Biology subjects

Kelly, S.

Publications and source records attributed to Kelly, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Age and sex alter the immune response in a chronic fibrosis model via changes in T cell and macrophage phenotype

The foreign body response (FBR) is an immune mediated event that occurs with every material implant. The extent of the fibrosis is dependent on many factors including the biomaterial design, tissue location, and host factors such as age, sex, ancestry, diet. There are known clinical outcomes of implants dependent on age and sex, including increased fibrosis and implant failure in aged and female patients. As the population ages, there is a growing need to understand how aging affects the FBR, and how preclinical models can capture this to guide biomaterial design. Here, we investigated how chronic fibrosis in a murine model of the FBR is altered by two biological factors: age and sex. We investigated changes in fibrosis using a volumetric muscle loss (VML) injury model coupled with polycaprolactone (PCL) or polyethylene (PE) microparticle implants. Fibrosis was quantified through gene expression, microscopic analysis of histologic sections, and the corresponding immune response measured via gene expression and flow cytometry data. We found gene expression differences with immune pathways enriched in female mice, and microscopy revealed collagen birefringence area increased in young male mice. Both the innate and adaptive immune response were altered by age and sex via T cell and macrophage phenotype, and the effects of aging differed between sexes. These results reveal both variables contribute to discrepant outcomes in both fibrosis and the local immune response to synthetic material implants. This demonstrates a clear need to understand and account for the influence of biological factors in biomaterial design.

bioengineering↗

bioRxiv2026

Singing and moving to the rhythm: song-entangled gestures in a vocal learning songbird

When humans speak, we use rhythmic hand and head gestures that are closely coordinated with the temporal structure of speech to emphasize words and phrases. Similarly, human singing is often accompanied by rhythmic body movements that are aligned with the timing and prosodic structure of the vocal sequence. This rhythmic synchronization of bodily gesture with vocal production is thought to be a shared feature of vocal-learning species. How do these two sensorimotor systems develop, coordinate, and synchronize with precise timing to support multimodal communication? The mechanisms underlying this rhythmic entrainment remain poorly understood, and no established animal model to date captures the human combination of speech and co-speech gesture. Here, we show that a vocal-learning songbird, the zebra finch, has evolved song-entrained head gestures. These co-song gestures rhythmically align with acoustically complex song syllables and are developed and produced independently of other innate, stereotyped, song-entangled courtship displays. Even when the song remains largely the same, co-song gestures can be dynamically modulated in rhythm, form, and/or extent across different social contexts. The rhythmic alignment requires auditory feedback, is under the control of a premotor song nucleus, and gradually develops during the sensitive period of vocal learning. Females respond differently when songs and co-song gestures are misaligned, suggesting a social function. This dynamic modulation of co-song gestures provides a behavioral window into brain and cognitive states, making the zebra finch a promising model for understanding the mechanisms underlying rhythmic synchronization of multi-sensorimotor systems and the origin and evolution of co-speech gestures in humans.

neuroscience↗

bioRxiv2026

Capillary-based Subcellular Sampling Uncovers the Stress Granule Proteome in Single Cells

Many diseases arise from dysfunction within specific organelles or biomolecular condensates, highlighting the value of analysing proteins at subcellular resolution to uncover new biological mechanisms. We report a novel capillary-based subcellular sampling workflow coupled with liquid chromatography-mass spectrometry (LC-MS) for proteomic analysis of defined subcellular regions of individual cells. We applied this methodology to stress granules (SGs), membrane-less biomolecular condensates that form in response to cellular stress (including viral infection), and are implicated in infection, neuropathology and cancer. Comprehensive characterisation of SG protein composition remains limited by technical challenges associated with bulk purification, including loss of spatial context, dynamic behaviour and contamination from cytosolic material. Using our novel method, we identified a high-confidence set of 405 SG-associated proteins, including 46 established SG residents alongside numerous previously unreported candidates. Functional enrichment analysis revealed pathways consistent with known SG biology, while comparison with an independent cytosolic proteome dataset demonstrated minimal overlap, supporting the specificity of the sampling strategy. Selected novel SG protein candidates (AHNAK2, DDX39B, NUDT1 and FKBP2) were validated using immunofluorescence microscopy. These findings establish capillary-based subcellular sampling as a viable approach for proteomic analysis of SGs with preserved spatial context and provide a framework for analysing other subcellular compartments. Table of contentsWe report an LC-MS-based capillary sampling workflow for proteomic analysis of subcellular structures within single cells. This methodology identified 405 high-confidence stress granule-associated proteins, including 46 previously established and numerous novel candidates. The approach demonstrated high specificity and preserved spatial context, expanding the capabilities of subcellular proteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/724230v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1fa0bb0org.highwire.dtl.DTLVardef@1158524org.highwire.dtl.DTLVardef@1d82812org.highwire.dtl.DTLVardef@2ee4d9_HPS_FORMAT_FIGEXP M_FIG C_FIG Figure made in Biorender.com.

cell biology↗

bioRxiv2026

Global population structure and phase variation of serotype 12F Streptococcus pneumoniae following the introduction of pneumococcal conjugate vaccine

BackgroundAfter the global deployment of pneumococcal conjugate vaccines (PCVs), serotype 12F has become the predominant serotype responsible for invasive pneumococcal disease (IPD) worldwide. As PCVs that include serotype 12F are gradually introduced, we aim to characterise the global population structure and genetic diversity of the 12F capsule locus using whole-genome sequencing. Capsule variants with vaccine evasion potential were further investigated by functional experiments. MethodsA global collection of pneumococcal serotype 12F genomes (n=806) from 37 countries across six continents were included in this study. To characterise the serotype 12F population, Global Pneumococcal Sequence Cluster (GPSC), in silico serotype, and antimicrobial resistance profile were inferred from whole-genome data for each isolate. The capsule biosynthesis (cps) locus was analysed for gene content variations that could alter polysaccharide capsule production or structure, thereby influencing recognition by vaccine-induced antibodies. These isolates were further investigated by assessing their capsule production using immunofluorescence assays and its susceptibility to vaccine-elicited antibody killing by opsonophagocytosis assays. FindingsThe global increase in serotype 12F was driven by both distinct pneumococcal lineages across different continents, and a globally-disseminated and multidrug-resistant lineage GPSC26. We identified six capsule variants in nine isolates that had disruptive mutations in cps genes including wze, wcil, wciJ and fnlA. Most (6/9) of the disruptive mutations were a result of strand-slippage mutations. A convergent strand-slippage mutation disrupting the glycosyltransferase gene wciJ was identified in four isolates from distinct lineages and countries. Despite the truncation, three of four isolates with available Quellung typing results still identified them as 12F, indicating the production of the capsule. We then created a genetically engineered lab strain with wciJ knockout and complemented with wciJ containing the strand-slipppage mutation. The knockout strain did not produce any capsule. In contrast, the lab strain with wciJ containing the strand-slippage mutation produced a mixed population of encapsulated and non-encapsulated pneumococci, even within the same chain of pneumococcal cells. This observation indicated encapsulated subpopulation possesses a functional WciJ and rapidly reversible strand-slippage mutation during replication. Opsonophagocytosis assays indicated that the clinical 12F strain with strand-slippage mutation in wciJ exhibited reduced susceptibility to vaccine-elicited serum killing, compared to a genetically closely related 12F clinical strain with an intact wciJ. However, substantial inter-individual antisera variation limits definitive interpretation. InterpretationOur work revealed the global rise of serotype 12F pneumococci has been driven by both regional-specific lineages, and a globally-disseminated and multidrug-resistant lineage GPSC26. We demonstrated that strand-slippage mutation is one of the major drivers of serotype 12F capsule variants and represents a novel mechanism enabling reversible on-off switching of capsule production. The ability to switch off capsule expression in a subpopulation may enable evasion of antibody-mediated killing but increase susceptibility to innate immune clearance. FundingBill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

genomics↗

bioRxiv2026

GLADE: Accurate inference of Gains, Losses, Ancestral genomes, and Duplication Events for comparative genomics

Changes in gene content through gain and loss play a key role in the adaptation and diversification of species. Accordingly, our ability to detect and accurately document the history of these changes is important for our understanding the evolutionary trajectories of life on Earth. Here we present GLADE, a tool that accurately reconstructs gene gains, losses, and duplications for a set of species under consideration and uses this information to infer ancestral gene contents for every speciation event in the species tree. GLADE requires as input only a standard OrthoFinder results directory, and outputs the full evolutionary history of every orthogroup, including branch-specific changes and reconstructed ancestral genomes. We benchmark GLADE using both real and simulated data and show that GLADE accurately identifies orthogroup gains, losses, and duplications, and reconstructs ancestral orthogroup sizes with higher precision and overall accuracy than any competitor method. To illustrate the utility of the method, we apply GLADE to a dataset of 78 mammalian genomes and uncover repeated contractions in orthogroups associated with tooth formation on branches leading to ant- and termite-eating mammals - revealing convergent genomic signatures underlying this dietary specialization. GLADE and accompanying documentation and tutorials are freely available at https://github.com/lauriebelch/GLADE/.

evolutionary biology↗

bioRxiv2026

FastSpeciesTree: Fast and Scalable Species tree Inference

The generation of species trees from genomic data is an important step in understanding evolutionary relationships across the tree of life. With the increasing availability of genomic data, species tree inference methods which can easily and rapidly produce accurate species trees from large datasets are required to enable downstream analyses of these resources. Here we present FastSpeciesTree, a fully automated species tree inference method designed to leverage advances in rapid pairwise sequence alignment and scalable phylogenetic inference methods. We show that FastSpeciesTree is able to produce species trees with accuracies equivalent to trees produced through manual-curation and best-practice approaches. We further demonstrate that FastSpeciesTree can produce these high accuracy phylogenies substantially faster than any competitor method. Finally, we show that FastSpeciesTree is capable of building a species tree comprising almost the entirety of the RefSeq databases available proteomes (n = 33285) in less than 12 hours using standard computing resources. FastSpeciesTree is executed as a single command and can be found at https://github.com/OrthoFinder/FastSpeciesTree.

evolutionary biology↗

bioRxiv2025

Single-Islet Proteomics Maps Pseudo-Temporal Islet Immune Responses and Dysfunction in Stage 1 Type 1 Diabetes

Progressive {beta}-cell dysfunction precedes the onset of type 1 diabetes (T1D), yet the molecular mechanisms driving early T1D development remain poorly understood. Although single-cell RNA-sequencing has uncovered transcript-level changes in human islet cells, it offers limited insight into the heterogeneity of distinct islet microenvironments. Here, we applied a single-islet proteomics workflow to profile intra-donor islet heterogeneity in three stage 1 T1D cases with matched non-diabetic controls and define in situ protein signatures of pseudo-temporal islet dysfunction. Intra-donor analyses of [~]100 individual islets per donor revealed highly consistent proteomic patterns reflecting pseudo-time progression of islet immune responses and {beta}-cell dysfunction. Several pathways, including extracellular matrix remodeling and mRNA processing, were identified as closely associated with progressive islet immune activation and loss of {beta}-cell function. These findings provide robust proteome-wide evidence of the progression of islet dysfunction, offer a valuable resource for investigating early mechanisms of T1D pathogenesis-- including novel candidates for functional studies--and underscore the utility of single-islet spatial proteomics for examining islet heterogeneity in T1D.

systems biology↗

bioRxiv2025

OrthoFinder: scalable phylogenetic orthology inference for comparative genomics

Here, we present a major advance of the OrthoFinder method. This extends OrthoFinders high accuracy comparative genomic framework to provide substantially enhanced scalability and accuracy. Specifically, we show that enhanced phylogenetic delineation of orthogroups provides a 7% relative increase in orthogroup inference accuracy. We further demonstrate that a new gene assignment method substantially reduces overall runtime RAM usage without compromising accuracy. The latest version of OrthoFinder is available at https://github.com/OrthoFinder/OrthoFinder.

bioinformatics↗

bioRxiv2025

HpBoRB, a helminth-derived CCP domain protein which binds RELMβ

Helminth infections persist by influencing host immunity through the release of immunomodulatory proteins which prevent immune ejection. The intestinal nematode Heligmosomoides polygyrus bakeri (Hpb) secretes multiple families of immunomodulatory proteins, many of which are composed of consecutive Complement Control Protein (CCP) domains. We hypothesized that further CCP domain proteins are secreted by the parasite to interact with the host. We identified an unusually large number of CCP domain-containing proteins in the genome of Hpb, and cloned a range of these for screening in an Avidity-based Extracellular Interaction Screening (AVEXIS) assay, focussing on interactions with host immune proteins. This screen confirmed the binding of known immunomodulators (HpBARI, TGM1) for their targets (ST2, TGFBR2) and identified a new interaction between a 2 CCP domain Hpb protein and mouse resistin-like molecule beta (RELM{beta}), a host protein demonstrated to have anti-helminth properties. This protein was named Binder of RELM{beta} (HpBoRB). This interaction was confirmed in ELISA, competition assays, size exclusion chromatography and surface plasmon resonance experiments, identifying a subnanomolar affinity interaction between HpBoRB and RELM{beta}. These data may indicate that Hpb interferes with the potent anti-helminth host protein RELM{beta} and adds to our knowledge of the host-parasite interactions mediated by Hpb secreted proteins.

immunology↗

bioRxiv2025

RECUR: Identifying recurrent amino acid substitutions from multiple sequence alignments

Identifying recurrent changes in biological sequences is important to multiple aspects of biological research -from understanding the molecular basis of convergent phenotypes, to pinpointing the causative sequence changes that give rise to antibiotic resistance and disease. Here, we present RECUR, a method for identifying recurrent amino acid substitutions from multiple sequence alignments that is fast, easy to use, and scalable to thousands of sequences. We demonstrate the utility and performance characteristics of RECUR on a data set of surface glycoprotein (S) protein sequences from SARS-CoV-2 - identifying widespread recurrent evolution throughout the protein. Structural analysis of the recurrently evolving sites revealed significant enrichment in the exposed receptor-binding S1 subunit and at the interface with the human angiotensin-converting enzyme 2 (hACE2), whereas recurrent substitutions were depleted at the trimeric interface of the S protein. Finally, in silico modelling showed that recurrent substitutions have primarily acted to stabilise the trimeric interface, but had no consistent effect at the hACE2 interface, suggesting that evolution at these sites has been shaped by opposing selection pressures - balancing the need to maintain or enhance hACE2 binding with pressures to diversify and evade host immune responses. A standalone implementation of the algorithm is available under the GPLv3 licence at https://github.com/OrthoFinder/RECUR.

bioinformatics↗

bioRxiv2025

Leaky evidence accumulation accounts for perceptual confidence and subjective duration

Perceptual consciousness is defined as the subjective experience associated with the processing of sensory cues from the environment. Subjective experience unfolds over time and is accompanied by a sense of confidence, yet the mechanisms underlying these two properties remain elusive. Here, we propose a computational mechanism that accounts not only for the onset of subjective experience but also its subjective duration and associated feeling of confidence. Our model assumes that a percept becomes conscious when an ongoing, leaky accumulation of sensory evidence surpasses a perceptual threshold and stops being conscious when it falls back below the threshold due to leakage. Crucially, this perceptual threshold can be lower than the decision threshold for reporting the percept. Moreover, our model derives confidence in accurately detecting sensory evidence from the maximum reached by the evidence accumulation process following stimulus onset. We conducted a preregistered study in which three distinct models of evidence accumulation were fitted to behavioral reports of detection, confidence, and subjective duration during a face detection task under temporal uncertainty. The leaky evidence accumulation model accounted for the observed behavioral data best and outperformed alternative models without leakage. In a follow-up experiment, we investigated the impact of leakage adaptation on detection and subjective duration. We found that changes in leakage induced by contextual variations of face duration influenced both detection rates and subjective duration, as predicted by our model. Altogether, our findings suggest that leaky evidence accumulation is a suitable candidate mechanism to explain qualitative aspects of subjective experience, including subjective duration and feelings of confidence.

neuroscience↗

bioRxiv2025

Elevated body temperature exacerbates arrhythmia and seizure-like activity in a zebrafish model of Timothy syndrome

Timothy syndrome (TS) is a multisystem disorder with autistic-like features, seizures and arrhythmia as the main symptoms. Most TS cases are caused by a de novo single amino acid substitution G406R in the CACNA1C gene that encodes the pore-forming subunit of the voltage-gated L-type calcium channel CaV1.2. We generated a zebrafish model of TS with a homologous amino acid substitution in the cacna1c-encoded protein. Unlike patients, heterozygous mutants showed only mild impairments with no changes in mortality. However, homozygous mutants showed increased mortality, arrhythmias, neural activity and sensitivity to pentylenetetrazole-induced seizure-like behavior. Mutants also exhibited microcephaly, cerebellar hypotrophy and abnormal development of GABAergic neuron populations, and transcriptomic analysis revealed dysregulated expression of neuropeptide genes (including bdnf and vgf). Consistent with the idea that CaV1.2 channels activate during fever, we found that heterozygous larvae manifested arrhythmia and seizure-like behavior when exposed to elevated environmental temperature, and homozygous larvae switched from bradycardia to tachycardia. These data provide a basis for using zebrafish to study the etiology of TS abnormalities and suggest t hat fever may be a particular risk for TS patients.

genetics↗

bioRxiv2024

Vaccine-induced and hybrid immunity against SARS-CoV-2 variants of concern in two cohorts in Queensland, Australia (2021-2022).

The spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for vaccine development, as antibodies generated against the spike protein are the most immunodominant and neutralizing against the virus. However, variants of concern (VOC), often containing multiple mutations within neutralizing epitopes, confer immune evasion of the response generated by current SARS-CoV-2 vaccines. To assess the immunogenicity and virus-neutralisation ability of antibodies induced by individual or combination of Vaxzevria (AstraZeneca: AZ) and Comirnaty (Pfizer: PZ) vaccines and of a hybrid immunity induced in people who were also infected with SARS-CoV-2, longitudinal sera samples of participants recruited through the David Serisier Research biobank (Mater Research Hospital) or at the University of Queensland were collected. ELISA with a panel of purified Spike proteins from ancestral, alpha, delta and omicron BA.1 and BA.2 VOCs showed significantly (by [~]2 to 6-fold) reduced IgG antibody titres against Spike proteins from Omicron BA.1 and BA.2 compared to ancestral strain regardless of the number of vaccinations or presence of infection. Neutralisation assays showed reduced activity against delta and omicron BA.1, BA.5 and BA.5 VOCs. However, the differences were in general less pronounced than in the ELISA assay and some were not statistically significant, particularly after four (two AZ and two PZ) vaccinations. We also generated by circular polymerase extension reaction an attenuated SARS-CoV-2 strain with deletion of all accessory genes, ORF 3, 6, 7 and 8, based on the ancestral (QLD02) virus backbone (QLD02{Delta}3678) and validated it in virus-neutralization assays with our panel of sera samples. We showed the attenuation of the QLD02{Delta}3678 virus in Vero E6 and human Caco2 cells. We demonstrated that neutralization assays with the wild-type QLD02 virus and QLD02{Delta}3678 virus were concordant, providing a safe platform for neutralisation assays in BSL2/PC2 settings.

microbiology↗

bioRxiv2024

Design of an Artificial Natural Killer Cell Mimicking System to Target Tumour Cells

NK cell mimics are assemblies of a cell membrane and a template that replicate biomimetic features and physicochemical properties, respectively. For the reported design, we used the cell membrane from human NK cell (KHYG-1) line and gelatin microspheres as a template. The gelatin microspheres were reinforced via DMTMM cross-linking in a water-in-oil emulsion to exhibit tunable Youngs modulus. These engineered NK cell mimics were found to be non-toxic, non-inflammatory, and capable of evading macrophage detection when tested with differentiated THP-1 cells. In vitro studies showed significant interaction/proximity of the mimics with cancer cells when tested in 2D cultures of breast cancer cells (MDA-MB-231), 3D spheroids of liver (HepG2) and colon (HT-29) cancer cell models, and a zebrafish breast cancer xenograft (MDA-MB-231) model. The NK cell mimics also evaded macrophage detection in a Kdrl:EGFP Spil: Ds Red zebrafish model. In a pilot assessment, loading and release of the sialyltransferase inhibitor (STI, 3Fax-Peracetyl Neu5Ac) using NK cell mimics significantly reduced -2,6 sialylation in 2D cultures of MDA-MB-231 cells, demonstrating the STIs intact functionality in inhibiting sialylation. These findings collectively underscore the promising potential of engineered NK cell mimics as versatile tools in cancer research and therapeutic applications.

bioengineering↗

bioRxiv2024

Metabolic engineering of stomatal precursor cells enhances photosynthetic water-use efficiency and vegetative growth under water-deficit conditions in Arabidopsis thaliana

Stomata are epidermal pores that control the exchange of gaseous CO2 and H2O between plants and their environment. Modulating stomatal density can alter this exchange, and thus presents a target for engineering improved crop productivity and climate resilience. Here, we show that stomatal density in Arabidopsis thaliana can be decreased by the expression of a water-forming NAD(P)H oxidase targeted to stomatal precursor cells. We demonstrate that this reduction in stomatal density occurs irrespective of whether the expressed enzyme is targeted to the cytosol, chloroplast stroma, or chloroplast intermembrane space of these cells. We reveal that this decrease in stomatal density occurs in the absence of any measurable impact on stomatal dynamics, or the efficiency or thermal sensitivity of photosynthesis. Consequently, overexpression plants exhibit a higher intrinsic water use efficiency due to an increase in CO2 fixed per unit water transpired. Finally, we demonstrate that this enhanced water-use efficiency translates to an improvement in vegetative growth and biomass accumulation under water-deficit conditions. Together, these results thus provide a novel approach for enhancing plant productivity through metabolic engineering of stomatal density.

plant biology↗

bioRxiv2024

Functional capacities drive recruitment of bacteria into plant root microbiota

Host-associated microbiota follow predictable assembly patterns but show significant variation at the bacterial isolate level depending on the host and environmental context. This variability poses challenges for studying, predicting, and engineering microbiomes. Here we examined how Arabidopsis, Barley, and Lotus plants recruit specific bacteria from highly complex synthetic communities (SynComs) composed of hundreds of bacterial isolates originating from these plants when grown in natural soil. We discovered that, despite their taxonomic diversity, bacteria enriched by these three plant species encode largely overlapping functions. A set of 266 functions common among all host-associated communities was identified at the foundation of the microbiotas functional potential. Analysis of the differences observed between root-associated communities revealed that functions recruited by Arabidopsis and Barley were primarily driven by the SynCom composition, while Lotus selected fewer isolates but with more diverse functionalities, akin to a Swiss army knife strategy. We analysed the variation at the functional level and found this can be explained by the combined functions of bacteria at the family level. Additionally, across major taxa, the isolates covering a broader range of their familys functional diversity achieved higher relative abundance in the root communities. Our work sheds light on key functions and principles guiding the recruitment of bacterial isolates into root microbiota, offering valuable insights for microbiome engineering and inoculant discovery at a previously inaccessible taxonomic level.

plant biology↗

bioRxiv2024

Increased chloroplast area in the rice bundle sheath through cell specific perturbation of brassinosteroid signalling

In the leaves of C3 species such as rice, mesophyll cells contain the largest compartment of photosynthetically active chloroplasts. In contrast, plants that use the derived and more efficient C4 photosynthetic pathway have a significant chloroplast compartment in both bundle sheath and mesophyll cells. Accordingly, the evolution of C4 photosynthesis from the ancestral C3 state requires an increase in the chloroplast compartment of the bundle sheath. Here we investigated the potential to increase chloroplast compartment size in rice bundle sheath cells by manipulating brassinosteroid signalling. Treatment with brassinazole, a brassinosteroid biosynthesis inhibitor, increased leaf chlorophyll content and increased the number but decreased the area of chloroplasts in bundle sheath cells. Constitutive overexpression of the BRASSINAZOLE RESISTANT 1 (OsBZR1) transcription factor increased bundle sheath chloroplast area by up to 45% but plants became chlorotic. However, when OsBZR1 was placed under the control of a bundle sheath specific promoter, the negative effects on growth and viability were removed whilst chloroplast area still increased. In summary, we report a role for brassinosteroids in controlling chloroplast area and number in rice and conclude that cell-specific manipulation of brassinosteroid signalling can be used to manipulate the chloroplast compartment in rice bundle sheath cells.

plant biology↗

bioRxiv2024

INCREASED CHLOROPLAST OCCUPANCY IN BUNDLE SHEATH CELLS OF RICE hap3H MUTANTS REVEALED BY CHLORO-COUNT, A NEW DEEP LEARNING-BASED TOOL

There is an increasing demand to boost photosynthesis in rice to increase yield potential. Chloroplasts are the site of photosynthesis, and increasing the number and size of these organelles in the in leaf is a potential route to elevate leaf-level photosynthetic activity. Notably, bundle sheath cells do not make a significant contribution to overall carbon fixation in rice and thus various attempts are being made to increase chloroplast content in this cell type. In this study we developed and applied a deep learning tool named Chloro-Count to demonstrate that loss of OsHAP3H function in rice increases chloroplast occupancy in bundle sheath cells by 50%. Although limited to a single season, when grown in the field Oshap3H mutants exhibited increased numbers of tillers and panicles as compared to controls or gain of function mutants. The implementation of Chloro-Count enabled precise quantification of chloroplasts in loss- and gain-of-function OsHAP3H mutants and facilitated a comparison between 2D and 3D quantification methods. In wild-type rice, as the dimensions of bundle sheath cells increase, the volume of individual chloroplasts also increases. However, the larger the chloroplasts the fewer there are per bundle sheath cell. This observation revealed that a mechanism operates in bundle sheath cells to restrict chloroplast occupancy as cell dimensions increase. That mechanism is unperturbed in Oshap3H mutants. The use of Chloro-Count also revealed that 2D quantification, upon which most previous studies have relied, is compromised by the positioning of chloroplasts within the cell. Chloro-Count is therefore a valuable tool for accurate and high-throughput quantification of chloroplasts that has enabled the robust characterization of OsHAP3H effects on chloroplast biogenesis in rice. Whereas previous studies have increased chloroplast occupancy in bundle sheath cells by increasing the size of individual chloroplasts, loss of OsHAP3H function leads to an increase in chloroplast numbers.

plant biology↗