Biology subjects

Field, B.

Publications and source records attributed to Field, B..

13 recordsLinked to original sources

bioRxiv2026

Alarmone ((p)ppGpp) signalling tunes Arabidopsis thaliana nuclear gene expression to shape the balance of plant immune outcomes

RSH enzymes (RelA/SpoT homologs) synthesize ppGpp (guanosine tetra-/pentaphosphate) in plastids, regulating organelle function. More importantly, whether ppGpp acts as a simple rheostat on immune output or determines which mode of defence a plant deploys remains poorly understood. Using Arabidopsis thaliana lines with high (RSH3OX) or null (rshq) ppGpp levels, we show that ppGpp controls nuclear-encoded defence genes in salicylic acid (SA) and jasmonic acid (JA) metabolism, including hormone-inactivating enzymes in RSH3OX and MeSA-to-SA conversion genes in rshq. Conversely, pathogen infection induces the ppGpp synthases RSH2 and RSH3, with induction preceding defence gene activation and requiring SA biosynthesis and a functional Type III secretion system. RSH3OX plants are hypersusceptible to Pseudomonas syringae pv. tomato DC3000, show impaired pattern-triggered immunity, and exhibit an accelerated decline in photosynthetic efficiency, while rshq plants show enhanced resistance to P. syringae but increased susceptibility to the necrotrophic fungus Botrytis cinerea. Using an inducible line to acutely elevate ppGpp, we confirm this susceptibility is directly caused by ppGpp levels, not a chronic developmental consequence of RSH3OX. Together, these results indicate that ppGpp governs the mode of immune execution rather than simply scaling its magnitude, acting as a chloroplast-based checkpoint linking organellar status to the nuclear defence transcriptome.

plant biology↗

bioRxiv2025

Guanosine tetraphosphate (ppGpp) signalling promotes high-light tolerance in Arabidopsis thaliana.

Guanosine tetraphosphate (ppGpp) is a hyperphosphorylated nucleotide originally discovered in prokaryotes and found in the chloroplasts of plants and algae. In plants, ppGpp signalling plays a role as a regulator of photosynthetic activity, which is important in acclimation to environmental stresses such as nitrogen limitation. However, the full range of stresses involving ppGpp signalling is not yet known. Here, we investigated the role of ppGpp accumulation in the acclimation of plants to high light. We found that the over-accumulation of ppGpp in transgenic lines that overexpress RSH3 (OX:RSH3) increases tolerance to high light intensity. Although ppGpp leads to higher non-photochemical energy dissipation (NPQ) than in wild-type plants, we show that NPQ is not critical for the enhanced high-light tolerance. Rather, our results show that ppGpp accumulation leads to broad changes in plant physiology that prime plants to resist and subsequently recover from high light exposure. ppGpp levels themselves also increase in response to high light exposure, suggesting that ppGpp signalling may play a physiological role in high light acclimation. Our work highlights the importance of ppGpp signalling in plant stress acclimation.

plant biology↗

bioRxiv2023

Molecular crosstalk between plant translation initiation complexes influences the outcome of virus infection

Successful subversion of translation initiation factors 4E and 4G determines the infection success of potyviruses, the largest group of viruses affecting plants. In the natural variability of many plant species, resistance to potyvirus infection is provided by polymorphisms at 4E and 4G that renders them inadequate for virus hijacking but still functional in translation initiation. In crops where such natural resistance alleles are limited, the genetic inactivation of 4E has been proposed for the engineering of potyvirus resistance. However, recent findings indicate that knockout 4E and 4G alleles may be deleterious for plant health and could jeopardize resistance efficiency in comparison to functional resistance proteins. Here, we explored the cause of these adverse effects by studying the role of the Arabidopsis eIF4E1, whose inactivation was previously reported as conferring resistance to the potyvirus clover yellow vein virus (ClYVV) while also promoting susceptibility to another potyvirus called turnip mosaic virus (TuMV). We report that eIF4E1 is required to maintain global plant translation and to restrict TuMV accumulation during infection, and its absence is associated with a favoured virus multiplication over host translation. Furthermore, our findings demonstrate that eIF4E1 plays a crucial role in inhibiting the TuMV-induced degradation of the translation initiation factor eIFiso4G1, thereby preventing the generation of a truncated protein. Finally, we demonstrate a role for eIFiso4G1 in TuMV accumulation and in supporting plant fitness during infection. These findings suggest that eIF4E1 counteracts the hijacking of the plant translational apparatus during TuMV infection and underscore the importance of preserving the functionality of translation initiation factors 4E and 4G when implementing potyvirus resistance strategies. Author summaryPlants are constantly under threat from viruses that can damage crops and reduce yield. Among these viruses, potyviruses are a major concern, and a small group of genes known as eIF4E are key factors in making a plant susceptible to them. To combat these viruses, it is possible to either use naturally-selected variants of eIF4E that provide resistance, or to disable the gene altogether. However, new research has shown that inactivating eIF4E genes may have unintended consequences for the plants development while compromise resistance to other potyviruses. To investigate this further, we focus in this work on the role of the Arabidopsis eIF4E1 whose inactivation confers resistance to one potyvirus, clover yellow vein virus (ClYVV). We looked why this same mutation at eIF4E1 makes the plants more susceptible to another potyvirus, turnip mosaic virus (TuMV). Our study reveals that eIF4E1 acts in safeguarding the plant translational machinery during TuMV infection. By preventing the degradation of the translation initiation protein eIFiso4G1, eIF4E1 enables the plant to maintain its normal translation activity and ultimately prevents the accumulation of virus proteins. Our findings provide valuable insights into how potyviruses hijack the plants translation process, and emphasizes the need of preserving the functionality of translation initiation factors when developing potyvirus resistances.

plant biology↗

bioRxiv2023

Post-translational regulation of photosynthetic activity via the TOR kinase in plants.

Chloroplasts are the powerhouse of the plant cell, yet they are resource-intensive and will cause photooxidative damage if their activity overshoots the demands of growth. The adjustment of chloroplast activity to match growth is therefore vital for stress acclimation. Here we identify a novel post-translational mechanism linking the conserved eukaryotic TOR kinase that promotes growth and the guanosine tetraphosphate (ppGpp) signaling pathway of prokaryotic origin that regulates chloroplast activity, and photosynthesis in particular. We show that RelA SpoT Homologue 3 (RSH3), a nuclear-encoded chloroplastic enzyme responsible for ppGpp biosynthesis, interacts directly with the TOR complex via a plant-specific N-terminal region (NTR) which is hyper-phosphorylated in a TOR-dependent manner. Downregulation of TOR activity reduces NTR phosphorylation, enhances ppGpp synthesis by RSH3, and causes a ppGpp-dependent decrease in photosynthetic capacity. Altogether we demonstrate that the TOR-RSH3 signaling axis is a novel and direct post-translational mechanism that allows chloroplast activity to be matched with plant growth, setting a new precedent for the regulation of organellar function by TOR. One sentence summaryThe TOR kinase post-translationally controls guanosine tetraphosphate signaling to regulate plant photosynthetic activity.

plant biology↗

bioRxiv2023

Insights into the function of the chloroplastic ribosome-associated GTPase HflX in Arabidopsis thaliana.

Ribosome-associated GTPases are conserved enzymes that participate in ribosome biogenesis and ribosome function. In bacteria, recent studies have identified HflX as a ribosome-associated GTPase that is involved in both ribosome biogenesis and recycling under stress conditions. Plants possess a chloroplastic HflX homolog, but its function remains unknown. Here, we characterised the role of HflX in the plant Arabidopsis thaliana. Our findings demonstrate that HflX does not have a detectable role in plant growth and development, nor does it play a distinct role in acclimation to several different stresses, including heat, manganese, cold, and salt stress. However, we found that HflX is required for plant resistance to chloroplast translational stress mediated by the antibiotic lincomycin. Our results suggest that HflX is a chloroplast ribosome-associated protein that may play a role in the surveillance of translation. These findings provide new insight into the function of HflX as a ribosome-associated GTPase in plants and highlight the importance of investigating conserved proteins in different organisms to gain a comprehensive understanding of their biological roles.

plant biology↗

bioRxiv2022

Structural Maintenance of Chromosome 3 interacts with the Topoisomerase VI complex and contributes to the oxidative stress response in Arabidopsis thaliana.

In plants adverse environmental conditions can induce the accumulation of reactive oxygen species, such as singlet oxygen or hydrogen peroxide, at the level of the photosynthetic apparatus. The coordinated action of nucleus-encoded genes is required for containing the deleterious effects of reactive oxygen species. The regulation of such genes follows a molecular signalling process between the chloroplast and the nucleus called retrograde signalling. Previously, we proposed that the Topoisomerase VI (Topo VI) complex participates in the singlet oxygen stress response by regulating the expression of specific subsets of nuclear genes. However, the underlying molecular mechanisms remain unresolved. In this study, we demonstrate that the Topo VI subunit BIN4 interacts with the cohesin subunit AtSMC3. We also show that, similarly to Topo VI mutants, a line suppressing AtSMC3 shows constitutive activation of singlet oxygen response genes and enhanced tolerance to photooxidative stress. Together, these results suggest that Topo VI and AtSMC3 control the expression of singlet oxygen response genes and are possibly involved in the acclimation of plants to photooxidative stress conditions.

plant biology↗

bioRxiv2021

A ppGpp-mediated brake on photosynthesis is required for acclimation to nitrogen limitation in Arabidopsis.

Guanosine pentaphosphate and tetraphosphate (together referred to as ppGpp) are hyperphosphorylated nucleotides found in bacteria and the chloroplasts of plants and algae. In plants and algae artificial ppGpp accumulation can inhibit chloroplast gene expression, and influence photosynthesis, nutrient remobilisation, growth, and immunity. However, it is so far unknown whether ppGpp is required for abiotic stress acclimation in plants. Here, we demonstrate that ppGpp biosynthesis is necessary for acclimation to nitrogen starvation in Arabidopsis. We show that ppGpp is required for remodeling the photosynthetic electron transport chain to downregulate photosynthetic activity and for protection against oxidative stress. Furthermore, we demonstrate that ppGpp is required for coupling chloroplastic and nuclear gene expression during nitrogen starvation. Altogether, our work indicates that ppGpp is a pivotal regulator of chloroplast activity for stress acclimation in plants.

plant biology↗

bioRxiv2021

A connectomic study of a petascale fragment of human cerebral cortex

We acquired a rapidly preserved human surgical sample from the temporal lobe of the cerebral cortex. We stained a 1 mm3 volume with heavy metals, embedded it in resin, cut more than 5000 slices at [~]30 nm and imaged these sections using a high-speed multibeam scanning electron microscope. We used computational methods to render the three-dimensional structure containing 57,216 cells, hundreds of millions of neurites and 133.7 million synaptic connections. The 1.4 petabyte electron microscopy volume, the segmented cells, cell parts, blood vessels, myelin, inhibitory and excitatory synapses, and 104 manually proofread cells are available to peruse online. Many interesting and unusual features were evident in this dataset. Glia outnumbered neurons 2:1 and oligodendrocytes were the most common cell type in the volume. Excitatory spiny neurons comprised 69% of the neuronal population, and excitatory synapses also were in the majority (76%). The synaptic drive onto spiny neurons was biased more strongly toward excitation (70%) than was the case for inhibitory interneurons (48%). Despite incompleteness of the automated segmentation caused by split and merge errors, we could automatically generate (and then validate) connections between most of the excitatory and inhibitory neuron types both within and between layers. In studying these neurons we found that deep layer excitatory cell types can be classified into new subsets, based on structural and connectivity differences, and that chandelier interneurons not only innervate excitatory neuron initial segments as previously described, but also each others initial segments. Furthermore, among the thousands of weak connections established on each neuron, there exist rarer highly powerful axonal inputs that establish multi-synaptic contacts (up to [~]20 synapses) with target neurons. Our analysis indicates that these strong inputs are specific, and allow small numbers of axons to have an outsized role in the activity of some of their postsynaptic partners.

neuroscience↗

bioRxiv2021

MoBiFC: development of a modular bimolecular fluorescence complementation toolkit for the analysis of chloroplast protein-protein interactions.

The bimolecular fluorescence complementation (BiFC) assay has emerged as one of the most popular methods for analysing protein-protein interactions (PPIs) in plant biology. This includes its increasing use as a tool for dissecting the molecular mechanisms of chloroplast function. However, the construction of chloroplast fusion proteins for BiFC can be difficult, and the availability and selection of appropriate controls is not trivial. Furthermore, the challenges of performing BiFC in restricted cellular compartments has not been specifically addressed. Here we describe the development of a flexible modular cloning-based toolkit (MoBiFC) for chloroplast BiFC and proximity labelling using synthetic biology principles. The approach facilitates the cloning process for chloroplast-targeted proteins, allows robust ratiometric quantification, and the toolkit comes with model positive and negative controls. Our study also highlights many potential pitfalls including the choice of fluorescent protein (FP) split, negative controls, cell type, and reference FP. Finally, we provide an example of how users can enrich the toolset by providing functional proximity labelling modules, and we discuss how MoBiFC could be further improved and extended to other compartments of the plant cell.

plant biology↗

bioRxiv2021

Guanosine tetraphosphate (ppGpp) accumulation inhibits chloroplast gene expression and promotes super grana formation in the moss Physcomitrium (Physcomitrella) patens

The nucleotides guanosine tetraphosphate and pentaphosphate (or ppGpp) are implicated in the regulation of chloroplast function in plants. ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana where it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little is known about the conservation or diversity of ppGpp signaling in other land plants. Here, we studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth. We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis. Our findings point to the existence of both highly conserved and more specific targets of ppGpp signalling in the land plants that may reflect different growth strategies.

plant biology↗

bioRxiv2020

ppGpp influences protein protection, growth and photosynthesis in Phaeodactylum tricornutum

O_LIChloroplasts retain elements of a bacterial stress response pathway that is mediated by the signalling nucleotides guanosine penta- and tetraphosphate, or (p)ppGpp. In the model flowering plant Arabidopsis, ppGpp acts as a potent regulator of plastid gene expression and influences photosynthesis, plant growth and development. However, little is known about ppGpp metabolism or its evolution in other photosynthetic eukaryotes. C_LIO_LIHere, we studied the function of ppGpp in the diatom P. tricornutum using transgenic lines containing an inducible system for ppGpp accumulation. We used these lines to investigate the effects of ppGpp on growth, photosynthesis, lipid metabolism and protein expression. C_LIO_LIWe demonstrate that ppGpp accumulation reduces photosynthetic capacity and promotes a quiescent-like state with reduced proliferation and ageing. Strikingly, using non-targeted proteomics, we discovered that ppGpp accumulation also leads to the coordinated upregulation of a protein protection response in multiple cellular compartments. C_LIO_LIOur findings highlight the importance of ppGpp as a fundamental regulator of chloroplast function across different domains of life, and lead to new questions about the molecular mechanisms and roles of (p)ppGpp signalling in photosynthetic eukaryotes. C_LI

plant biology↗

bioRxiv2019

Quantification of guanosine tetraphosphate and other nucleotides in plants and algae using stable isotope-labelled internal standards.

Guanosine tetraphosphate (G4P) and guanosine pentaphosphate (G5P) are signalling nucleotides found in bacteria and photosynthetic eukaryotes that are implicated in a wide-range of processes including stress acclimation, developmental transitions and growth control. Measurements of G4P/G5P levels are essential for studying the diverse roles of these nucleotides. However, G4P/G5P quantification is particularly challenging in plants and algae due to lower cellular concentrations, compartmentation and high metabolic complexity. Despite recent advances the speed and accuracy of G4P quantification in plants and algae can still be improved. Here, we report a new approach for rapid and accurate G4P quantification, applicable to plants and algae, which relies on the use of synthesised stable isotope-labelled as internal standards. We anticipate that this approach will accelerate research into the function of G4P signaling in plants, algae and other organisms.

plant biology↗

bioRxiv2018

Golden magic: RSH enzymes for (p)ppGpp metabolism in the diatom Phaeodactylum tricornutum.

The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the golden-coloured marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae.\n\nHighlightWe discover RSH enzymes for (p)ppGpp metabolism in the diatom Phaeodactylum tricornutum and show that they have surprising functional and structural features, and belong to novel red-plastid lineage RSH clades.

plant biology↗