Biology subjects

Bozhkov, P. V.

Publications and source records attributed to Bozhkov, P. V..

7 recordsLinked to original sources

bioRxiv2026

Organ identity shapes autophagy dynamics and selectivity in plants

Plants, with their unique evolutionary trajectory and complex physiological adaptations, have developed organ-specific mechanisms to cope with various environmental stresses. Autophagy, an essential catabolic process, plays an important role in maintaining plant growth, immunity, and overall fitness. In this study, we reveal the spatio-temporal dynamics of autophagic responses in Arabidopsis thaliana roots and shoots under different stress conditions, including AZD8055 treatment and carbon and nitrogen depletion. Our findings demonstrate that roots exhibit stronger autophagic activity than shoots under all three conditions, highlighting the unique adaptations of these two organs. Furthermore, we dissect the selectivity of autophagy in targeting organelles, revealing immediate, delayed, and no uptake categories. Additionally, we observe organelles coexisting within autophagic bodies, shedding light on the complexity of cargo selection. This study enhances our understanding of plant specific autophagy dynamics, emphasizing its role in sustaining the source-sink functions and offering insights into plant adaptation to diverse stressors.

plant biology↗

bioRxiv2023

Seed Longevity is Controlled by Metacaspases

To survive extreme desiccation, seeds enter dormancy that can last millennia. This dormancy involves the accumulation of protective but structurally disordered storage proteins through unknown adjustments of proteolytic surveillance mechanisms. Mutation of all six types II metacaspases (MCAs)-II in the model plant Arabidopsis revealed their essential role in modulating these proteolytic mechanisms. MCA-II mutant seeds fail to properly target at the endoplasmic reticulum (ER) the AAA ATPase Cell Division Cycle 48 (CDC48) to dispose of misfolded proteins. MCA-IIs cleave a CDC48 adaptor, the ubiquitination regulatory X domain-containing (PUX) responsible for localizing CDC48 to the lipid droplets. When cleaved, CDC48-PUX is inactivated and allows a lipid droplet-to-ER shuttling of CDC48, an important step in the regulation of seeds lifespan. In sum, we uncover antagonism between proteolytic pathways bestowing longevity. One-Sentence SummaryMetacaspase proteases confer seed longevity by antagonizing CDC48 activity.

plant biology↗

bioRxiv2023

Structure-function analysis of a calcium-independent metacaspase reveals a novel proteolytic pathway for lateral root emergence

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. Despite the extensive study of metacaspases in the two decades since their discovery, the structure-function relationship of metacaspases remains poorly understood. Furthermore, previous studies on their function have been thwarted by the redundancy in gene copy number and potential phenotypic suppression of genetic mutations, especially in plants. Here, we have solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) that belongs to a particular sub-group that does not require calcium ions for activation. Compared to crystal structures of other metacaspases and caspases, the AtMCA-IIf active site is structurally similar and poses a conundrum for the catalytic mechanism of the cysteine-histidine dyad. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors. Several hits with a minimal thioxodihydropyrimidine-dione (TDP) structure were identified, some being specific inhibitors of AtMCA-IIf. We provide a mechanistic basis for inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) was effective at inhibiting lateral root emergence in vivo, likely through the inhibition of metacaspases that are specifically expressed in the endodermal cells overlaying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in various other species, such as important human pathogens including those causing neglected diseases.

plant biology↗

bioRxiv2021

SPIRO-the automated Petri plate imaging platform designed by biologists, for biologists

Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time-consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photos of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIROs excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open-source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared to manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof-of-concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing and photoblastic response.

plant biology↗

bioRxiv2020

Apoptosis in plants: from semantic appeal to empirical rejection

BackgroundAnimals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell-death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant "apoptosis-like programmed cell death" (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis; in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies. ResultsHere, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40-55{degrees}C and 85{degrees}C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85{degrees}C, the only striking morphological difference between cell deaths induced by 40-55{degrees}C or 85{degrees}C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents. ConclusionsWe conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways.

plant biology↗

bioRxiv2020

Tudor staphylococcal nuclease acts as a docking platform for stress granule components in Arabidopsis thaliana

Adaptation to stress depends on the modulation of gene expression. Regulation of mRNA stability and degradation in stress granules (SGs), - cytoplasmic membraneless organelles composed of messenger ribonucleoprotein (mRNP) complexes, - plays an important role in fine-tuning of gene expression. In addition, SG formation can modulate stress signaling pathways by protein sequestration. Molecular composition, structure, and function of SGs in plants remain obscure. Recently, we established Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) as integral component of SGs in Arabidopsis thaliana. Here, we combined purification of TSN interactome with cell biology, reverse genetics and bioinformatics to study composition and function of SGs in plants. We found that under both normal (in the absence of stress) and stress conditions TSN interactome is enriched in the homologues of known mammalian and yeast SG proteins, in addition to novel or plant-specific SG components. We estimate that upon stress perception, approximately half of TSN interactors are recruited to SGs de novo, in a stress-dependent manner, while another half represent a dense protein-protein interaction network pre-formed before onset of stress. Almost all TSN-interacting proteins are moderately or highly disordered and approximately 20% of them are predisposed for liquid-liquid phase separation (LLPS). This suggests that plant SGs, similarly to mammalian and yeast counterparts, are multicomponent viscous liquid droplets. Finally, we have discovered that evolutionary conserved SNF1-related protein kinase 1 (SnRK1) interacts with TSN in heat-induced SGs and that SnRK1 activation critically depends on the presence of TSN and formation of SGs. Altogether, our results establish TSN as a docking platform for SG-associated proteins and important stress signal mediator in plants.

plant biology↗

bioRxiv2019

Chemical screening pipeline for identification of specific plant autophagy modulators

Autophagy is a major catabolic process in eukaryotes with a key role in homeostasis, programmed cell death and aging. In plants, autophagy is also known to regulate agriculturally important traits such as stress resistance, longevity, vegetative biomass and seed yield. Despite its significance, there is still a shortage of reliable tools modulating plant autophagy. Here we describe the first robust pipeline for identification of specific plant autophagy-modulating compounds. Our screening protocol comprises four phases: (i) high-throughput screening of chemical compounds in cell cultures of Nicotiana tabacum; (ii) confirmation of the identified hits in planta using Arabidopsis thaliana; (iii) further characterization of the effect using conventional molecular biology methods; (iv) verification of chemical specificity on autophagy in planta. The methods detailed here streamline identification of specific plant autophagy modulators and aid in unravelling the molecular mechanisms of plant autophagy.

plant biology↗