Biology subjects

Honys, D.

Publications and source records attributed to Honys, D..

8 recordsLinked to original sources

bioRxiv2025

HSP90-mediated stress resilience in male gametophyte of Arabidopsis thaliana

Despite the accumulation of protective heat shock proteins (HSPs) during the male gametophyte development, pollen grains are highly sensitive to elevated temperatures. We performed transcriptomic analysis of five pollen developmental stages isolated from plants under normal and heat stress (H) conditions; uni-nuclear (UN), early bi-cellular (EB), bi-cellular (BC), tri-cellular (TC), and mature pollen (MPG). We show that the majority of genes that are up- or down-regulated under HS are specific for each stage, except BC stage that also exhibited the highest number of differentially expressed genes (DEGs) (>4000). This emphasize a complex, stage-dependent heat stress response, possibly dependent on HSP levels. Additionally, promoter motif analysis revealed that heat shock elements (HSEs) exhibit a stage- specific pattern of enrichment, which peaked in MP. To explore stage-specific influences of HSP90s in pollen development, we characterized a knockdown RNAi line, under normal and stress conditions in early- and late-stage RNAi lines using stage-specific promoters pJASON (JA90R) and pLAT52 (L90R). The hsp90 background leads to lower germination rate in both RNAi lines that is more pronounced under heat stress caused by significant alterations in heat stress control via impaired ABA signalling or ER stress response. Heat stress conditions also lead to a high percentage of nuclei shape and orientation defects in the L90R line pollen, pointing to the higher sensitivity of late stage development. We show this defect is linked to the down-regulation of DNA metabolism genes. Our complex dataset provides insight into stage-specific stress response on the level of single cell undergoing developmental changes.

plant biology↗

bioRxiv2025

Molecular and Phenotypic Characterization of Telomere Repeat Binding (TRBs) Proteins in Moss: Evolutionary and Functional Perspectives

Telomere repeat binding (TRB) proteins are plant-specific proteins with a unique domain structure distinct from telomere-binding proteins in animals and yeast. While extensively studied in seed plants, their role in early-diverging plant lineages remain largely unexplored. Here, we investigate TRB proteins in a model moss, Physcomitrium patens, to assess their evolutionary conservation and functional significance. Functional analysis using single knockout mutants revealed that individual PpTRB genes are essential for normal development, with mutants exhibiting defects in the two-dimensional (protonemal) stage and, more prominently, in the formation of three-dimensional (gametophore) structures. Some double mutants displayed telomere shortening, a phenotype also observed in TRB-deficient seed plants, indicating a conserved role for TRBs in telomere maintenance. Transcriptome profiling of TRB mutants revealed altered expression of genes associated with transcriptional regulation and stimulus response in protonema. Subcellular localization studies across various plant cell types confirmed that PpTRBs, like their seed plant counterparts, localize prevalently to the plant nucleus and mutually interact. In bryophytes, TRBs form a monophyletic group that mirrors the species phylogeny, whereas in seed plants, TRBs have diversified into two distinct monophyletic groups. Our findings provide the first comprehensive characterization of TRB proteins in non-vascular plants and demonstrate their conserved roles in telomere maintenance, with additional implications for plant development and gene regulation across land plant lineages. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/659030v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1fea36dorg.highwire.dtl.DTLVardef@9b5569org.highwire.dtl.DTLVardef@488d0org.highwire.dtl.DTLVardef@1b22c6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2025

Arabidopsis seed stored mRNAs and translation regulation during post-harvest ripening and imbibition.

Seed germination marks the critical transition from dormancy to active growth, driven by environmental cues and water availability. This study explores translational regulation during germination by comparing two Arabidopsis thaliana accessions with contrasting dormancy phenotypes: Columbia (Col), which germinates readily, and Cape Verde Islands (Cvi), which exhibits deep dormancy. Using sucrose gradient centrifugation, we isolated monosomal and polysomal fractions from freshly harvested (FH), after-ripened (AR), and imbibed (IM) seeds. RNA-seq analysis revealed stage- and genotype-specific gene expression, with Col IM seeds displaying the highest number of expressed genes. We identified [~]14,000 mRNAs in FH seeds, increasing to 19,000 in Col and 17,000 in Cvi upon imbibition. Of these, 9,000 were shared, while 3,000 were accession-specific in monosomes. Enrichment analysis highlighted molecular pathways associated with translation and dormancy release. Analysis of RNA modifications identified N1-methyladenosine (m1A) as the predominant modification, with Col seeds exhibiting higher m1A levels than Cvi, peaking at three months post-harvest. m6A sequencing revealed distinct modification patterns between accessions, with the highest abundance of m6A-modified transcripts in IM seeds. Positional analysis of m6A peaks suggested a link to differential gene expression between Col and Cvi. Proteomic analysis identified [~]15,000 proteins, with translation-related proteins enriched in IM seeds. Notable differences between Col and Cvi were observed in both monosomal and polysomal fractions. RNA-binding proteins exhibited similar profiles in FH and AR stages but diverged significantly in IM seeds. Col-specific proteins were enriched in 40S ribosomes, processing bodies, and RNA-binding complexes. These findings provide new insights into the molecular and translational dynamics underlying seed germination, advancing our understanding of dormancy release and early seedling establishment.

plant biology↗

bioRxiv2024

Implementation of Ribo-BiFC method to plant systems using a split mVenus approach

Translation is a fundamental process for every living organism. In plants, the rate of translation is tightly modulated during development and in response to environmental cues. However, it is difficult to measure the actual translation state of the tissues in vivo. Here, we report the implementation of an in vivo translation marker based on bimolecular fluorescence complementation, the Ribo-BiFC. We combined method originally developed for fruit-fly with an improved low background split-mVenus BiFC system previously described in plants. We labelled Arabidopsis thaliana small subunit ribosomal protein (RPS) and large subunit ribosomal protein (RPL) with fragments of the mVenus fluorescent protein. Upon the assembly of the 80S ribosome, the mVenus fragments complemented and were detected by fluorescent microscopy. We show that these recombinant proteins are in close proximity in the tobacco epidermal cells, although the signal is reduced when compared to BiFC signal from known interactors. This Ribo-BiFC method system can be used in stable transgenic lines to enable visualisation of translational rate in plant tissues and could be used to study translation dynamics and its changes during plant development, under abiotic stress or in different genetic backgrounds.

plant biology↗

bioRxiv2024

GOLEM: A tool for visualizing the distribution of Gene regulatOry eLEMents within the plant promoters with a focus on male gametophyte

BackgroundThe regulation of gene expression during tissue development is very complex. A key mechanism of gene regulation is the recognition of regulatory motifs, also known as cis-regulatory elements (CREs), by various proteins in gene promoter regions. Localization of these motifs near the transcription start site (TSS) or translation start site (ATG) is crucial for transcription initiation and rate. Transcription levels of individual genes, regulated by these motifs, can vary significantly across tissues and developmental stages, especially in processes like sexual reproduction. However, the precise localization and visualization of regulatory motifs in relation to gene expression in specific tissues can be challenging. ResultsHere, we introduce a program called GOLEM (Gene regulatOry eLEMents) which enables users to precisely locate any motif of interest with respect to TSS or ATG within the relevant plant genomes across the plant Tree of Life (Marchantia, Physcomitrium, Amborella, Oryza, Zea, Solanum and Arabidopsis). The visualization of the motifs is performed with respect to the transcript levels of particular genes in leaves and male reproductive tissues and can be compared with genome-wide distribution regardless of the transcription level. Additionally, genes with specific CREs at defined positions and high expression in selected tissues can be exported for further analysis. GOLEMs functionality is illustrated by its application to conserved motifs (e.g. TATA-box, ABRE, I-box, and TC-element), as well as to male gametophyte-related motifs (e.g. LAT52, MEF2, ARR10_core, and DOF_core). ConclusionGOLEM is a freely available tool (https://golem.ncbr.muni.cz) for tracking the precise localization and distribution of any CREs of interest in plant gene promoters. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/606583v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@13c1fc9org.highwire.dtl.DTLVardef@4b034dorg.highwire.dtl.DTLVardef@c5e28aorg.highwire.dtl.DTLVardef@19c0ce4_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

Completing the TRB family: newly characterized members show ancient evolutionary origins and distinct localization, yet similar interactions

Telomere repeat binding proteins (TRBs) belong to a family of proteins possessing a Myb-like domain which binds to telomeric repeats. Three members of this family (TRB1, TRB2, TRB3) from Arabidopsis thaliana have already been described as associated with terminal telomeric repeats (telomeres) or short interstitial telomeric repeats in gene promoters (telo-boxes). They are also known to interact with several protein complexes: telomerase, Polycomb repressive complex 2 (PRC2) E(z) subunits and the PEAT complex (PWOs-EPCRs-ARIDs-TRBs). Here we characterize two novel members of the TRB family (TRB4 and TRB5). Our wide phylogenetic analyses have shown that TRB proteins evolved in the plant kingdom after the transition to a terrestrial habitat in Streptophyta, and consequently TRBs diversified in seed plants. TRB4-5 share common TRB motifs while differing in several others and seem to have an earlier phylogenetic origin than TRB1-3. Their common Myb-like domains bind long arrays of telomeric repeats in vitro, and we have determined the minimal recognition motif of all TRBs as one telo-box. Our data indicate that despite the distinct localization patterns of TRB1-3 and TRB4-5 in situ, all members of TRB family mutually interact and also bind to telomerase/PRC2/PEAT complexes. Additionally, we have detected novel interactions between TRB4-5 and EMF2 and VRN2, which are Su(z)12 subunits of PRC2.

plant biology↗

bioRxiv2020

LARP6C regulates selective mRNA translation to promote pollen tube guidance in Arabidopsis thaliana.

In angiosperms, non-motile sperm cells are delivered to the ovules for fertilization via a guided growth of the pollen tube. RNA binding proteins are key regulators that control mRNA fate post-transcriptionally and thus essential for normal cell function. But very little is known on the mechanistic bases of mRNA regulations and trans-acting factors governing male-female signalling. Here we demonstrate that the evolutionarily conserved RNA binding protein LARP6C is necessary for pollen tube guidance and fertilization. larp6c loss-of-function mutants exhibit male induced fertility defects as mutant pollen tubes frequently are unable to find ovules for fertilization. In mature pollen, LARP6C localises with a pollen specific poly(A) binding protein to cytoplasmic foci likely containing mRNPs. With RNA immunoprecipitation and sequencing, we demonstrate that LARP6C is associated in vivo with mRNAs required for pollen tube guidance or polarized cell growth. We further demonstrate using in vitro and in planta transient assays that LARP6C binds 5-UTR box motifs to orchestrate the balance between translation, decay and storage of its mRNA targets. We propose a model where LARP6C maintains its mRNA target in translationally silent state likely to promote localized translation and guided pollen tube growth upon paracrine signalling.

plant biology↗

bioRxiv2020

Comparative transcriptomic analysis reveals conserved transcriptional programs underpinning organogenesis and reproduction in land plants

The evolution of plant organs, including leaves, stems, roots, and flowers, mediated the explosive radiation of land plants, which shaped the biosphere and allowed the establishment of terrestrial animal life. Furthermore, the fertilization products of angiosperms, seeds serve as the basis for most of our food. The evolution of organs and immobile gametes required the coordinated acquisition of novel gene functions, the co-option of existing genes, and the development of novel regulatory programs. However, our knowledge of these events is limited, as no large-scale analyses of genomic and transcriptomic data have been performed for land plants. To remedy this, we have generated gene expression atlases for various organs and gametes of 10 plant species comprising bryophytes, vascular plants, gymnosperms, and flowering plants. Comparative analysis of the atlases identified hundreds of organ- and gamete-specific gene families and revealed that most of the specific transcriptomes are significantly conserved. Interestingly, the appearance of organ-specific gene families does not coincide with the corresponding organs appearance, suggesting that co-option of existing genes is the main mechanism for evolving new organs. In contrast to female gametes, male gametes showed a high number and conservation of specific genes, suggesting that male reproduction is highly specialized. The expression atlas capturing pollen development revealed numerous transcription factors and kinases essential for pollen biogenesis and function. To provide easy access to the expression atlases and these comparative analyses, we provide an online database, www.evorepro.plant.tools, that allows the exploration of expression profiles, organ-specific genes, phylogenetic trees, co-expression networks, and others.

evolutionary biology↗