Biology subjects

Urbanowicz, B. R.

Publications and source records attributed to Urbanowicz, B. R..

9 recordsLinked to original sources

bioRxiv2026

A Comprehensive Epidermal Map from a Poplar Single-Cell Shoot Atlas Reveals New Trichome-Specific Genes

Poplar (Populus spp.) is a model system for tree biology. Specifically, P. tremula x P. alba INRA 717-1B4 (hereafter "poplar 717") has become an important platform for functional genomics and synthetic biology due to its rapid growth and ease of transgenesis. Here, we present a single-cell RNA-seq atlas of the poplar 717 shoot, including apical meristem, primary and secondary stems, and three stages of leaf development. Analysis of ca. 159,000 cells resolved 40 transcriptionally distinct clusters representing 7 major cell types, providing a high-resolution view of shoot development and tissue organization. We focused on the epidermis which constituted >15% of cells in the shoot atlas for in-depth characterization of epidermal heterogeneity. By integrating known marker genes with transcriptomic signatures consistent with established poplar leaf phytochemistry, we annotated epidermal cell subclusters corresponding to developmental stages, spatial location, and specialized cell types, including a distinct population of non-glandular trichomes. Coupling the single-cell RNA-seq atlas with bulk transcriptome data from glabrous mutants enabled the identification of novel trichome markers. Experimental validation of a representative trichome-specific promoter established a tool with potential to support cell type-targeted-metabolic engineering. We provide the poplar 717 atlas to the community through the BioPoplar Atlas Viewer (http://bio-poplar-atlas.com), providing a platform to explore the poplar transcriptome at single-cell resolution and a foundation for data-driven cell type-aware genetic engineering strategies in poplar.

plant biology↗

bioRxiv2026

Glydentify: An explainable deep learning platform for glycosyltransferase donor substrate prediction

Glycosyltransferases (GTs) are a large family of enzymes that catalyze glycosidic linkages formation between chemically diverse donor and acceptor molecules to regulate diverse cellular processes across all domains of life. Despite their importance, the activated sugar donors (donor substrates) used by most GTs remain unidentified, limiting our understanding of GT functions. To address this challenge, we developed Glydentify, a deep learning framework that predicts donor usage across GT-A and GT-B fold glycosyltransferases. Trained on large-scale UniProt annotations, Glydentify integrates protein sequence embeddings learned from protein language models with chemical features derived from molecular encoders trained on extensive chemical datasets. The resulting models achieve high predictive performance, with precision-recall AUCs (PR-AUC) of 0.86 for GT-A and 0.91 for GT-B, surpassing general enzyme-substrate predictors while requiring minimal manual curation. We employed Glydentify to predict the donor specificity of uncharacterized plant GTs and experimentally tested the predictions using in vitro biochemical assays. Furthermore, we demonstrate that the model utilizes a combination of evolutionary, structural, and biochemical features to predict donor specificity through residue attention score analysis. Together, these results establish Glydentify as a robust, explainable framework for decoding donor-glycosyltransferase relationships and highlight its potential as a broadly applicable framework for modeling enzyme classes that act on chemically diverse substrates.

bioinformatics↗

bioRxiv2025

Loss of flavonol 3-O-glucosyltransferase activity confers soybean resistance to leaf-chewing insects

Caterpillars and beetles are among the most economically damaging defoliating insects, and their economic damage is predicted to increase in the coming decades. Hence the use of genetically derived resistance to supplement other pest control strategies is warranted. In soybean (Glycine max (L.) Merr.), a major determinant for resistance is the quantitative trait locus, QTL-M. Glyma07g14530, the gene underlying QTL-M, encodes a feeding-inducible flavonol 3-O-glycosyltransferase (F3GlcT or UGT78D2) that glucosylates kaempferol, as well as quercetin, myricetin, and isorhamnetin. The resistant allele has a premature stop codon in it, thus preventing the glucosylation and sequestration of flavonols in the vacuole, leading to a concomitant accumulation of proanthocyanidins and manifestation of resistance. Expressing the dominant (susceptible) allele in resistant plants restores susceptibility and silencing the susceptible allele results in resistance. The discovery and characterization of GmF3GlcT helps clarify the role of flavonoids in resistance to leaf-chewing insects and facilitates the development of insect-resistant cultivars that ultimately can lower production costs and reduce insecticide applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/679769v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@10e2e77org.highwire.dtl.DTLVardef@ca905org.highwire.dtl.DTLVardef@12cb46corg.highwire.dtl.DTLVardef@1f9328b_HPS_FORMAT_FIGEXP M_FIG C_FIG The loss of flavonol 3-O-glucosyltransferase in soybean reduces feeding damage from defoliating insects and is accompanied by a concomitant increase in proanthocyanidins (red arrowhead). SignificanceUnraveling the biochemical and genetic basis of soybean resistance to leaf-chewing insects facilitates the development of new, naturally insect resistant varieties. Such varieties contribute to on-farm profitability and reduced concerns over pesticide residues in the field.

plant biology↗

bioRxiv2025

Biochemical and structural characterization of glycosyltransferase family 61 proteins reveal a key determinant of sugar donor specificity

Xylan, the most abundant non-cellulosic polymer in plant cell walls, is structurally diverse, especially in grasses where it is heavily substituted with arabinofuranose and further modified by various residues. Common substitutions across species include glucuronic and 4-O-methyl-glucuronic acid. Arabinose and xylose sidechains are synthesized by glycosyltransferase family 61 (GT61) proteins, many of which remain uncharacterized in plants, with limited structural and mechanistic understanding. In this study, we identified two novel GT61 enzymes in Sorghum bicolor, functioning as xylan arabinosyltransferase (SbXAT) and xylan xylosyltransferase (SbXXT). We resolved the crystal structure of SbXAT, which exhibits a GT-B fold with two Rossmann-like domains linked by a cleft that accommodates the catalytic site. Structural comparison with a predicted SbXXT model revealed a substrate-binding residue critical for sugar donor specificity, validated through site-directed mutagenesis and enzymatic assays. These findings enhance understanding of xylan biosynthesis and provide a foundation for engineering glycosyltransferases and predicting their functions.

biochemistry↗

bioRxiv2025

Modulation of the GT Family 47 clade B gene affects arabinan deposition in elaters of Marchantia polymorpha

The plant cell wall polymer -1,5-linked arabinan is associated with important functions in plant physiology, such as cell wall flexibility and desiccation tolerance in angiosperms. However, its biosynthetic mechanism remains obscure. A putative arabinan arabinosyltransferase (AraT) is the Arabidopsis ARABINAN-DEFICIENT1 in GT family 47 clade B (GT47B), but its role is inferred by its mutant cell wall phenotypes, and not through in vitro assays. With seven other genes in the clade, investigating the function of the members in GT47B is hampered by the high genetic redundancy in Arabidopsis. Instead, we probe the function of the only two genes in GT47B in the liverwort model organism Marchantia polymorpha, named MpARAD-Like 1 (MpARADL1) and MpARADL2. Mparadl1 and Mparadl2 loss-of-function mutants and overexpression lines were generated, and their cell wall composition was probed using Comprehensive Microarray Polymer Profiling (CoMPP), glycosyl linkage analysis and immunolabelling. Interestingly, Mparadl2 mutants have much less of the 1,5--L-arabinan epitope in elaters since LM6 antibody recognition showed a notable reduction. However, 5-linked Araf levels in the Marchantia thallus tested with glycosyl linkage analysis are comparable between mutants and wild type, suggesting that there is another enzyme forming 5-Araf linkages in Marchantia. Our attempts to obtain the biochemical activity of these enzymes through the expression and purification of AtARAD1, MpARADL1 and MpARADL2 proteins in the HEK293 cell heterologous expression system were unsuccessful. Therefore, we suspect that an evolutionarily conserved, and clade-specific mechanism is required to confer solubility for purification and subsequent activity characterisation of GT47B proteins. Previous studies have shown that co-expression of protein interaction partners can enhance protein solubility in the HEK293 cell system, but co-expression of MpARADL2 with AtARAD1 and MpARADL1 did not yield soluble proteins, prompting further investigation into the protein-protein interactors of GT47B proteins.

plant biology↗

bioRxiv2024

Decoding substrate specificity determining factors in glycosyltransferase-B enzymes - Insights from machine learning models

Substrate specificity is an essential characteristic of any enzymes function and an understanding of the factors that determine this specificity is crucial for enzyme engineering. Unlike the structure of an enzyme which is directly impacted by its sequence, substrate specificity as an enzyme attribute involves a rather indirect relationship with sequence as it also depends on structural aspects that dictate substrate accessibility and active site dynamics. In this study, we explore the performance of classifier-based machine learning models trained on curated sequence and structural data for a class of glycosyltransferases (GTs), namely GT-Bs, to understand their substrate specificity determining factors. GTs enable the transfer of sugar moieties to other biomolecules such as oligosaccharides or proteins and are found in all kingdoms of life. In plants, GTs participate in the biosynthesis of plant cell wall biopolymers (eg: hemicelluloses and pectins) and are an integral part of the enzymatic machinery that enables the storage of carbon and energy as plant biomass. To elucidate the substrate specificity of uncharacterized GT-Bs, we constructed multi-label machine learning models (Support Vector Classifier, K-Nearest Neighbors, Gaussian Naive-Bayes, Random Forest) that incorporate both sequence and structural features. These models achieve good predictive accuracies on test datasets. However, despite our use of structural information, we highlight that there is further scope for improvement in training these models to draw interpretable relationships between sequence, structure and substrate specificity determining motifs in GT-Bs.

bioinformatics↗

bioRxiv2024

Deep structural analysis of rhamnogalacturonan-I (RG-I) in Arabidopsis reveals new insights into pectin diversity

Pectic polysaccharides are an integral part of primary plant cell walls, where they are perfrom crucial structural and biological functions. Pectin is generally divided into four distinct structural categories, including homogalacturonan, xylogalacturonan, rhamnogalacturonan I (RG-I) and rhamnogalacturonan II. Among the four main pectin domains, the structural intricacies of rhamnogalacturonan-I (RG-I) remain the least understood, especially in widely employed plant models. We employed multiple complementary analytical techniques to present a detailed structural analysis of RG-I in the model system Arabidopsis thaliana. Using highly purified RG-I from different tissues, we conducted comparative linkage and NMR analyses, complemented by mass spectrometry of enzymatically digested RG-I oligosaccharides. Our findings present the most comprehensive structural overview of Arabidopsis RG-I to date, revealing novel structural features. Notably, we identified O-acetylation of rhamnose backbone residues as a predominant feature, a modification previously unreported in this species. The combined results present a comprehensive structural overview of Arabidopsis thaliana RG-I that will serve as a roadmap for studying pectin biosynthesis and function.

biochemistry↗

bioRxiv2023

Identification of a putative rhamnogalacturonan-II CMP-beta-Kdo transferase through a callus-based gene editing method which overcomes embryo lethality.

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate crosslinking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect crosslinking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here we developed a method using CRISPR/Cas9-mediated gene to generate callus carrying loss-of-function mutations in the MPG2 gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We show that in the callus mutant cell walls, RG-II does not crosslink normally because it lacks 3-deoxy-D-manno- octulosonic acid (Kdo) and thus cannot form the -L-Rhap-(1[->]5)--D-kdop-(1[->] sidechain. We suggest that MGP2 encodes an inverting CMP-{beta}-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization.

plant biology↗

bioRxiv2018

The most abundant cyst wall proteins of Acanthamoeba castellanii are cellulose-binding lectins from three gene families that localize to distinct structures in cyst walls

Acanthamoeba castellanii, cause of keratitis and blindness, is an emerging pathogen because of its association with contact lens use. The cyst wall contributes to pathogenesis as cysts are resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Here we used structured illumination microscopy (SIM) and probes for glycopolymers to show that purified cyst walls of A. castellanii retain endocyst and ectocyst layers and conical structures (ostioles) that connect them. Mass spectrometry showed candidate cyst wall proteins (CWPs) are dominated by three families of lectins (named here Luke, Leo, and Jonah), because each binds to microcrystalline cellulose +/- chitin. Luke lectins contain two or three carbohydrate-binding modules (CBM49), which were first identified in a tomato cellulase. Leo lectins have two unique domains with eight Cys residues each (8-Cys) +/- a Thr-, Lys-, and His-rich spacer. Jonah lectins contain one or three choice-of-anchor A (CAA) domains previously of unknown function. Representative members of each family were tagged with green fluorescent protein (GFP) and expressed under their own promoters in transfected parasites. A representative Jonah lectin with one CAA domain is made early during encystation and localizes to the ectocyst layer. In contrast, Leo and Luke lectins are made later and localize to the endocyst layer and ostioles. Probes for CWPs (anti-GFP antibodies) and for glycopolymers (maltose-binding protein-fusions with CWPs) suggest Jonah lectin and the glycopolymers to which it binds are accessible in the ectocyst layer, while Luke and Leo lectins and their epitopes are mostly inaccessible in the ectocyst layer and ostioles. In summary, the most abundant A. castellanii CWPs are three sets of lectins, which have conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique carbohydrate-binding modules (8-Cys of Jonah).\n\nAuthor summaryFifty years ago, the cyst wall of Acanthamoeba castellanii was shown to contain cellulose and have an ectocyst layer, an endocyst layer, and conical ostioles that attach them. The goals here were to identify abundant cyst wall proteins (CWPs) and begin to determine how the wall is assembled. We used wheat germ agglutinin to show cyst walls also contain chitin fibrils. When trophozoites are starved of nutrients, they become immotile and make CWPs and glycopolymers in dozens of small vesicles. The primordial cyst wall is composed of a single, thin layer containing cellulose, chitin, and an abundant CWP we called Jonah. The primordial wall also has small, flat ostioles that contain another abundant CWP we called Luke. Jonah (the best candidate for diagnostic antibodies) is accessible in the ectocyst layer of mature cyst walls, while Luke and a third abundant CWP we termed Leo are present but mostly inaccessible in the endocyst layer and ostioles. While A. castellanii cyst walls contain cellulose (like plants) and chitin (like fungi), the glycopolymers are made in vesicles rather than at the plasma membrane, and the CWPs (Luke, Leo, and Jonah lectins) are unique to the protist.

microbiology↗