Biology subjects

Dinneny, J. R.

Publications and source records attributed to Dinneny, J. R..

18 recordsLinked to original sources

bioRxiv2026

Fluorescent Biosensors for Cryogenic Correlative Light and Electron Microscopy

Cryogenic electron tomography provides nanometer-scale views of subcellular structures but lacks essential physiological context, such as the metabolic state or ion concentration. To bridge this gap, we incorporated fluorescent biosensors into a cryogenic correlative light and electron microscopy workflow, enabling direct measurement of physiological conditions alongside structural data. Here, we focused on establishing and calibrating biosensors for calcium, pH, and osmotic stress at cryogenic conditions through an in vitro workflow in which biosensors were frozen in defined chemical environments and then imaged ratiometrically. We then applied these biosensors across a range of sample types: an osmotic stress sensor in Saccharomyces cerevisiae, a calcium sensor in human U2OS cells, and a pH sensor in the bacterium Halothiobacillus neapolitanus. This approach links localized physiological changes to the 3D structures of macromolecules and their organization in situ.

biophysics↗

bioRxiv2026

Spatial and developmental reprogramming enables root growth under high salinity in the extremophyte Schrenkiella parvula

High salinity severely restricts root growth in most plants, yet the extremophyte model Schrenkiella parvula maintains growth under otherwise inhibitory conditions through a previously unrecognized developmental reorganization of the primary root. Under high salinity, the elongation zone rapidly reorganizes into two distinct states: the bulged and gap zones. These zones form in response to ionic stress and emerge during a transient growth pause followed by resumed tip growth and localized lateral root emergence from the gap zone. Jasmonic acid (JA) is necessary to initiate this developmental transition, while cell-layer-resolved hormone profiling revealed spatiotemporally coordinated JA and auxin accompanying the maintenance of the distinct zones. The zone-specific expression profiling resolved transcriptomic networks spanning core development and broad or zone-specific stress responses, revealing spatial regulation of hormone signaling, cell-wall remodeling, and osmotic and oxidative stress pathways. Raman spectroscopy, metabolite profiling, and cellular imaging supported localized regulation of water availability, ionic balance, and suppression of ROS accumulation and cell death. These networks also identified orthologous genes co-opted for novel functions potentially used to sustain growth. Together, these findings reveal a spatially coordinated mechanism for maintaining root growth under salt stress and provide a framework for discovering genetic mechanisms that optimize growth under stress.

plant biology↗

bioRxiv2026

Rhizobacterial Biosensors Spatially Map Natural and Engineered Sucrose Exudation

Root exudation mediates the delivery of plant primary and secondary metabolites into soil, where they regulate plant-microbe interactions and terrestrial carbon cycling. Conventional exudate analyses quantify total root-released carbon yet obscure the spatial origin and rhizosphere influence of individual compounds. Here, we develop a rhizobacterial biosensor platform, named Suc-MAPP, to map local exudate profiles along the surface of colonized root tissues. Focusing on sucrose, we engineered sfGFP-based, sucrose-responsive gene circuits in Pseudomonas putida KT2440 for live imaging of exudate concentrations in the micromolar range. These biosensors reveal spatially structured sucrose exudation patterns across eudicots and monocots and implicate photoassimilated source-sink dynamics as a major determinant. We further apply this platform to phenotype exudation modulated by synthetic gene circuitry in Arabidopsis thaliana, identifying genetic design rules for graded sucrose release and quantifying how engineered export sculpts rhizosphere assembly of a defined bacterial community. Together, these results establish programmable rhizobacterial biosensors as tools to spatially resolve plant-environment carbon exchange in situ and provide a framework for extending this approach to diverse exudate targets.

synthetic biology↗

bioRxiv2025

Cellulose Synthase Complex and Remorin Nanodomains Mediate Stress Resilience Through Cell Wall-Plasma Membrane Attachments

HighlightsO_LIThe plasma membrane forms attachments to the plant cell wall that are revealed by hyperosmotic shock and correlate with tolerance to stress. C_LIO_LICellulose Synthase Complex (CSC) clusters and REMORIN (REM) nanodomains localize to cell wall-plasma membrane attachment sites. C_LIO_LICSC density at the plasma membrane determines the extent of cell wall-plasma membrane attachment under hyperosmotic stress. C_LIO_LIREMs rapidly form nanodomains under hyperosmotic stress and are associated with SHOU4/4L, CSC exocytosis inhibitors that limit CSC density at the plasma membrane. C_LI The outer cell surface of an organism is the frontline for detecting and responding to environmental stimuli. In plants, this interface consists of the plasma membrane that lies beneath the cell wall and remains associated with it through attachment sites. These wall-membrane attachments become evident upon hyperosmotic shock, when severe water loss causes the membrane to retract from the wall. Despite their long-standing observation, the molecular identity and function of these attachments remain poorly understood. Here, we identified two mechanisms governing wall-membrane attachments: one dependent on the Cellulose Synthase Complex (CSC), whose density at the plasma membrane positively correlates with resistance to hyperosmotic stress, and the other on REMORIN (REM), which acts antagonistically to the CSC mechanism. Using proximity-labeling proteomics, we identified SHOU4/4L as REM-associated proteins that mediate this antagonism. Together, our findings reveal how wall-membrane attachments are patterned to mediate plant cell resilience under water stress.

plant biology↗

bioRxiv2024

Maize genetic diversity identifies moisture-dependent root-branch signaling pathways

Plants grow complex root systems to extract unevenly distributed resources from soils. Spatial differences in soil moisture are perceived by root tips leading to the patterning of new root branches towards available water, a process called hydropatterning. Little is known about hydropatterning behavior and its genetic basis in crops plants. Here, we develop an assay to measure hydropatterning in maize and reveal substantial differences between tropical/subtropical and temperate maize breeding germplasm that likely resulted from divergent selection. Genetic dissection of hydropatterning confirmed the regulatory role of auxin and revealed that the gaseous hormone ethylene acts to locally inhibit root branching from air-exposed tissues. These findings demonstrate the crop relevance of hydropatterning and establish its genetic basis.

plant biology↗

bioRxiv2024

Diversification of gene expression across extremophytes and stress-sensitive species in the Brassicaceae

Plants are composed of diverse cell types that facilitate adaptations to the environment, yet cross-species comparisons of such response programs at single-cell resolution remain scarce. To explore this diversity, here we profiled >200,000 root cells from five Brassicaceae species, including stress-sensitive species (Arabidopsis thaliana, Sisymbrium irio), extremophytes (Eutrema salsugineum, Schrenkiella parvula), and the polyploid crop Camelina sativa under control, NaCl, and abscisic acid (ABA) treatments. We developed a computational pipeline to characterize the conservation and divergence of cell-type gene expression across the Brassicaceae, revealing that approximately half of previously defined Arabidopsis cell-type markers fail to maintain conserved expression in one or more non-Arabidopsis species. Therefore, we curated a refined set of pan-Brassicaceae markers and identified orthologs whose expression profiles have diverged across lineages. Using in situ hybridization, we mapped distinct cortex subpopulations to specific cortical layers across species, and found flavonoid biosynthesis programs preferentially localized to the inner cortex layer, linking cortex-layer specification to metabolic specialization. Cell-type contributions to stress responses differed among species and across treatments, with lineage-specific losses of responsiveness occurring less frequently but evolutionarily more favored than lineage-specific gains. In C. sativa, sub-genomes contributed equally to stress responses, and homeologs with divergent responses typically lacked other signatures of functional divergence. Together, this work establishes a foundational root single-cell atlas and an analytical framework for multi-species comparative transcriptomics, providing insights into how stress responses diversify across cell types, stress-sensitive to stress-adapted species, and in crop lineages.

plant biology↗

bioRxiv2022

Synthetic genetic circuits enable reprogramming of plant roots

The shape of a plants root system influences its ability to reach essential nutrients in the soil and to acquire water during drought. Progress in engineering plant roots to optimize water and nutrient acquisition has been limited by our capacity to design and build genetic programs that alter root growth in a predictable manner. Here, we construct synthetic genetic circuits to control gene expression with high spatial precision across root tissues. These circuits produce specific patterns of gene expression by combining the activity of multiple native promoters using logical operations. We then use the circuits to predictably alter root structure. This work demonstrates the ability of synthetic genetic circuits to control gene expression across tissues and offers an exciting means to reprogram plant growth.

synthetic biology↗

bioRxiv2021

Uncovering natural variation in root system architecture and growth dynamics using a robotics-assisted phenomics platform

The plant kingdom contains a stunning array of complex morphologies easily observed above ground, but largely unexplored below-ground. Understanding the magnitude of diversity in root distribution within the soil, termed root system architecture (RSA), is fundamental to determining how this trait contributes to species adaptation in local environments. Roots are the interface between the soil environment and the shoot system and therefore play a key role in anchorage, resource uptake, and stress resilience. Previously, we presented the GLO-Roots (Growth and Luminescence Observatory for Roots) system to study the RSA of soil-grown Arabidopsis thaliana plants from germination to maturity (Rellan-Alvarez et al. 2015). In this study, we present the automation of GLO-Roots using robotics and the development of image analysis pipelines in order to examine the natural variation of RSA in Arabidopsis over time. This dataset describes the developmental dynamics of 93 accessions and reveals highly complex and polygenic RSA traits that show significant correlation with climate variables.

plant biology↗

bioRxiv2021

Identification of green lineage osmotic stress pathways

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the green alga Chlamydomonas reinhardtii to establish a foundational understanding of evolutionarily conserved osmotic-stress signaling pathways in the green lineage through transcriptomics, phosphoproteomics, and functional genomics approaches. Five genes acting across diverse cellular pathways were found to be essential for osmotic-stress tolerance in Chlamydomonas including cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs of these genes in the multicellular land plant Arabidopsis thaliana have conserved functional roles in stress tolerance and reveal a novel PROFILIN-dependent actin remodeling stage of acclimation that ensures cell survival and tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.

plant biology↗

bioRxiv2021

Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Methods that dynamically monitor these intracellular effects are currently lacking. Here, we leveraged the environmental sensitivity and structural plasticity of intrinsically disordered regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use in environmentally-responsive molecular tools.

cell biology↗

bioRxiv2020

Systematic characterization of gene function in a photosynthetic organism

Photosynthetic organisms are essential for human life, yet most of their genes remain functionally uncharacterized. Single-celled photosynthetic model systems have the potential to accelerate our ability to connect genes to functions. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. 78% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles allow us to place known and previously uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism, and ciliogenesis. We illustrate the value of this resource by validating novel phenotypes and gene functions, including the discovery of three novel components of a defense pathway that counteracts actin cytoskeleton inhibitors released by other organisms. The data also inform phenotype discovery in land plants: mutants in Arabidopsis thaliana genes exhibit similar phenotypes to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

systems biology↗

bioRxiv2020

Divergence in a stress-associated gene regulatory network underlies differential growth control in the Brassicaceae family

The phytohormone abscisic acid (ABA) is a central regulator of acclimation during environmental stress. While some plants exhibit tremendous stress resilience, it has been unclear whether differences in ABA response underlie such adaptations. Here we establish a cross-species gene regulatory network (GRN) for ABA to identify broadly conserved, core components of the ABA signaling network and peripheral pathways exhibiting species-specific connectivity. Genes that are broadly conserved in the network share promoter architecture and patterns of gene expression. Networks associated with growth hormones exhibited highly divergent wiring of their ABA network leading to changes in the physiological outcome of signaling. Together our study provides a model for understanding how GRN subcircuits deploy different growth regulatory states across ecologically diverse species. One Sentence SummaryComparative studies reveal core and peripheral stress-mediated gene networks driving divergent growth control in plants.

plant biology↗

bioRxiv2020

A plant lipocalin is required for retinal-mediated de novo root organogenesis

Branching of root systems enables the exploration and colonization of the soil environment. In Arabidopsis roots, de novo organogenesis of lateral roots is patterned by an oscillatory mechanism called the root clock, which is dependent on metabolites derived from the {beta}-carotene pathway1, 2. Retinoids are {beta}-carotene-derived regulators of organogenesis in the animal kingdom. To determine if retinoids function in plant development, we conducted time-lapse imaging of a chemical reporter for retinoid binding proteins. We found that it oscillates with a comparable frequency to the root clock and accurately predicts sites of lateral root organogenesis. Exogenous application of retinal to wild-type plants is sufficient to induce root clock oscillations and lateral root organogenesis. A homology search yielded a potential Arabidopsis homolog, TEMPERATURE INDUCED LIPOCALIN (TIL) to vertebrate retinoid binding proteins. Genetic analysis indicates that TIL is necessary for normal lateral root development and a til mutant has decreased retinal sensitivity. TIL expression in a heterologous system conferred retinal binding activity, suggesting that it may directly interact with this molecule. Together, these results demonstrate an essential role for retinal and for plant retinal binding proteins in lateral root organogenesis.

plant biology↗

bioRxiv2020

Disruption of CYCLOPHILIN 38 function reveals a photosynthesis-dependent systemic signal controlling lateral root emergence

Photosynthesis in leaves generates the fixed-carbon resources and essential metabolites that support sink tissues, such as roots [1]. One of these products, sucrose, is known to promote primary root growth, but it is not clear what other molecules may be involved and whether other stages of root system development are affected by photosynthate levels [2]. Through a mutant screen to identify pathways regulating root system architecture, we identified a mutation in the CYCLOPHILIN 38 (CYP38) gene, which causes an accumulation of pre-emergent stage lateral roots, with a minor effect on primary root growth. CYP38 was previously reported to maintain the stability of Photosystem II (PSII) in chloroplasts [3]. CYP38 expression is enriched in the shoot and grafting experiments show that the gene acts non-cell autonomously to promote lateral root emergence. Growth of wild-type plants under low light conditions phenocopied the cyp38 lateral root emergence phenotype as did the inhibition of PSII-dependent electron transport or NADPH production. Importantly, the cyp38 root phenotype is not rescued by exogenous sucrose, suggesting the involvement of another metabolite. Auxin (IAA) is an essential hormone promoting root growth and its biosynthesis from tryptophan is dependent on reductant generated during photosynthesis [4,5]. Both WT seedlings grown under low light and cyp38 mutants have highly diminished levels of IAA in root tissues. The cyp38 lateral root defect is rescued by IAA treatment, revealing that photosynthesis promotes lateral root emergence partly through IAA biosynthesis. Metabolomic profiling shows that the accumulation of several defense-related metabolites are also photosynthesis-dependent, suggesting that the regulation of a number of energy-intensive pathways are down-regulated when light becomes limiting.

plant biology↗

bioRxiv2019

Cytokinin functions as an asymmetric and anti-gravitropic signal in lateral roots

Directional organ growth allows the plant root system to strategically cover its surroundings. Intercellular auxin transport is aligned with the gravity vector in the primary root tips, facilitating downward organ bending at the lower root flank. Here we show that cytokinin signaling functions as a lateral root specific anti-gravitropic component, promoting the radial distribution of the root system. We performed a genome-wide association study and revealed that signal peptide processing of Cytokinin Oxidase 2 (CKX2) affects its enzymatic activity and, thereby, determines the degradation of cytokinins in natural Arabidopsis thaliana accessions. Cytokinin signaling interferes with growth at the upper lateral root flank and thereby prevents downward bending. Our interdisciplinary approach revealed that two phytohormonal cues at opposite organ flanks counterbalance each others negative impact on growth, suppressing organ growth towards gravity and allow for radial expansion of the root system.

plant biology↗

bioRxiv2017

A microbially derived tyrosine sulfated peptide mimics a plant peptide hormone

O_LIThe biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces a sulfated peptide named RaxX, which shares similarity to peptides in the PSY (plant peptide containing sulfated tyrosine) family. We hypothesize that RaxX functionally mimics the growth stimulating activity of PSY peptides.\nC_LIO_LIRoot length was measured in Arabidopsis and rice treated with synthetic RaxX peptides. We also used comparative genomic analysis and Reactive Oxygen Species (ROS) burst assay to evaluate the activity of RaxX and PSY peptides.\nC_LIO_LIHere we found that a synthetic sulfated RaxX derivative comprising 13 residues (RaxX13-sY), highly conserved between RaxX and PSY, induces root growth in Arabidopsis and rice in a manner similar to that triggered by PSY. We identified residues that are required for activation of immunity mediated by the rice XA21 receptor but that are not essential for root growth induced by PSY. Finally, we showed that a Xanthomonas strain lacking raxX is impaired in virulence.\nC_LIO_LIThese findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide.\nC_LI

plant biology↗

bioRxiv2017

Growth is required for perception of water availability to pattern plant root branches

Water availability is a potent regulator of development in plants and acts as a positional cue to induce root branching through a process termed hydropatterning. The mechanism by which roots perceive the spatial distribution of water to position lateral branches is unknown. Here we reveal that a root's developmental competence for hydropatterning is limited to the root tip, where tissue growth occurs. Mathematical modeling suggests that water uptake during growth creates spatial biases in tissue water potential, and we show that these gradients predict the position of future lateral branches. By altering growth dynamics with exogenous chemical and environmental treatments, we demonstrate that growth is necessary to allow roots to distinguish environments with relatively high or low water availability and pattern branching accordingly. Furthermore, we show that these cues regulate a number of other physiologically important pathways. Our work supports a sense-by-growth mechanism governing lateral root hydropatterning, in which water availability cues are rendered interpretable through growth-sustained water movement.

plant biology↗

bioRxiv2016

Time dependent genetic analysis links field and controlled environment phenotypes in the model C4 grass Setaria

Vertical growth of plants is a dynamic process that is influenced by genetic and environmental factors and has a pronounced effect on overall plant architecture and biomass composition. We have performed twelve controlled growth trials of an interspecific Setaria italica x Setaria viridis recombinant inbred line population to assess how the genetic architecture of plant height is influenced by developmental queues, water availability and planting density. The nondestructive nature of plant height measurements has enabled us to monitor vertical growth throughout the plant life cycle in both field and controlled environments. We find that plant height is reduced under water limitation and high density planting and affected by growth environment (field vs. growth chamber). The results support a model where plant height is a heritable, polygenic trait and that the major genetic loci that influence plant height function independent of growth environment. The identity and contribution of loci that influence height changes dynamically throughout development and the reduction of growth observed in water limited environments is a consequence of delayed progression through the genetic program which establishes plant height in Setaria. In this population, alleles inherited from the weedy S. viridis parent act to increase plant height early, whereas a larger number of small effect alleles inherited from the domesticated S. italica parent collectively act to increase plant height later in development.

plant biology↗