formation and (ii) directional growth responses such as gravitropism. Here we identify a mechanism, by which auxin signaling directly targets PIN auxin transporters. Via the cell-surface ABP1-TMK1 receptor module, auxin rapidly induces phosphorylation and thus stabilization of PIN2. Following gravistimulation, initial auxin asymmetry activates autophosphorylation of the TMK1 kinase. This induces TMK1 interaction with and phosphorylation of PIN2, stabilizing PIN2 at the lower root side, thus reinforcing asymmetric auxin flow for root bending. Upstream of TMK1 in this regulation, ABP1 acts redundantly with the root-expressed ABP1-LIKE auxin receptor ABL3. Such positive feedback between cell-surface auxin signaling and PIN-mediated polar auxin transport is fundamental for robust root gravitropism and presumably also for other self-organizing developmental phenomena.

plant biology↗

bioRxiv2021

The EXO70B2 exocyst subunit contributes to papillae and encasement formation in anti-fungal defence in Arabidopsis

In the reaction to non-adapted Blumeria graminis f. sp. hordei (Bg), Arabidopsis thaliana leaf epidermal cells deposit cell wall reinforcements called papillae or seal fungal haustoria in encasements, both of which involve intensive exocytosis. A plant syntaxin SYP121/PEN1 has been found to be of key importance for the timely formation of papillae, and the vesicle tethering complex exocyst subunit EXO70B2 has been found to contribute to their morphology. Here, we identify a specific role for the EXO70B2-containing exocyst complex in the papillae membrane domains important for the callose deposition and GFP-SYP121 delivery to the focal attack sites, as well as its contribution to encasement formation. The mRuby2-EXO70B2 co-localises with the exocyst core subunit SEC6 and GFP-SYP121 in the membrane domain of papillae, and both proteins have the capacity to directly interact. The exo70B2/syp121 double mutant has a reduced number of papillae and haustorial encasements in response to Bg, indicating an additive role of the exocyst in SYP121 coordinated non-host resistance. In summary, we report cooperation between the plant exocyst and a SNARE protein in penetration resistance against non-adapted fungal pathogens. HighlightThe exocyst complex containing EXO70B2 subunit has specific role in papilla and encasement formation, implemented in coordination with the pathway regulated by the SYP121 SNARE complex in defence.

cell biology↗

bioRxiv2020

The Arabidopsis TTL3 protein interconnects brassinosteroid signalling and cytoskeleton during lateral root development

Lateral roots are essential components of the plant edaphic interface, contributing to water and nutrient uptake, biotic and abiotic interactions, stress survival, and plant anchorage. We have identified the TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 3 (TTL3) being related to lateral root emergence and later development. TTL3 interacts with microtubules and potentially interconnects cytoskeletal function with the brassinosteroid signalling pathway. Loss of function of TTL3 leads to a reduced number of emerged lateral roots due to delayed development of lateral root primordia. Lateral root growth of the ttl3 mutant is less sensitive to BR treatment. Timing and spatial distribution of TTL3 expression is consistent with its role in development of lateral root primordia before their emergence and subsequent development into lateral roots. TTL3 is a novel component of the root system morphogenesis regulatory network.

developmental 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↗