Genetically-encoded fluorescence-based sensors have emerged as an essential tool for measuring the abundance of heme, revealing its trafficking pathways, and probing its signalling and regulatory role in cells. A number of different sensor designs have been described in the literature, and these typically report on the abundance of exchangeable heme via an intensity modulation of the emission from fluorescent-protein reporters. Here, we show that multi-photon fluorescence-lifetime imaging microscopy (MP-FLIM) can be used to monitor the response of heme sensors in transfected-HEK293 cells. The adoption of a multi-photon approach could extend heme quantification further to deep-tissue imaging in the future, where it could also reduce phototoxicity as the non-linear excitation of fluorescent reporters is confined to the focal volume.

Blood feeding (hematophagy) is widespread across Diptera (true flies), yet the underlying genetic basis remains poorly understood. Using phylogenomics, we show that four gene families associated with neuromodulation, immune responses, embryonic development, and iron metabolism have undergone independent expansions within mosquitoes and sandflies. Our findings illuminate the underlying genetic basis for blood-feeding adaptations in these important disease vectors.

Although hormone biology is critical for plant breeding, complex phenotypic outputs make it difficult to compare across species. We used transient expression of hormone biosensors and transcriptomics to simplify this output and quantify gibberellic acid (GA) and auxin responses across multiple cereal crop genotypes and tissues. We show the GPS2 biosensor detects exogenous GA in maize, barley, sorghum, and wheat. Measuring across GA dosages, we detect tissue- and genotype-specific differences in GA sensor response with an unexpected drop in GPS2 output in the maize d1 GA biosynthesis mutant after GA treatment, likely reflecting differences in GA response across samples. We used RNA sequencing followed by ortholog prediction and Gene Ontology-term enrichment analysis to measure GA responses in leaves and floral tissues from maize wildtype, d1, and barley Golden Promise. We determine that cross-tissue, cross-genotype, and cross-species GA responses include downregulation of GA-INSENSITIVE DWARF1 and upregulation of α-Expansin1, independent of GA biosynthesis. We identify F-Box proteins, hexokinase, and AMPK/SNF1 protein kinase orthologs as unexpected cross-species GA-responsive genes. We then compared transient expression of DR5, DR5v2, and DII-mDII auxin reporters in barley and maize and find DR5v2 and DII-mDII are functional auxin reporters in both species.

Animals have endogenous timekeeping mechanisms to measure time and adjust their physiology to cyclical environmental changes. These biological clocks rely on neural networks to orchestrate circadian rhythmicity. Some clock neurons undergo a daily remodeling of their morphology-known as circadian structural plasticity-that is expected to impact their connectivity and function. This process remains poorly defined at the subcellular level, preventing a full understanding of how it relates to other cyclical properties. In this study, we generated 3D electron microscopy reconstructions of adult Drosophila brains at three key time points to examine ultrastructural changes in the terminals of core clock neurons. We found that neuronal varicosities are the organizational units underlying these structural changes, as they contain the functional elements required for neuronal communication (active zones and dense-core vesicles) and for their metabolic support (mitochondria). Varicosities appear to change in number during the day-night cycle while exhibiting differences in the number of active zones, in the accumulation of dense-core vesicles, both full and fused, and in the number, shape, and size of the mitochondria. These results suggest an interplay between structural and functional plasticity that was not appreciated to date. We propose that circadian plasticity of presynaptic varicosities modulates the influence of specific clock neurons onto the circadian network. Given the conservation across timekeeping mechanisms, ultrastructural changes might underlie circadian shifts in neuronal connectivity across species.

Coumarin is a bioactive molecule that impacts various plant developmental processes, including germination, root growth and shoot elongation. Despite the long-standing awareness of coumarin’s effect on plant growth, its mode of action remains elusive. We investigated the capacity of coumarin to enhance hypocotyl elongation in light-grown Arabidopsis seedlings, demonstrating that this effect is mediated by the stimulation of cell elongation rather than cell division. Functional analyses revealed that the promotion of cell elongation occurs independently of ethylene and gibberellic acid, while emphasizing the critical roles of auxin and brassinosteroids in this process. The auxin dependency was evidenced by the reduced response to coumarin treatment in both the tir1-1/afb2-1/afb3-1 triple and slr-1 gain-of-function mutant. The persistence of coumarin’s effects in the presence of yucasin suggests that de novo auxin biosynthesis is not essential for its activity. In contrast, brassinosteroid biosynthesis is critical as coumarin failed to restore the stunted hypocotyl phenotype when brassinosteroid biosynthesis was inhibited by propiconazole. Furthermore, the brassinosteroid receptor mutant bri1-116 exhibited insensitivity to coumarin and the bak1-4 signalling mutant showed a markedly diminished response, underscoring the necessity of both brassinosteroids and a functional brassinosteroid signalling pathway for coumarin-induced hypocotyl elongation.

Plant bodies are built from immobile cells, making the regulation of cell expansion essential for growth, development, and adaptation. In roots, cell elongation executes the movement of the root tips through the soil. This process is tightly controlled by numerous signaling pathways. Among these, gibberellin and auxin signaling stand out for their contrasting effects on root growth, interacting through complex cross talk at multiple regulatory levels. Here, we reveal the molecular basis of the auxin-gibberellin cross talk in the model plant Arabidopsis thaliana. We show that the auxin signaling pathway steers the expression of GIBBERELLIN 2-OXIDASES (GA2OXs), key gibberellin-deactivating enzymes in the root elongation zone (EZ). GA2OXs are negative regulators of root cell elongation; GA2OX8 overexpression decreases gibberellin levels and inhibits root cell elongation; in contrast, the ga2ox heptuple mutant roots show elevated gibberellin levels in the EZ and grow longer roots. Intriguingly, shoot-derived auxin can regulate GA2OX6 and GA2OX8 expression in roots, linking systemic auxin signaling to local gibberellin level modulation. Together, our findings identify GA2OX6 and GA2OX8 enzymes as key mediators of auxin-gibberellin cross talk, providing insights into their roles in root elongation. These results expand our understanding of how auxin integrates with gibberellin signaling to coordinate root development and growth dynamics.

The nuclear TIR1/AFB-Aux/IAA auxin pathway plays a crucial role in regulating plant growth and development. Specifically, the IAA17/AXR3 protein participates in Arabidopsis thaliana root development, response to auxin and gravitropism. However, the mechanism by which AXR3 regulates cell elongation is not fully understood. We combined genetical and cell biological tools with transcriptomics and determination of auxin levels and employed live cell imaging and image analysis to address how the auxin response pathways influence the dynamics of root growth. We revealed that manipulations of the TIR1/AFB-Aux/IAA pathway rapidly modulate root cell elongation. While inducible overexpression of the AXR3-1 transcriptional inhibitor accelerated growth, overexpression of the dominant activator form of ARF5/MONOPTEROS inhibited growth. In parallel, AXR3-1 expression caused loss of auxin sensitivity, leading to transcriptional reprogramming, phytohormone signaling imbalance and increased levels of auxin. Furthermore, we demonstrated that AXR3-1 specifically perturbs nuclear auxin signaling, while the rapid auxin response remains functional. Our results shed light on the interplay between the nuclear and cytoplasmic auxin pathways in roots, revealing their partial independence but also the dominant role of the nuclear auxin pathway during the gravitropic response of Arabidopsis thaliana roots.

Arthropod herbivory poses a serious threat to crop yield, prompting plants to employ intricate defense mechanisms against pest feeding. The generalist pest 2-spotted spider mite (Tetranychus urticae) inflicts rapid damage and remains challenging due to its broad target range. In this study, we explored the Arabidopsis (Arabidopsis thaliana) response to T. urticae infestation, revealing the induction of abscisic acid (ABA), a hormone typically associated with abiotic stress adaptation, and stomatal closure during water stress. Leveraging a Forster resonance energy transfer (FRET)-based ABA biosensor (nlsABACUS2-400n), we observed elevated ABA levels in various leaf cell types postmite feeding. While ABA's role in pest resistance or susceptibility has been debated, an ABA-deficient mutant exhibited increased mite infestation alongside intact canonical biotic stress signaling, indicating an independent function of ABA in mite defense. We established that ABA-triggered stomatal closure effectively hinders mite feeding and minimizes leaf cell damage through genetic and pharmacological interventions targeting ABA levels, ABA signaling, stomatal aperture, and density. This study underscores the critical interplay between biotic and abiotic stresses in plants, highlighting how the vulnerability to mite infestation arising from open stomata, crucial for transpiration and photosynthesis, reinforces the intricate relationship between these stress types.

Fatalska et al.1 use an interdisciplinary strategy to elucidate how an intrinsically disordered regulatory subunit of protein phosphatase 1 binds trimeric eIF2 and positions the phosphatase-substrate complex for dephosphorylation. As validation, they show that a disease mutation abolishes the interaction.

The phytohormone gibberellic acid (GA) is critical for environmentally sensitive plant development including germination, skotomorphogenesis, and flowering. The Förster resonance energy transfer biosensor GIBBERELLIN PERCEPTION SENSOR1, which permits single-cell GA measurements in vivo, has been used to observe a GA gradient correlated with cell length in dark-grown, but not light-grown, hypocotyls. We sought to understand how light signaling integrates into cellular GA regulation. Here, we show how the E3 ligase CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) and transcription factor ELONGATED HYPOCOTYL 5 (HY5) play central roles in directing cellular GA distribution in skoto- and photomorphogenic hypocotyls, respectively. We demonstrate that the expression pattern of the GA biosynthetic enzyme gene GA20ox1 is the key determinant of the GA gradient in dark-grown hypocotyls and is a target of COP1 signaling. We engineered a second generation GPS2 biosensor with improved orthogonality and reversibility. GPS2 revealed a previously undetectable cellular pattern of GA depletion during the transition to growth in the light. This GA depletion partly explains the resetting of hypocotyl growth dynamics during photomorphogenesis. Achieving cell-level resolution has revealed how GA distributions link environmental conditions with morphology and morphological plasticity. The GPS2 biosensor is an ideal tool for GA studies in many conditions, organs, and plant species.

Differential growth is central to eukaryotic morphogenesis. We showed using cellular imaging, simulations, and perturbations that light-induced differential growth in a curved organ, the Arabidopsis thaliana apical hook, emerges from the longitudinal expansion of subepidermal cells, acting in parallel with a differential in the material properties of epidermal cell walls that resist expansion. The greater expansion of inner hook cells that results in apical hook opening is gated by wall alkalinity and auxin, both of which are depleted upon illumination. We further identified mechanochemical feedback from wall mechanics to light stimulated auxin depletion, which may contribute to gating hook opening under mechanical restraint. These results highlight how plant cells coordinate growth among tissue layers by linking mechanics and hormonal gradients with the cell wall remodeling required for differential growth.

Formation of the apical hook in etiolated dicot seedlings results from differential growth in the hypocotyl apex and is tightly controlled by environmental cues and hormones, among which auxin and gibberellins (GAs) play an important role. Cell expansion is tightly regulated by the cell wall, but whether and how feedback from this structure contributes to hook development are still unclear. Here, we show that etiolated seedlings of the Arabidopsis (Arabidopsis thaliana) quasimodo2-1 (qua2) mutant, defective in pectin biosynthesis, display severe defects in apical hook formation and maintenance, accompanied by loss of asymmetric auxin maxima and differential cell expansion. Moreover, qua2 seedlings show reduced expression of HOOKLESS1 (HLS1) and PHYTOCHROME INTERACTING FACTOR4 (PIF4), which are positive regulators of hook formation. Treatment of wild-type seedlings with the cellulose inhibitor isoxaben (isx) also prevents hook development and represses HLS1 and PIF4 expression. Exogenous GAs, loss of DELLA proteins, or HLS1 overexpression partially restore hook development in qua2 and isx-treated seedlings. Interestingly, increased agar concentration in the medium restores, both in qua2 and isx-treated seedlings, hook formation, asymmetric auxin maxima, and PIF4 and HLS1 expression. Analyses of plants expressing a Förster resonance energy transfer-based GA sensor indicate that isx reduces accumulation of GAs in the apical hook region in a turgor-dependent manner. Lack of the cell wall integrity sensor THESEUS 1, which modulates turgor loss point, restores hook formation in qua2 and isx-treated seedlings. We propose that turgor-dependent signals link changes in cell wall integrity to the PIF4-HLS1 signaling module to control differential cell elongation during hook formation.

During nutrient scarcity, plants can adapt their developmental strategy to maximize their chance of survival. Such plasticity in development is underpinned by hormonal regulation, which mediates the relationship between environmental cues and developmental outputs. In legumes, endosymbiosis with nitrogen-fixing bacteria (rhizobia) is a key adaptation for supplying the plant with nitrogen in the form of ammonium. Rhizobia are housed in lateral root-derived organs termed nodules that maintain an environment conducive to Nitrogenase in these bacteria. Several phytohormones are important for regulating the formation of nodules, with both positive and negative roles proposed for gibberellin (GA). In this study, we determine the cellular location and function of bioactive GA during nodule organogenesis using a genetically encoded second-generation GA biosensor, GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula. We find endogenous bioactive GA accumulates locally at the site of nodule primordia, increasing dramatically in the cortical cell layers, persisting through cell divisions, and maintaining accumulation in the mature nodule meristem. We show, through misexpression of GA-catabolic enzymes that suppress GA accumulation, that GA acts as a positive regulator of nodule growth and development. Furthermore, increasing or decreasing GA through perturbation of biosynthesis gene expression can increase or decrease the size of nodules, respectively. This is unique from lateral root formation, a developmental program that shares common organogenesis regulators. We link GA to a wider gene regulatory program by showing that nodule-identity genes induce and sustain GA accumulation necessary for proper nodule formation.

Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signalling biosensor by engineering one of the DELLA proteins, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this novel degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signalling activity in the SAM. We show that high GA signalling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organisation of internodes, thus contributing to internode specification in the SAM.

Mosquitoes are the deadliest vectors of diseases. They impose a huge health burden on human populations spreading parasites as disparate as protozoans (malaria), viruses (yellow fever and more) and nematodes (filariasis) that cause life-threatening conditions. In recent years, mating has been proposed as a putative target for population control. Mosquitoes mate mid-air, in swarms initiated by males and triggered by a combination of internal and external stimuli. As the number of females in a swarm is limited, there is intense competition among males, and they ‘retune’ their physiology for this demanding behaviour. There is limited knowledge on the ‘genetic reprogramming’ required to enable swarming. Interestingly, recent evidence indicates that the upregulation of circadian clock genes may be involved in the swarming of malaria mosquitoes of the genus Anopheles . Here, we use whole-head RNA-seq to identify gene expression changes in Aedes aegypti males that are engaged in swarming in a laboratory setting. Our results suggest that in preparation to swarming, males tend to lower some housekeeping functions while increasing remodelling of the cytoskeleton and neuronal connectivity; the transcription of circadian clock genes is unaffected.

Arthropod herbivory possess a significant threat to crop yield, prompting plants to employ intricate defense mechanisms against pest feeding. The generalist pest, Tetranychus urticae , inflicts rapid damage and remains a challenge due to its broad target range. In this study, we explored Arabidopsis thaliana’s response to T. urticae infestation, revealing the induction of abscisic acid (ABA), a hormone typically associated with abiotic stress adaptation, including stomatal closure during water stress. Leveraging a FRET-based ABA biosensor (nlsABACUS2-400n), we observed elevated ABA levels in various leaf cell types post-mite feeding. While ABA’s role in pest resistance or susceptibility has been debated, an ABA-deficient mutant exhibited increased mite infestation, alongside intact canonical biotic stress signalling, indicating an independent function of ABA in mite defense. Through genetic and pharmacological interventions targeting ABA levels, ABA signalling, stomatal aperture, and density, we established that ABA-triggered stomatal closure effectively hinders mite feeding and minimizes leaf cell damage. This study underscores the critical interplay between biotic and abiotic stresses in plants, highlighting how the vulnerability to mite infestation arising from open stomata, crucial for transpiration and photosynthesis, underscores the intricate relationship between these two stress types.

Serine/arginine-rich (SR) proteins are conserved splicing regulators that play important roles in plant stress responses, namely those mediated by the abscisic acid (ABA) hormone. The Arabidopsis thaliana SR-like protein SR45 is a described negative regulator of the ABA pathway during early seedling development. How the inhibition of growth by ABA signaling is counteracted to maintain plant development under stress conditions remains largely unknown. Here, we show that SR45 overexpression reduces Arabidopsis sensitivity to ABA during early seedling development. Biochemical and confocal microscopy analyses of transgenic plants expressing fluorescently tagged SR45 revealed that exposure to ABA dephosphorylates the protein at multiple amino acid residues and leads to its accumulation, due to SR45 stabilization via reduced ubiquitination and proteasomal degradation. Using phosphomutant and phosphomimetic transgenic Arabidopsis lines, we demonstrate the functional relevance of ABA-mediated dephosphorylation of a single SR45 residue, T264, in antagonizing SR45 ubiquitination and degradation to promote its function as a repressor of seedling ABA sensitivity. Our results reveal a mechanism that negatively autoregulates ABA signaling and allows early plant growth under stress via posttranslational control of the SR45 splicing factor.

EDITORIAL article Front. Mol. Biosci., 17 January 2023Sec. Structural Biology Volume 10 - 2023 | https://doi.org/10.3389/fmolb.2023.1125791

Many animals use Earth’s magnetic field (also known as the geomagnetic field) for navigation 1 . The favoured mechanism for magnetosensitivity involves a blue-light-activated electron-transfer reaction between flavin adenine dinucleotide (FAD) and a chain of tryptophan residues within the photoreceptor protein CRYPTOCHROME (CRY). The spin-state of the resultant radical pair, and therefore the concentration of CRY in its active state, is influenced by the geomagnetic field 2 . However, the canonical CRY-centric radical-pair mechanism does not explain many physiological and behavioural observations 2 – 8 . Here, using electrophysiology and behavioural analyses, we assay magnetic-field responses at the single-neuron and organismal levels. We show that the 52 C-terminal amino acid residues of Drosophila melanogaster CRY, lacking the canonical FAD-binding domain and tryptophan chain, are sufficient to facilitate magnetoreception. We also show that increasing intracellular FAD potentiates both blue-light-induced and magnetic-field-dependent effects on the activity mediated by the C terminus. High levels of FAD alone are sufficient to cause blue-light neuronal sensitivity and, notably, the potentiation of this response in the co-presence of a magnetic field. These results reveal the essential components of a primary magnetoreceptor in flies, providing strong evidence that non-canonical (that is, non-CRY-dependent) radical pairs can elicit magnetic-field responses in cells.

The phytohormone gibberellin (GA) is critical for environmentally sensitive plant development including germination, skotomorphogenesis and flowering. The FRET biosensor GIBBERELLIN PERCEPTION SENSOR1, which permits single-cell GA measurements in vivo , was previously used to observe a GA gradient correlated with cell length in dark-grown but not light-grown hypocotyls. We sought to understand how light signalling integrates into cellular GA regulation. Here we show how the E3 ligase COP1 and transcription factor HY5 play central roles in directing cellular GA distribution in skoto- and photomorphogenic hypocotyls, respectively. We demonstrate that the expression pattern of biosynthetic enzyme GA20ox1 is the key determinant of the GA gradient in dark-grown hypocotyls and is a target of COP1 signalling. We engineered a second generation GPS2 biosensor with improved orthogonality and reversibility to show the cellular pattern of GA depletion during the transition to growth in the light. This GA depletion partly explains the resetting of hypocotyl growth dynamics during photomorphogenesis. Achieving cell-level resolution has revealed how GA distributions link environmental conditions with morphology and morphological plasticity and the GPS2 biosensor is an ideal tool for GA studies in further conditions, organs and plant species.