Biology subjects

Hodgens, C.

Publications and source records attributed to Hodgens, C..

3 recordsLinked to original sources

bioRxiv2025

Regulation of Vacuole Fusion in Stomata by Dephosphorylation of the HOPS subunit VPS39

Understanding how plants regulate water loss is important for improving crop productivity. Tight control of stomatal opening and closing is essential for the uptake of CO2 while mitigating water vapor loss. The opening of stomata is regulated in part by homotypic vacuole fusion, which is mediated by conserved homotypic vacuole protein sorting (HOPS) and vacuolar SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) complexes. HOPS tethers apposing vacuole membranes and promotes the formation of trans-SNARE complexes to mediate fusion. In yeast, HOPS dissociates from the assembled SNARE complex to complete vacuole fusion, but little is known about this process in plants. HOPS-specific subunits VACUOLE PROTEIN SORTING39 (VPS39) and VPS41 are required for homotypic plant vacuole fusion, and a computational model predicted that post-translational modifications of HOPS may be needed for plant stomatal vacuole fusion. Here, we characterized a viable T-DNA insertion allele of VPS39 which demonstrated a critical role of VPS39 in stomatal vacuole fusion. We found that VPS39 has increased levels of phosphorylation when stomata are closed versus open, and that VPS39 function in stomata and embryonic development requires dynamic changes in phosphorylation. Our data are consistent with VPS39 phosphorylation altering vacuole dynamics in response to environmental cues, similar to well-established phosphorylation cascades that regulate ion transport during stomatal opening. SIGNIFICANCE STATEMENTVacuole fusion is important for stomata opening but how it is regulated in response of stomata opening signals is not characterized. This research demonstrated the role of the HOPS complex in vacuole fusion in stomata, and it identified phosphorylation sites in the HOPS subunit VPS39 that are critical for vacuole fusion. One Ser residue was enriched in closed stomata and represents a putative site for control of vacuole fusion downstream of stomata opening signals.

plant biology↗

bioRxiv2023

Model-based inference of a plant-specific dual role for HOPS in regulating guard cell vacuole fusion

Stomata are the pores on a leaf surface that regulate gas exchange. Each stoma consists of two guard cells whose movements regulate pore opening and thereby control CO2 fixation and water loss. Guard cell movements depend in part on the remodeling of vacuoles, which have been observed to change from a highly fragmented state to a fused morphology during stomata opening. This change in morphology requires a membrane fusion mechanism that responds rapidly to environmental signals, allowing plants to respond to diurnal and stress cues. With guard cell vacuoles being both large and responsive to external signals, stomata represent a unique system in which to delineate mechanisms of membrane fusion. Fusion of vacuole membranes is a highly conserved process in eukaryotes, with key roles played by two multi-subunit complexes: HOPS (homotypic fusion and vacuolar protein sorting) and SNARE (soluble NSF attachment protein receptor). HOPS is a vacuole tethering factor that is thought to chaperone SNAREs from apposing vacuole membranes into a fusion-competent complex capable of rearranging membranes. To resolve a counter-intuitive observation regarding the role of HOPS in regulating plant vacuole morphology, we derived a quantitative model of vacuole fusion dynamics and used it to generate testable predictions about HOPS-SNARE interactions. We derived our model by applying simulation-based inference to integrate prior knowledge about molecular interactions with limited, qualitative observations of emergent vacuole phenotypes. By constraining the model parameters to yield the emergent outcomes observed for stoma opening - as induced by two distinct chemical treatments - we predicted a dual role for HOPS and identified a stalled form of the SNARE complex that differs from phenomena reported in yeast. We predict that HOPS has contradictory actions at different points in the fusion signaling pathway, promoting the formation of SNARE complexes, but limiting their activity. Author summaryPlants "breathe" through pores in their leaves where each pore is formed by two specialized cells called guard cells. To open these pores, guard cells change in volume. This volume change is controlled by water-filled organelles called vacuoles that morph from multiple small entities to a few large ones capable of taking up more water to reshape the cell. Specialized proteins in vacuole membranes make this change happen by pulling vacuoles together until they fuse. Some of these proteins reside in membranes, but others must be drawn to the membrane from the cells cytoplasm. Specific lipid molecules in the membrane play an important role in recruiting those proteins to the vacuole membrane. We previously made an unexpected finding that removing this lipid induces plant vacuole fusion. To make sense of this observation, we used a mathematical model to piece together our knowledge of the proteins involved in this process and what we know about the chemical treatments that cause vacuoles to morph. Using computer simulations, we uncovered new rules about how molecules interact in membranes to accomplish the task of vacuole fusion in plants. We think the rules uncovered through mathematical modeling allow plants to respond quickly to environmental cues.

systems biology↗

bioRxiv2017

indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events

Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be easily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

plant biology↗