Noninvasive In Planta Live Measurements of H2O2 and Glutathione Redox Potential with Fluorescent roGFPs-Based Sensors.

In this protocol, we present a noninvasive in planta bioimaging technique for the analysis of hydrogen peroxide (H2O2) and glutathione redox potential in adult Arabidopsis thaliana plants. The technique is based on the use of stereo fluorescence microscopy to image A. thaliana plants expressing the two genetically encoded fluorescent sensors roGFP2-Orp1 and Grx1-roGFP2. We provide a detailed step-by-step protocol for performing low magnification imaging with mature plants grown in soil or hydroponic systems. This protocol aims to serve the scientific community by providing an accessible approach to noninvasive in planta bioimaging and data analysis.

Rapid alkalinization factor 22 has a structural and signalling role in root hair cell wall assembly.

Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types.

Non-pharmacological therapies for pain management in paediatric intensive care units: a protocol for a scoping review.

INTRODUCTION: In critically ill children, pain management is complex owing to cognitive development and the nature of hospitalisation in paediatric intensive therapy units. Although there are many protocols and guidelines for pain control via pharmacological interventions, non-pharmacological practices should also be explored and disseminated for their potential benefit. METHODS AND ANALYSIS: A systematic literature search will be performed using the following databases: Academic Search Premier, Cumulative Index to Nursing and Allied Health Literature, Cochrane Library, Excerpta Medica Database, Virtual Health Library, Medical Literature Analysis and Retrieval System Online, ScienceDirect, Scopus, Web of Science Core Collection, Theses from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Dart Europe, Open Access Theses and Dissertations and grey literature from Google Scholar. The research will consider quantitative and qualitative studies, mixed-method studies, systematic reviews, text articles, opinion articles, letters to editors and editorials in any language and from any database. The following will be eligible for inclusion: (1) newborns, infants, children and adolescents; and (2) non-pharmacological therapies used for pain in paediatric intensive care. ETHICS AND DISSEMINATION: This study does not require ethical approval. The results of this research will be disseminated through social media channels and podcasts about pain in children. TRIAL REGISTRATION NUMBER: This protocol has been registered with the Open Science Framework (DOI 10.17605/OSF.IO/DZHKT).

Smart selection of soil microbes for resilient and sustainable viticulture.

The grapevine industry is of high economic importance in several countries worldwide. Its growing market demand led to an acceleration of the entire production processes, implying increasing use of water resources at the expense of environmental water balance and the hydrological cycle. Furthermore, in recent decades climate change and the consequent expansion of drought have further compromised water availability, making current agricultural systems even more fragile from ecological and economical perspectives. Consequently, farmers' income and welfare are increasingly unpredictable and unstable. Therefore, it is urgent to improve the resilience of vineyards, and of agro-ecosystems in general, by developing sustainable and environmentally friendly farming practices by more rational biological and natural resources use. The PRIMA project PROSIT addresses these challenges by characterizing and harnessing grapevine-associated microbiota to propose innovative and sustainable agronomic practices. PROSIT aims to determine the efficacy of natural microbiomes transferred from grapevines adapted to arid climate to commonly cultivated grapevine cultivars. In doing so it will test those natural microbiome effects on drought tolerance. This multidisciplinary project will utilize in vitro culture techniques, bioimaging, microbiological tests, metabolomics, metabarcoding and epigenetic analyses. These will be combined to shed light on molecular mechanisms triggered in plants by microbial associations upon water stress. To this end it is hoped that the project will serve as a blueprint not only for studies uncovering the microbiome role in drought stress in a wide range of species, but also for analyzing its effect on a wide range of stresses commonly encountered in modern agricultural systems.

Mitochondria-derived reactive oxygen species are the likely primary trigger of mitochondrial retrograde signaling in Arabidopsis.

Besides their central function in respiration, plant mitochondria play a crucial role in maintaining cellular homeostasis during stress by providing "retrograde" feedback to the nucleus. Despite the growing understanding of this signaling network, the nature of the signals that initiate mitochondrial retrograde regulation (MRR) in plants remains unknown. Here, we investigated the dynamics and causative relationship of a wide range of mitochondria-related parameters for MRR, using a combination of Arabidopsis fluorescent protein biosensor lines, in vitro assays, and genetic and pharmacological approaches. We show that previously linked physiological parameters, including changes in cytosolic ATP, NADH/NAD+ ratio, cytosolic reactive oxygen species (ROS), pH, free Ca2+, and mitochondrial membrane potential, may often be correlated with-but are not the primary drivers of-MRR induction in plants. However, we demonstrate that the induced production of mitochondrial ROS is the likely primary trigger for MRR induction in Arabidopsis. Furthermore, we demonstrate that mitochondrial ROS-mediated signaling uses the ER-localized ANAC017-pathway to induce MRR response. Finally, our data suggest that mitochondrially generated ROS can induce MRR without substantially leaking into other cellular compartments such as the cytosol or ER lumen, as previously proposed. Overall, our results offer compelling evidence that mitochondrial ROS elevation is the likely trigger of MRR.

Chemical induction of hypocotyl rooting reveals extensive conservation of auxin signalling controlling lateral and adventitious root formation.

Upon exposure to light, etiolated Arabidopsis seedlings form adventitious roots (AR) along the hypocotyl. While processes underlying lateral root formation are studied intensively, comparatively little is known about the molecular processes involved in the initiation of hypocotyl AR. AR and LR formation were studied using a small molecule named Hypocotyl Specific Adventitious Root INducer (HYSPARIN) that strongly induces AR but not LR formation. HYSPARIN does not trigger rapid DR5-reporter activation, DII-Venus degradation or Ca2+ signalling. Transcriptome analysis, auxin signalling reporter lines and mutants show that HYSPARIN AR induction involves nuclear TIR1/AFB and plasma membrane TMK auxin signalling, as well as multiple downstream LR development genes (SHY2/IAA3, PUCHI, MAKR4 and GATA23). Comparison of the AR and LR induction transcriptome identified SAURs, AGC kinases and OFP transcription factors as specifically upregulated by HYSPARIN. Members of the SAUR19 subfamily, OFP4 and AGC2 suppress HYS-induced AR formation. While SAUR19 and OFP subfamily members also mildly modulate LR formation, AGC2 regulates only AR induction. Analysis of HYSPARIN-induced AR formation uncovers an evolutionary conservation of auxin signalling controlling LR and AR induction in Arabidopsis seedlings and identifies SAUR19, OFP4 and AGC2 kinase as novel regulators of AR formation.

Compartment-specific Ca2+ imaging in the green alga Chlamydomonas reinhardtii reveals high light-induced chloroplast Ca2+ signatures.

To investigate the role of intracellular Ca2+ signaling in the perception and response mechanisms to light in unicellular microalgae, the genetically encoded ratiometric Ca2+ indicator Yellow Cameleon (YC3.6) was expressed in the model organism for green algae Chlamydomonas reinhardtii, targeted to cytosol, chloroplast, and mitochondria. Through in vivo single-cell confocal microscopy imaging, light-induced Ca2+ signaling was investigated in different conditions and different genotypes, including the photoreceptors mutants phot and acry. A genetically encoded H2 O2 sensor was also adopted to investigate the possible role of H2 O2 formation in light-dependent Ca2+ signaling. Light-dependent Ca2+ response was observed in Chlamydomonas reinhardtii cells only in the chloroplast as an organelle-autonomous response, influenced by light intensity and photosynthetic electron transport. The absence of blue and red-light photoreceptor aCRY strongly reduced the light-dependent chloroplast Ca2+ response, while the absence of the blue photoreceptor PHOT had no significant effects. A correlation between high light-induced chloroplast H2 O2 gradients and Ca2+ transients was drawn, supported by H2 O2 -induced chloroplast Ca2+ transients in the dark. In conclusion, different triggers are involved in the light-induced chloroplast Ca2+ signaling as saturation of the photosynthetic electron transport, H2 O2 formation, and aCRY-dependent light perception.

Plant electrophysiology with conformable organic electronics: Deciphering the propagation of Venus flytrap action potentials.

Electrical signals in plants are mediators of long-distance signaling and correlate with plant movements and responses to stress. These signals are studied with single surface electrodes that cannot resolve signal propagation and integration, thus impeding their decoding and link to function. Here, we developed a conformable multielectrode array based on organic electronics for large-scale and high-resolution plant electrophysiology. We performed precise spatiotemporal mapping of the action potential (AP) in Venus flytrap and found that the AP actively propagates through the tissue with constant speed and without strong directionality. We also found that spontaneously generated APs can originate from unstimulated hairs and that they correlate with trap movement. Last, we demonstrate that the Venus flytrap circuitry can be activated by cells other than the sensory hairs. Our work reveals key properties of the AP and establishes the capacity of organic bioelectronics for resolving electrical signaling in plants contributing to the mechanistic understanding of long-distance responses in plants.

Missense variants in RPH3A cause defects in excitatory synaptic function and are associated with a clinically variable neurodevelopmental disorder.

PURPOSE: RPH3A encodes a protein involved in the stabilization of GluN2A subunit of N-methyl-D-aspartate (NMDA)-type glutamate receptors at the cell surface, forming a complex essential for synaptic plasticity and cognition. We investigated the effect of variants in RPH3A in patients with neurodevelopmental disorders. METHODS: By using trio-based exome sequencing, GeneMatcher, and screening of 100,000 Genomes Project data, we identified 6 heterozygous variants in RPH3A. In silico and in vitro models, including rat hippocampal neuronal cultures, have been used to characterize the effect of the variants. RESULTS: Four cases had a neurodevelopmental disorder with untreatable epileptic seizures [p.(Gln73His)dn; p.(Arg209Lys); p.(Thr450Ser)dn; p.(Gln508His)], and 2 cases [p.(Arg235Ser); p.(Asn618Ser)dn] showed high-functioning autism spectrum disorder. Using neuronal cultures, we demonstrated that p.(Thr450Ser) and p.(Asn618Ser) reduce the synaptic localization of GluN2A; p.(Thr450Ser) also increased the surface levels of GluN2A. Electrophysiological recordings showed increased GluN2A-dependent NMDA ionotropic glutamate receptor currents for both variants and alteration of postsynaptic calcium levels. Finally, expression of the Rph3AThr450Ser variant in neurons affected dendritic spine morphology. CONCLUSION: Overall, we provide evidence that missense gain-of-function variants in RPH3A increase GluN2A-containing NMDA ionotropic glutamate receptors at extrasynaptic sites, altering synaptic function and leading to a clinically variable neurodevelopmental presentation ranging from untreatable epilepsy to autism spectrum disorder.

Plant Ca2+-ATPases: From biochemistry to signalling.

Calcium (Ca2+)-ATPases are ATP-dependent enzymes that transport Ca2+ ions against their electrochemical gradient playing the fundamental biological function of keeping the free cytosolic Ca2+ concentration in the submicromolar range to prevent cytotoxic effects. In plants, type IIB autoinhibited Ca2+-ATPases (ACAs) are localised both at the plasma membrane and at the endomembranes including endoplasmic reticulum (ER) and tonoplast and their activity is primarily regulated by Ca2+-dependent mechanisms. Instead, type IIA ER-type Ca2+-ATPases (ECAs) are present mainly at the ER and Golgi Apparatus membranes and are active at resting Ca2+. Whereas research in plants has historically focused on the biochemical characterization of these pumps, more recently the attention has been also addressed on the physiological roles played by the different isoforms. This review aims to highlight the main biochemical properties of both type IIB and type IIA Ca2+ pumps and their involvement in the shaping of cellular Ca2+ dynamics induced by different stimuli.

Widely spaced and divergent inverted repeats become a potent source of chromosomal rearrangements in long single-stranded DNA regions.

DNA inverted repeats (IRs) are widespread across many eukaryotic genomes. Their ability to form stable hairpin/cruciform secondary structures is causative in triggering chromosome instability leading to several human diseases. Distance and sequence divergence between IRs are inversely correlated with their ability to induce gross chromosomal rearrangements (GCRs) because of a lesser probability of secondary structure formation and chromosomal breakage. In this study, we demonstrate that structural parameters that normally constrain the instability of IRs are overcome when the repeats interact in single-stranded DNA (ssDNA). We established a system in budding yeast whereby >73 kb of ssDNA can be formed in cdc13-707fs mutants. We found that in ssDNA, 12 bp or 30 kb spaced Alu-IRs show similarly high levels of GCRs, while heterology only beyond 25% suppresses IR-induced instability. Mechanistically, rearrangements arise after cis-interaction of IRs leading to a DNA fold-back and the formation of a dicentric chromosome, which requires Rad52/Rad59 for IR annealing as well as Rad1-Rad10, Slx4, Msh2/Msh3 and Saw1 proteins for nonhomologous tail removal. Importantly, using structural characteristics rendering IRs permissive to DNA fold-back in yeast, we found that ssDNA regions mapped in cancer genomes contain a substantial number of potentially interacting and unstable IRs.

Long-distance turgor pressure changes induce local activation of plant glutamate receptor-like channels.

In Arabidopsis thaliana, local wounding and herbivore feeding provoke leaf-to-leaf propagating Ca2+ waves that are dependent on the activity of members of the glutamate receptor-like channels (GLRs). In systemic tissues, GLRs are needed to sustain the synthesis of jasmonic acid (JA) with the subsequent activation of JA-dependent signaling response required for the plant acclimation to the perceived stress. Even though the role of GLRs is well established, the mechanism through which they are activated remains unclear. Here, we report that in vivo, the amino-acid-dependent activation of the AtGLR3.3 channel and systemic responses require a functional ligand-binding domain. By combining imaging and genetics, we show that leaf mechanical injury, such as wounds and burns, as well as hypo-osmotic stress in root cells, induces the systemic apoplastic increase of L-glutamate (L-Glu), which is largely independent of AtGLR3.3 that is instead required for systemic cytosolic Ca2+ elevation. Moreover, by using a bioelectronic approach, we show that the local release of minute concentrations of L-Glu in the leaf lamina fails to induce any long-distance Ca2+ waves.

Mitochondrial H2S donor AP39 induces stomatal closure by modulating guard cell mitochondrial activity.

Hydrogen sulfide (H2S) is a gaseous signaling molecule involved in numerous physiological processes in plants, including gas exchange with the environment through the regulation of stomatal pore width. Guard cells (GCs) are pairs of specialized epidermal cells that delimit stomatal pores and have a higher mitochondrial density and metabolic activity than their neighboring cells. However, there is no clear evidence on the role of mitochondrial activity in stomatal closure induction. In this work, we showed that the mitochondrial-targeted H2S donor AP39 induces stomatal closure in a dose-dependent manner. Experiments using inhibitors of the mitochondrial electron transport chain (mETC) or insertional mutants in cytochrome c (CYTc) indicated that the activity of mitochondrial CYTc and/or complex IV are required for AP39-dependent stomatal closure. By using fluorescent probes and genetically encoded biosensors we reported that AP39 hyperpolarized the mitochondrial inner potential (Δψm) and increased cytosolic ATP, cytosolic hydrogen peroxide levels, and oxidation of the glutathione pool in GCs. These findings showed that mitochondrial-targeted H2S donors induce stomatal closure, modulate guard cell mETC activity, the cytosolic energetic and oxidative status, pointing to an interplay between mitochondrial H2S, mitochondrial activity, and stomatal closure.

MCU proteins dominate in vivo mitochondrial Ca2+ uptake in Arabidopsis roots.

Ca2+ signaling is central to plant development and acclimation. While Ca2+-responsive proteins have been investigated intensely in plants, only a few Ca2+-permeable channels have been identified, and our understanding of how intracellular Ca2+ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca2+ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca2+ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca2+ transport in planta and that this mechanism is the major route for fast Ca2+ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca2+ imaging in living root tips and the stimulation of Ca2+ transients of different amplitudes, we demonstrated that mitochondrial Ca2+ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca2+ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca2+ entry route in planta and link mitochondrial Ca2+ transport with phytohormone signaling.

Signaling by plant glutamate receptor-like channels: What else!

Plant glutamate receptor-like channels (GLRs) are transmembrane proteins that allow the movement of several ions across membranes. In the model plant Arabidopsis, there are 20 GLR isoforms grouped in three clades and, since their discovery, it was hypothesized that GLRs were mainly involved in signaling processes. Indeed, in the last years, several pieces of evidence demonstrate different signaling roles played by GLRs, related to pollen development, sexual reproduction, chemotaxis, root development, regulation of stomatal aperture, and response to pathogens. Recently, GLRs have gained attention for their role in long-distance electric and calcium signaling. In this review, we resume the evidence about the role of GLRs in signaling processes. This role is mostly linked to the GLRs involvement in the regulation of ion fluxes across membranes and, in particular, of calcium, which represents a key second messenger in plant cell responses to both endogenous and exogenous stimuli.

Green Tea Catechins, (-)-Catechin Gallate, and (-)-Gallocatechin Gallate are Potent Inhibitors of ABA-Induced Stomatal Closure.

Stomatal movement is indispensable for plant growth and survival in response to environmental stimuli. Cytosolic Ca2+ elevation plays a crucial role in ABA-induced stomatal closure during drought stress; however, to what extent the Ca2+ movement across the plasma membrane from the apoplast to the cytosol contributes to this process still needs clarification. Here the authors identify (-)-catechin gallate (CG) and (-)-gallocatechin gallate (GCG), components of green tea, as inhibitors of voltage-dependent K+ channels which regulate K+ fluxes in Arabidopsis thaliana guard cells. In Arabidopsis guard cells CG/GCG prevent ABA-induced: i) membrane depolarization; ii) activation of Ca2+ permeable cation (ICa ) channels; and iii) cytosolic Ca2+ transients. In whole Arabidopsis plants co-treatment with CG/GCG and ABA suppressed ABA-induced stomatal closure and surface temperature increase. Similar to ABA, CG/GCG inhibited stomatal closure is elicited by the elicitor peptide, flg22 but has no impact on dark-induced stomatal closure or light- and fusicoccin-induced stomatal opening, suggesting that the inhibitory effect of CG/GCG is associated with Ca2+ -related signaling pathways. This study further supports the crucial role of ICa channels of the plasma membrane in ABA-induced stomatal closure. Moreover, CG and GCG represent a new tool for the study of abiotic or biotic stress-induced signal transduction pathways.

Characterization of canavanine-resistance of cat1 and vhc1 deletions and a dominant any1 mutation in fission yeast.

Positive and counter-selectable markers have been successfully integrated as a part of numerous genetic assays in many model organisms. In this study, we investigate the mechanism of resistance to arginine analog canavanine and its applicability for genetic selection in Schizosaccharomyces pombe. Deletion of both the arginine permease gene cat1 and SPBC18H10.16/vhc1 (formerly mistakenly called can1) provides strong drug resistance, while the single SPBC18H10.16/vhc1 deletion does not have an impact on canavanine resistance. Surprisingly, the widely used can1-1 allele does not encode for a defective arginine permease but rather corresponds to the any1-523C>T allele. The strong canavanine-resistance conferred by this allele arises from an inability to deposit basic amino acid transporters on the cellular membrane. any1-523C>T leads to reduced post-translational modifications of Any1 regulated by the Tor2 kinase. We also demonstrate that any1-523C>T is a dominate allele. Our results uncover the mechanisms of canavanine-resistance in fission yeast and open the opportunity of using cat1, vhc1 and any1 mutant alleles in genetic assays.

Distinct Functions of the Atypical Terminal Hydrophilic Domain of the HKT Transporter in the Liverwort Marchantia polymorpha.

K+/Na+ homeostasis is important for land plants, particularly under salt stress. In this study, the structure and ion transport properties of the high-affinity K+ transporter (HKT) of the liverwort Marchantia polymorpha were investigated. Only one HKT gene, MpHKT1, was identified in the genome of M. polymorpha. Phylogenetic analysis of HKT proteins revealed that non-seed plants possess HKTs grouped into a clade independent of the other two clades including HKTs of angiosperms. A distinct long hydrophilic domain was found in the C-terminus of MpHKT1. Complementary DNA (cDNA) of truncated MpHKT1 (t-MpHKT1) encoding the MpHKT_Δ596-812 protein was used to examine the functions of the C-terminal domain. Both MpHKT1 transporters fused with enhanced green fluorescent protein at the N-terminus were localized to the plasma membrane when expressed in rice protoplasts. Two-electrode voltage clamp experiments using Xenopus laevis oocytes indicated that MpHKT1 mediated the transport of monovalent alkali cations with higher selectivity for Na+ and K+, but truncation of the C-terminal domain significantly reduced the transport activity with a decrease in the Na+ permeability. Overexpression of MpHKT1 or t-MpHKT1 in M. polymorpha conferred accumulation of higher Na+ levels and showed higher Na+ uptake rates, compared to those of wild-type plants; however, phenotypes with t-MpHKT1 were consistently weaker than those with MpHKT1. Together, these findings suggest that the hydrophilic C-terminal domain plays a unique role in the regulation of transport activity and ion selectivity of MpHKT1.

Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters.

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.