Biology subjects

Ehrhardt, D. W.

Publications and source records attributed to Ehrhardt, D. W..

6 recordsLinked to original sources

bioRxiv2025

Sorghum Metabolic Atlas: Large-Scale Mapping of Subcellular Enzyme Localization in Sorghum bicolor

Plant metabolism drives traits essential for productivity and resilience, yet understanding metabolic networks requires subcellular, cellular, and tissue-level spatial context that remains limited, particularly in crop species. Experimentally-derived subcellular localization data for enzymes are sparse, constraining analyses of metabolic organization in the cell. We developed a high-throughput protoplast transformation and fluorescent protein (FP) tagging system optimized for Sorghum bicolor, a climate-resilient C4 crop. Using this platform, we experimentally determined the subcellular localization of 234 metabolic enzymes spanning 184 pathways. The sorghum enzymes we characterized localize to 12 subcellular compartments. Comparison with computational predictions highlights variable accuracy across compartments, and cross-species comparison with Arabidopsis thaliana shows partial agreement with available experimental data. All data are accessible through the Sorghum Metabolic Atlas (www.sorghummetabolicatlas.org) web platform, enabling search, visualization, and download. This study presents a large-scale experimental dataset of enzyme localization in sorghum, providing a resource for studies of plant metabolic organization and comparative analyses.

plant biology↗

bioRxiv2024

Cortical microtubules act as a template to organize nano-scale patterning of exocytosis

The microtubule cytoskeleton organizes exocytosis to enable cellular morphogenesis, but how non-centrosomal arrays control exocytotic site positioning remains poorly understood. Using elongating plant cells as a model, we developed quantitative methods to move beyond coarse correlation and reveal the precise spatial relationship between cortical microtubules and secretion. We identify KEULE, an essential SEC/MUNC protein, as a dynamic exocytosis marker that forms clusters with stereotyped assembly and disassembly kinetics at discrete secretion sites. Combining confocal microscopy with super-resolution analysis and Bayesian inference, we quantified microtubule-exocytosis positioning at nanometer precision. This analysis revealed that microtubules create [~]180 nm enrichment zones flanked by [~]520 nm depletion zones, generating a spatial pattern that replicates the cortical array structure. Unexpectedly, Bayesian inference showed strong evidence for a flat enrichment profile within these zones rather than peaked distributions. This flat profile, combined with enrichment zone widths exceeding the [~]50 nm reach of known tethering proteins, challenges a direct vesicle capture mechanism. Instead, our data support a membrane domain mechanism where microtubules organize lipid/protein composition to create preferred exocytosis territories. These findings establish quantitative spatial rules for how non-centrosomal microtubule arrays organize secretion.

cell biology↗

bioRxiv2023

Thermo-sensitive condensation of SUF4 tunes flowering time in Arabidopsis thaliana

Intrinsically disordered proteins (IDP) lack stable tertiary structures, which allows them to change conformation and function under different physicochemical conditions. This may be highly advantageous for plants, which often use changes in their environment to elicit a variety of responses, including developmental events. For instance, some plants delay flowering in the fall and require exposure to winter temperatures as a cue to initiate flowering the following spring. Many of the genes involved in temperature-dependent flowering have been extensively studied in Arabidopsis, yet how plants coordinate their molecular states with seasonal temperature change is poorly understood. Here, we explore the role of temperature-sensitive phase separation of the IDP and flowering-time regulator, SUPPRESSOR OF FRIGIDA 4 (SUF4), in modulating flowering time. SUF4 has a well-defined role in regulating temperature-dependent flowering time by activating the master floral suppressor FLOWERING LOCUS C (FLC). We show that in plant nuclei, SUF4 is a temperature-sensitive protein that assembles into biomolecular condensates in warm temperatures (20{degrees}C). When temperatures cool (4{degrees}C), SUF4 nuclear condensates disassemble, causing SUF4 to disperse within the nucleoplasm. Additionally, we demonstrate that the number of SUF4 condensates quantitatively correlates with flowering time. Progressive alterations to the amino acid composition of SUF4s disordered region cause likewise progressive changes in temperature-dependent condensation both in vitro and in vivo, FLC transcription, and the onset of flowering. Lastly, we observe that SUF4 condensates coincide with the accumulation of other key flowering-time proteins (FRIGIDA and ELF7). These findings indicate that condensation of SUF4 likely plays a pivotal role in promoting flowering, possibly by concentrating and stabilizing regulatory factors needed for the transcriptional activation of FLC through temperature-dependent phase separation. This research suggests that in plants, IDPs can sense environmental cues and regulate critical developmental processes. One-Sentence SummaryEvidence suggest that Arabidopsis uses temperature-sensitive condensation to regulate FLC transcription and flowering time.

plant biology↗

bioRxiv2023

Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei

Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.

plant biology↗

bioRxiv2023

Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi Network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the interactome of the Transport Protein Particle II (TRAPPII) complex, required for TGN structure and function. We identified physical and genetic interactions between TRAPPII and shaggy-like kinases (GSK3/AtSKs). Kinase assays and pharmacological inhibition provided in vitro and in vivo evidence that AtSKs target the TRAPPII-specific subunit AtTRS120/TRAPPC9. GSK3/AtSK phosphorylation sites in AtTRS120/TRAPPC9 were mutated, and the resulting AtTRS120 phosphovariants subjected to a variety of single and multiple stress conditions in planta. The non-phosphorylatable TRS120 mutant exhibited enhanced adaptation to multiple stress conditions and to osmotic stress whereas the phosphomimetic version was less resilient. Higher order inducible trappii atsk mutants had a synthetically enhanced defect in root gravitropism. Our results suggest that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the TRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

plant biology↗

bioRxiv2017

CLASP stabilizes microtubule plus ends after severing to facilitate light-induced microtubule reorientation

Central to building and reorganizing cytoskeletal arrays is the creation of new polymers. While nucleation has been the major focus of study for new microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. As severing produces new plus ends behind the stabilizing GTP-cap, an important and unanswered question is how these are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing and find that CLASP is required for rapid cortical array reorientation. In clasp mutants both rescue of shrinking plus ends and the regrowth of plus ends immediately after severing are reduced, computational modeling reveals that it is the specific stabilization of severed ends that explains CLASPs function in promoting microtubule amplification by severing and cortical array reorientation.

cell biology↗