Biology subjects

Szymanski, D. B.

Publications and source records attributed to Szymanski, D. B..

10 recordsLinked to original sources

bioRxiv2025

Rapid ethylene-triggered protein complex remodeling in dark grown hypocotyls

Protein-level cellular dynamics, including multimerization, play a crucial role in rapid adaptation of plants during developmental transitions and environmental stresses. The gaseous hormone ethylene is a key regulator of rapid cellular growth modulation. During soil emergence, etiolated seedlings undergo crucial morphological changes to their apical hooks and hypocotyls, with ethylene inhibiting hypocotyl axial elongation while promoting radial expansion. Ethylene triggers these growth responses within two hours; however, the protein machineries and cellular processes that mediate morphogenesis control are not known. Here, we used quantitative proteomics and co-fractionation mass spectrometry to test for rapid ethylene-dependent changes in protein abundance and protein complex composition. Protein multimerization responses were numerous and diverse. There were instances of protein complex assembly and disassembly, with varying degrees of completeness. Small-scale validation tests indicate that the identified proteins play a role in hypocotyl development and suggest that this novel approach to gene discovery identifies potential targets for ethylene-mediated growth regulation and enhanced seedling adaptability during early development.

plant biology↗

bioRxiv2025

Comparative multi-omics profiling of Gossypium hirsutum and Gossypium barbadense fibers at high temporal resolution reveals key differences in polysaccharide composition and associated glycosyltransferases

Among the two allopolyploid cultivated species of cotton, Gossypium barbadense is known for its superior quality fiber compared to G. hirsutum. Length and strength are key determinants of the fiber quality. Although the mature fibers are dried cell walls consisting mainly cellulose, the dynamic remodeling of pectin, xyloglucan, and xylan polysaccharides during fiber growth significantly impact the final fiber quality. Comprehensive knowledge about polysaccharides and their biosynthesis during fiber development of the cultivated species is important for improving fiber quality. In this study, comparative large-scale glycome, transcriptome and proteome profiling were conducted daily on fibers of both cotton species covering critical stages of fiber development spanning primary cell wall synthesis and the transition to secondary wall synthesis. Interspecific comparisons revealed that a delayed accumulation of cellulose content, as well as the occurrence of lower levels and differential compositions of non-fucosylated/fucosylated xyloglucans, homogalacturonans, and highly branched Rhamnogalacturonan-I polysaccharides might be contributing to longer fiber phenotypes of G. barbadense relative to G. hirsutum. Our study also suggests, differential temporal compositions of heteroxylans might contribute to variation in cellulose microfibril arrangement and strength of fiber exists between the two species of cotton. Comparative transcriptomic analysis identified differentially expressed polysaccharide-synthesizing glycosyltransferases that might underlie fiber quality differences between the two species. Transcripts encoding many cell wall localized expansins were more abundant in G. barbadense than in G. hirsutum. Overall, these findings extend our knowledge regarding the molecular factors that contribute to fiber quality and provide insights for targeted cotton fiber improvement. SIGNIFICANCE STATEMENTComparative multi-omics profiling of two major commercial cotton species, Gossypium hirsutum and Gossypium barbadense revealed substantial differences in polysaccharide structures and expressed polysaccharide-synthesizing glycosyltransferases, that potentially contribute to differences in fiber length and strength. The molecular details elucidated in the present study contribute to the goal of improving fiber quality and its commercial value.

plant biology↗

bioRxiv2025

Proteomics-based models of gene expression and cellular control of cotton fiber development

The shapes and material properties of cotton seed coat trichoblasts are the basis of a multibillion-dollar natural fiber industry. As such, these highly specialized cells are low-hanging fruit for intentional trait engineering. However, broad successes will require more mechanistic knowledge about their systems-level cellular controls. This time-series study integrates daily measurements of purified fiber transcriptomes and proteomes with multiscale fiber phenotyping datasets that span the same developmental interval. Abundance profiles of the subcellular proteomes are the foundation of the analyses. This resource article provides direct information concerning which homoeologs operate and informative depictions of how compartmentalized cellular systems change during developmental transitions. Prediction accuracy was partially validated by analysis of the protein expression group 11, which contained multiple known secondary cell wall cellulose synthases and dozens of unknown proteins and an averaged profile that was strongly correlated with a sharp state transition in cellulose microfibril alignment and increased cellulose content. The dataset as a whole can serve as a hypothesis-generating machine to guide future experiments that relate to cell shape and growth rate control, reversible tissue formation, and cell wall remodeling. Integration of mRNA and protein abundance revealed widespread evidence for post-transcriptional control. In addition, there were hundreds of transcriptionally controlled genes with differing timepoints of transition. This latter gene set can be used to more reliably analyze transcriptional control networks and to generate collections of gene expression drivers for cotton fiber research. The protein and transcript data are organized into user-friendly tables and a web interface that can be searched using any plant ortholog of interest based on developmental time, abundance, annotations, or phenotypic association.

plant biology↗

bioRxiv2024

A cell fractionation and quantitative proteomics pipeline to enable functional analyses of cotton fiber development

Cotton fibers are aerial trichoblasts that employ a highly polarized diffuse growth mechanism to emerge from the developing ovule epidermis. After executing a complicated morphogenetic program, the cells reach lengths over 2 cm and serve as the foundation of a multi-billion-dollar textile industry. Important traits such as fiber diameter, length, and strength are defined by the growth patterns and cell wall properties of individual cells. At present, the ability to engineer fiber traits is limited by our lack of understanding regarding the primary controls governing the rate, duration, and patterns of cell growth. To gain insights into the compartmentalized functions of proteins in cotton fiber cells, we developed a label-free liquid chromatography mass spectrometry method for systems level analyses of fiber proteome. Purified fibers from a single locule were used to fractionate the fiber proteome into apoplast (APOT), membrane-associated (p200), and crude cytosolic (s200) fractions. Subsequently, proteins were identified, and their localizations and potential functions were analyzed using combinations of size exclusion chromatography, statistical and bioinformatic analyses. This method had good coverage of the p200 and apoplast fractions, the latter of which was dominated by proteins associated with particulate membrane-enclosed compartments. The apoplastic proteome was diverse, the proteins were not degraded, and some displayed distinct multimerization states compared to their cytosolic pool. This quantitative proteomic pipeline can be used to improve coverage and functional analyses of the cotton fiber proteome as a function of developmental time or differing genotypes.

plant biology↗

bioRxiv2024

A high-resolution model of gene expression during Gossypium hirsutum (cotton) fiber development

Cotton fiber development relies on complex and intricate biological processes to transform newly differentiated fiber initials into the mature, extravagantly elongated cellulosic cells that are the foundation of this economically important cash crop. Here we extend previous research into cotton fiber development by employing controlled conditions to minimize variability and utilizing time-series sampling and analyses to capture daily transcriptomic changes from early elongation through the early stages of secondary wall synthesis (6 to 24 days post anthesis; DPA). A majority of genes are expressed in fiber, largely partitioned into two major coexpression modules that represent genes whose expression generally increases or decreases during development. Differential gene expression reveals a massive transcriptomic shift between 16 and 17 DPA, corresponding to the onset of the transition phase that leads to secondary wall synthesis. Subtle gene expression changes are captured by the daily sampling, which are discussed in the context of fiber development. Coexpression and gene regulatory networks are constructed and associated with phenotypic aspects of fiber development, including turgor and cellulose production. Key genes are considered in the broader context of plant secondary wall synthesis, noting their known and putative roles in cotton fiber development. The analyses presented here highlight the importance of fine-scale temporal sampling on understanding developmental processes and offer insight into genes and regulatory networks that may be important in conferring the unique fiber phenotype.

cell biology↗

bioRxiv2024

Daily glycome and transcriptome profiling reveals polysaccharide structures and glycosyltransferases critical for cotton fiber growth

Cotton fiber length and strength are key determinants of its quality. Dynamic changes in the pectin, xyloglucan, xylan, and cellulose polysaccharide epitopes content during fiber growth contribute to complex remodeling of fiber cell wall (CW) and quality. Detailed knowledge about polysaccharide compositional and structural alteration in the fiber during fiber elongation and strengthening is vastly limited. Here, large-scale glycome profiling coupled with fiber phenotype and transcriptome profiling was conducted on fiber collected daily covering the most critical fiber developmental window. High temporal resolution profiling allowed us to identify specific polysaccharide epitopes associated with distinct fiber phenotypes that might contribute to fiber quality. This study revealed the critical role of highly branched RG-I pectin epitopes such as, {beta}-1,4-linked-galactans, {beta}-1,6-linked-galactans, and arabinogalactans, in addition to earlier reported homogalacturonans and xyloglucans in the formation of cotton-fiber-middle-lamella and contributing to fiber plasticity and elongation. We also propose the essential role of heteroxylans (Xyl-MeGlcA and Xyl-3Ar), as a guiding factor for secondary CW cellulose-microfibril arrangement, thus contributing to fiber strength. Correlation analysis of glycome and transcriptome data identified several key putative glycosyltransferases involved in synthesizing the critical polysaccharide epitopes. Novel details discovered here provide a foundation to identify molecular factors that dictate important fiber traits.

plant biology↗

bioRxiv2021

A Co-Fractionation Mass Spectrometry-based Prediction of Protein Complex Assemblies in the Developing Rice Aleurone-subaleurone

Multiprotein complexes execute and coordinate diverse cellular processes such as organelle biogenesis, vesicle trafficking, cell signaling, and metabolism. Knowledge about their composition and localization provides useful clues about the mechanisms of cellular homeostasis and systems-level control. This is of great biological importance and practical significance in heterotrophic rice endosperm and aleurone-subaleurone tissues that are a primary source of seed vitamins and stored energy. Dozens of protein complexes have been implicated in the synthesis, transport, and storage of seed proteins, lipids, vitamins, and minerals. Mutations in protein complexes that control RNA transport result in aberrant endosperm with shrunken and floury phenotypes, significantly reducing seed yield and quality. The purpose of this research is to broadly predict protein complex composition in the aleurone-subaleurone layers of developing rice seeds using co-fractionation mass spectrometry. Following orthogonal chromatographic separations of biological replicates, thousands of protein elution profiles were subjected to distance-based clustering to enable a large-scale determination of multimerization state and complex composition. Predictions included evolutionarily conserved proteins across diverse functional categories, including novel heteromeric RNA binding protein complexes that influence seed quality. This effective and open-ended proteomics pipeline provides useful clues about systems-level controls in the early stage of rice seed development. One-sentence summaryA co-fractionation mass spectrometry pipeline predicts compositions of cytosolic protein complexes present in the early stages of rice seed development.

plant biology↗

bioRxiv2021

Protocol for mapping the spatial variability in cell wall mechanical bending behavior in living leaf pavement cells

An integrated, experimental-computational approach is presented to analyze the variation of elastic bending behavior in the primary cell wall of living Arabidopsis thaliana pavement cells and to measure turgor pressure in the cells quantitatively under different osmotic conditions. Mechanical properties, size and geometry of cells and internal turgor pressure greatly influence their morphogenesis. Computational models of plant morphogenesis require values for wall elastic modulus and turgor pressure but very few experiments were designed to validate the results using measurements that deform the entire thickness of the cell wall. Because new wall material is deposited from inside the cell, full-thickness deformations are needed to quantify relevant changes associated with cell development. The approach here uses laser scanning confocal microscopy to measure the three-dimensional geometry of a single pavement cell, and indentation experiments equipped with high magnification objective lens to probe the local mechanical responses across the same cell wall. These experimental results are matched iteratively using a finite element model of the experiment to determine the local mechanical properties, turgor pressure, and cell height. The resulting modulus distribution along the periclinal wall is shown to be nonuniform. These results are consistent with the characteristics of plant cell walls which have a heterogeneous organization. This research and the resulting model will provide a reference for future work associated with the heterogeneity and anisotropy of mechanical properties of plant cell walls in order to understand morphogenesis of the primary cell walls during growth and to predict quantitatively the magnitudes/directions of cell wall forces. One sentence summaryThe distribution of elastic modulus of the periclinal cell walls of living Arabidopsis epidermis is nonuniform as measured by bending the entire thickness of the wall. HighlightsO_LIExperimental characterization of the spatial distribution of elastic bending behavior across the periclinal wall C_LIO_LIQuantification of the turgor pressure of the living plant epidermal cells validated with osmotic treatments C_LIO_LIQuantification of the effect of cell geometry on the measured mechanical response C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/432478v1_ufig1.gif" ALT="Figure 1"> View larger version (117K): org.highwire.dtl.DTLVardef@46fcfcorg.highwire.dtl.DTLVardef@8b0ab0org.highwire.dtl.DTLVardef@6aad31org.highwire.dtl.DTLVardef@1909d6a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Real-time conversion of tissue-scale mechanical forces into an interdigitated growth pattern

The leaf epidermis is a dynamic biomechanical shell that integrates growth across spatial scales to influence organ morphology. Pavement cells, the fundamental unit of this tissue, morph irreversibly into highly lobed cells that drive planar leaf expansion. Here we define how tissue-scale cell wall tensile forces and the microtubule-cellulose synthase systems pattern interdigitated growth in real-time. A morphologically potent subset of cortical microtubules span the periclinal and anticlinal cell faces to pattern cellulose fibers that generate a patch of anisotropic wall. The result is local polarized growth that is mechanically coupled to the adjacent cell via a pectin-rich middle lamella, and this drives lobe formation. Finite element pavement cell models revealed cell wall tensile stress as an upstream patterning element that links cell- and tissue-scale biomechanical parameters to interdigitated growth. Cell lobing in leaves is evolutionarily conserved, occurs in multiple cell types, and is associated with important agronomic traits. Our general mechanistic models of lobe formation provide a foundation to analyze the cellular basis of leaf morphology and function.

cell biology↗

bioRxiv2020

The BEACH Domain-Containing Protein SPIRRIG Modulates Actin-Dependent Root Hair Development in Coordination with the WAVE/SCAR and ARP2/3 Complexes

Root hairs are single cell protrusions that enable roots to optimize nutrient and water acquisition. They attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a BEACH domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations to root hairs at distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi vesicles and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced in spi and the position of root hair emergence was disrupted in brk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development through pathways that might intersect with W/SC.

plant biology↗