How do plants feel the heat and survive?

Climate change is increasingly affecting the quality of life of organisms on Earth. More frequent, extreme, and lengthy heat waves are contributing to the sixth mass extinction of complex life forms in the Earth's history. From an anthropocentric point of view, global warming is a major threat to human health because it also compromises crop yields and food security. Thus, achieving agricultural productivity under climate change calls for closer examination of the molecular mechanisms of heat-stress resistance in model and crop plants. This requires a better understanding of the mechanisms by which plant cells can sense rising temperatures and establish effective molecular defenses, such as molecular chaperones and thermoprotective metabolites, as reviewed here, to survive extreme diurnal variations in temperature and seasonal heat waves.

RNA Sequencing of Arabidopsis thaliana Seedlings after Non-Thermal Plasma-Seed Treatment Reveals Upregulation in Plant Stress and Defense Pathways.

Not all agricultural practices are sustainable; however, non-thermal plasma treatment of seeds may be an eco-friendly alternative to improve macroscopic plant growth parameters. Despite the numerous successful results of plasma-seed treatments reported in the literature, there is a large gap in our understanding of how non-thermal plasma treatments affect seeds, especially due to the plethora of physical, chemical, and biological variables. This study uses RNA sequencing to characterize the changes in gene transcription in Arabidopsis thaliana (L.) Heynh. seeds 6 days after exposure to surface dielectric barrier discharge plasma treatment. Here, we provide an overview of all pathways that are differentially expressed where few genes are upregulated and many genes are downregulated. Our results reveal that plasma treatment time is a parameter that can activate different pathways in plant defense. An 80 s treatment upregulates the glucosinolate pathway, a defense response to insects and herbivores to deter feeding, whereas a shorter treatment of 60 s upregulates the phenylpropanoid pathway, which reinforces the cell wall with lignin and produces antimicrobial compounds, a defense response to bacterial or fungal plant pathogens. It seems that plasma elicits a wounding response from the seed in addition to redox changes. This suggests that plasma treatment can be potentially applied in agriculture to protect plants against abiotic and biotic stresses without discharging residues into the environment.

Quantitative proteomic analysis to capture the role of heat-accumulated proteins in moss plant acquired thermotolerance.

At dawn of a scorching summer day, land plants must anticipate upcoming extreme midday temperatures by timely establishing molecular defences that can keep heat-labile membranes and proteins functional. A gradual morning pre-exposure to increasing sub-damaging temperatures induces heat-shock proteins (HSPs) that are central to the onset of plant acquired thermotolerance (AT). To gain knowledge on the mechanisms of AT in the model land plant Physcomitrium patens, we used label-free LC-MS/MS proteomics to quantify the accumulated and depleted proteins before and following a mild heat-priming treatment. High protein crowding is thought to promote protein aggregation, whereas molecular chaperones prevent and actively revert aggregation. Yet, we found that heat priming (HP) did not accumulate HSP chaperones in chloroplasts, although protein crowding was six times higher than in the cytosol. In contrast, several HSP20s strongly accumulated in the cytosol, yet contributing merely 4% of the net mass increase of heat-accumulated proteins. This is in poor concordance with their presumed role at preventing the aggregation of heat-labile proteins. The data suggests that under mild HP unlikely to affect protein stability. Accumulating HSP20s leading to AT, regulate the activity of rare and specific signalling proteins, thereby preventing cell death under noxious heat stress.

A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ-dependent manner in Catharanthus roseus.

Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.

Fluorescent fatty acid conjugates for live cell imaging of peroxisomes.

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways, including fatty acid oxidation, degradation of amino acids, and biosynthesis of ether lipids. Consequently, peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions, including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic, tissue-specific, and context-dependent nature of their biogenesis and function, live cell imaging of peroxisomes is essential for studying peroxisome regulation, as well as for the diagnosis of PBD-linked abnormalities. However, the peroxisomal imaging toolkit is lacking in many respects, with no reporters for substrate import, nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12, generating peroxisome-specific reagents, PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity, bright fluorescence in the red and far-red spectrum, and fast non-cytotoxic staining, making them ideal tools for live cell, whole organism, or tissue imaging of peroxisomes. Finally, we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines.

Design of an Arabidopsis thaliana reporter line to detect heat-sensing and signaling mutants.

BACKGROUND: Global warming is a major challenge for plant survival and growth. Understanding the molecular mechanisms by which higher plants sense and adapt to upsurges in the ambient temperature is essential for developing strategies to enhance plant tolerance to heat stress. Here, we designed a heat-responsive Arabidopsis thaliana reporter line that allows an in-depth investigation of the mechanisms underlying the accumulation of protective heat-shock proteins (HSPs) in response to high temperature. METHODS: A transgenic Arabidopsis thaliana reporter line named "Heat-Inducible Bioluminescence And Toxicity" (HIBAT) was designed to express from a conditional heat-inducible promoter, a fusion gene encoding for nanoluciferase and D-amino acid oxidase, whose expression is toxic in the presence of D-valine. HIBAT seedlings were exposed to different heat treatments in presence or absence of D-valine and analyzed for survival rate, bioluminescence and HSP gene expression. RESULTS: Whereas at 22 °C, HIBAT seedlings grew unaffected by D-valine, and all survived iterative heat treatments without D-valine, 98% died following heat treatments on D-valine. The HSP17.3B promoter was highly specific to heat as it remained unresponsive to various plant hormones, Flagellin, H2O2, osmotic stress and high salt. RNAseq analysis of heat-treated HIBAT seedlings showed a strong correlation with expression profiles of two wild type lines, confirming that HIBAT does not significantly differ from its Col-0 parent. Using HIBAT, a forward genetic screen revealed candidate loss-of-function mutants, apparently defective either at accumulating HSPs at high temperature or at repressing HSP accumulation at non-heat-shock temperatures. CONCLUSION: HIBAT is a valuable candidate tool to identify Arabidopsis mutants defective in the response to high temperature stress. It opens new avenues for future research on the regulation of HSP expression and for understanding the mechanisms of plant acquired thermotolerance.

A single gene encodes isopentenyl diphosphate isomerase isoforms targeted to plastids, mitochondria and peroxisomes in Catharanthus roseus.

Isopentenyl diphosphate isomerases (IDI) catalyze the interconversion of the two isoprenoid universal C5 units, isopentenyl diphosphate and dimethylally diphosphate, to allow the biosynthesis of the large variety of isoprenoids including both primary and specialized metabolites. This isomerisation is usually performed by two distinct IDI isoforms located either in plastids/peroxisomes or mitochondria/peroxisomes as recently established in Arabidopsis thaliana mainly accumulating primary isoprenoids. By contrast, almost nothing is known in plants accumulating specialized isoprenoids. Here we report the cloning and functional validation of an IDI encoding cDNA (CrIDI1) from Catharanthus roseus that produces high amount of monoterpenoid indole alkaloids. The corresponding gene is expressed in all organs including roots, flowers and young leaves where transcripts have been detected in internal phloem parenchyma and epidermis. The CrIDI1 gene also produces long and short transcripts giving rise to corresponding proteins with and without a N-terminal transit peptide (TP), respectively. Expression of green fluorescent protein fusions revealed that the long isoform is targeted to both plastids and mitochondria with an apparent similar efficiency. Deletion/fusion experiments established that the first 18-residues of the N-terminal TP are solely responsible of the mitochondria targeting while the entire 77-residue long TP is needed for an additional plastid localization. The short isoform is targeted to peroxisomes in agreement with the presence of peroxisome targeting sequence at its C-terminal end. This complex plastid/mitochondria/peroxisomes triple targeting occurring in C. roseus producing specialized isoprenoid secondary metabolites is somehow different from the situation observed in A. thaliana mainly producing housekeeping isoprenoid metabolites.

Characterization and subcellular localization of geranylgeranyl diphosphate synthase from Catharanthus roseus.

The enzyme geranylgeranyl diphosphate synthase (GGPS: EC 2.5.1.1, EC 2.5.1.10, EC 2.5.1.29) catalyses the formation of geranylgeranyl diphosphate (GGPP) from isopentenyl diphosphate and dimethylallyl diphosphate via three successive condensation reactions. A full-length nucleotide sequence of GGPS (named CrGGPS) was cloned from the medicinal plant Catharanthus roseus. The deduced polypeptide has 383 amino acids with a calculated mass of 41.6 kDa and possesses prenyltransferase signatures characteristic of plant type II GGPS. The enzyme was characterized by functional complementation in carotenoid accumulating strains of Escherichia coli. When cultures of Catharanthus cell lines were treated with methyljasmonate, no specific increase in transcript levels were observed. In plants, GGPS are encoded by a small multigene family and the isoforms have been shown to be localized in three different subcellular compartments: chloroplast, endoplasmic reticulum and mitochondria. We investigated the subcellular distribution of CrGGPS through transient transformations of C. roseus cells with a yellow fluorescent protein-fused construct. Our results clearly indicate that CrGGPS is located to plastids within stroma and stromules.

How do humans and plants feel the heat?

The 2021 Nobel prize was awarded for the discovery of the animal thermosensory channel TRPV1. We highlight notable shared features with the higher plant thermosensory channel CNGC2/4. Both channels respond to temperature-induced changes in plasma membrane fluidity, leading to hyperphosphorylation of the HSF1 transcription factor via a specific heat-signaling cascade.

Catabolism of Glucosinolates into Nitriles Revealed by RNA Sequencing of Arabidopsis thaliana Seedlings after Non-Thermal Plasma-Seed Treatment.

Non-thermal plasma-seed treatments could be an environmentally friendly method to modulate plant properties. Since it remains unclear how plasmas affect seeds, RNA sequencing was used here to analyze gene transcription changes in 7-day-old Arabidopsis thaliana (L.) Heynh. seedlings grown from surface dielectric barrier discharge plasma-treated seeds. In a previous study, seeds were analyzed 6 days after plasma exposure and a plant stress and defense response was observed. Here, we performed a pathway analysis on differentially expressed genes and our results revealed again an increased expression of plant stress and defense, specifically glucosinolate pathway-related compounds. The main difference was that a different part of the plant defense response changed at 7 days, which was not previously observed at 6 days. With a 24-h delayed extraction time point, the glucosinolates were selectively broken down into nitriles among all of the glucosinolates catabolic products. Although information about nitriles is limited, it protects plants against biotic stresses and has variable toxicity depending on the interacting organism. More work needs to be performed to better understand which plasma seed treatment parameters affect plant defense; however, these preliminary findings suggest that an optimized plasma treatment could be used to elicit a plant defense response.

Heat Shock Signaling in Land Plants: From Plasma Membrane Sensing to the Transcription of Small Heat Shock Proteins.

Heat stress events are major factors limiting crop productivity. During summer days, land plants must anticipate in a timely manner upcoming mild and severe temperature. They respond by accumulating protective heat-shock proteins (HSPs), conferring acquired thermotolerance. All organisms synthetize HSPs; many of which are members of the conserved chaperones families. This review describes recent advances in plant temperature sensing, signaling, and response. We highlight the pathway from heat perception by the plasma membrane through calcium channels, such as cyclic nucleotide-gated channels, to the activation of the heat-shock transcription factors (HSFs). An unclear cellular signal activates HSFs, which act as essential regulators. In particular, the HSFA subfamily can bind heat shock elements in HSP promoters and could mediate the dissociation of bound histones, leading to HSPs transcription. Although plants can modulate their transcriptome, proteome, and metabolome to protect the cellular machinery, HSP chaperones prevent, use, and revert the formation of misfolded proteins, thereby avoiding heat-induced cell death. Remarkably, the HSP20 family is mostly tightly repressed at low temperature, suggesting that a costly mechanism can become detrimental under unnecessary conditions. Here, the role of HSP20s in response to HS and their possible deleterious expression at non-HS temperatures is discussed.

Resveratrol and related stilbene derivatives induce stress granules with distinct clearance kinetics.

Stress granules (SGs) are ribonucleoprotein functional condensates that form under stress conditions in all eukaryotic cells. Although their stress-survival function is far from clear, SGs have been implicated in the regulation of many vital cellular pathways. Consequently, SG dysfunction is thought to be a mechanistic point of origin for many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Additionally, SGs are thought to play a role in pathogenic pathways as diverse as viral infection and chemotherapy resistance. There is a growing consensus on the hypothesis that understanding the mechanistic regulation of SG physical properties is essential to understanding their function. Although the internal dynamics and condensation mechanisms of SGs have been broadly investigated, there have been fewer investigations into the timing of SG formation and clearance in live cells. Because the lifetime of SG persistence can be a key factor in their function and tendency toward pathological dysregulation, SG clearance mechanisms deserve particular attention. Here we show that resveratrol and its analogues piceatannol, pterostilbene, and 3,4,5,4'-tetramethoxystilbene induce G3BP-dependent SG formation with atypically rapid clearance kinetics. Resveratrol binds to G3BP, thereby reducing its protein-protein association valency. We suggest that altering G3BP valency is a pathway for the formation of uniquely transient SGs.

Effect of hydroxychloroquine with or without azithromycin on the mortality of coronavirus disease 2019 (COVID-19) patients: a systematic review and meta-analysis.

BACKGROUND: Hydroxychloroquine or chloroquine with or without azithromycin have been widely promoted to treat coronavirus disease 2019 (COVID-19) following early in vitro antiviral effects against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVE: The aim of this systematic review and meta-analysis was to assess whether chloroquine or hydroxychloroquine with or without azithromycin decreased COVID-19 mortality compared with the standard of care. DATA SOURCES: PubMed, Web of Science, Embase Cochrane Library, Google Scholar and MedRxiv were searched up to 25 July 2020. STUDY ELIGIBILITY CRITERIA: We included published and unpublished studies comparing the mortality rate between patients treated with chloroquine or hydroxychloroquine with or without azithromycin and patients managed with standard of care. PARTICIPANTS: Patients ≥18 years old with confirmed COVID-19. INTERVENTIONS: Chloroquine or hydroxychloroquine with or without azithromycin. METHODS: Effect sizes were pooled using a random-effects model. Multiple subgroup analyses were conducted to assess drug safety. RESULTS: The initial search yielded 839 articles, of which 29 met our inclusion criteria. All studies except one were conducted on hospitalized patients and evaluated the effects of hydroxychloroquine with or without azithromycin. Among the 29 articles, three were randomized controlled trials, one was a non-randomized trial and 25 were observational studies, including 11 with a critical risk of bias and 14 with a serious or moderate risk of bias. After excluding studies with critical risk of bias, the meta-analysis included 11 932 participants for the hydroxychloroquine group, 8081 for the hydroxychloroquine with azithromycin group and 12 930 for the control group. Hydroxychloroquine was not significantly associated with mortality: pooled relative risk (RR) 0.83 (95% CI 0.65-1.06, n = 17 studies) for all studies and RR = 1.09 (95% CI 0.97-1.24, n = 3 studies) for randomized controlled trials. Hydroxychloroquine with azithromycin was associated with an increased mortality (RR = 1.27; 95% CI 1.04-1.54, n = 7 studies). We found similar results with a Bayesian meta-analysis. CONCLUSION: Hydroxychloroquine alone was not associated with reduced mortality in hospitalized COVID-19 patients but the combination of hydroxychloroquine and azithromycin significantly increased mortality.

Diabetes, hypertension, body mass index, smoking and COVID-19-related mortality: a systematic review and meta-analysis of observational studies.

OBJECTIVES: We conducted a systematic literature review and meta-analysis of observational studies to investigate the association between diabetes, hypertension, body mass index (BMI) or smoking with the risk of death in patients with COVID-19 and to estimate the proportion of deaths attributable to these conditions. METHODS: Relevant observational studies were identified by searches in the PubMed, Cochrane library and Embase databases through 14 November 2020. Random-effects models were used to estimate summary relative risks (SRRs) and 95% CIs. Certainty of evidence was assessed using the Cochrane methods and the Grading of Recommendations, Assessment, Development and Evaluations framework. RESULTS: A total of 186 studies representing 210 447 deaths among 1 304 587 patients with COVID-19 were included in this analysis. The SRR for death in patients with COVID-19 was 1.54 (95% CI 1.44 to 1.64, I2=92%, n=145, low certainty) for diabetes and 1.42 (95% CI 1.30 to 1.54, I2=90%, n=127, low certainty) for hypertension compared with patients without each of these comorbidities. Regarding obesity, the SSR was 1.45 (95% CI 1.31 to 1.61, I2=91%, n=54, high certainty) for patients with BMI ≥30 kg/m2 compared with those with BMI <30 kg/m2 and 1.12 (95% CI 1.07 to 1.17, I2=68%, n=25) per 5 kg/m2 increase in BMI. There was evidence of a J-shaped non-linear dose-response relationship between BMI and mortality from COVID-19, with the nadir of the curve at a BMI of around 22-24, and a 1.5-2-fold increase in COVID-19 mortality with extreme obesity (BMI of 40-45). The SRR was 1.28 (95% CI 1.17 to 1.40, I2=74%, n=28, low certainty) for ever, 1.29 (95% CI 1.03 to 1.62, I2=84%, n=19) for current and 1.25 (95% CI 1.11 to 1.42, I2=75%, n=14) for former smokers compared with never smokers. The absolute risk of COVID-19 death was increased by 14%, 11%, 12% and 7% for diabetes, hypertension, obesity and smoking, respectively. The proportion of deaths attributable to diabetes, hypertension, obesity and smoking was 8%, 7%, 11% and 2%, respectively. CONCLUSION: Our findings suggest that diabetes, hypertension, obesity and smoking were associated with higher COVID-19 mortality, contributing to nearly 30% of COVID-19 deaths. TRIAL REGISTRATION NUMBER: CRD42020218115.

Strictosidine activation in Apocynaceae: towards a "nuclear time bomb"?

BACKGROUND: The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine beta-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in Catharanthus roseus or ajmaline in Rauvolfia serpentina. However, very few reports focused on the MIA physiological functions. RESULTS: In this study we showed that a strictosidine pool existed in planta and that the strictosidine deglucosylation product(s) was (were) specifically responsible for in vitro protein cross-linking and precipitation suggesting a potential role for strictosidine activation in plant defence. The spatial feasibility of such an activation process was evaluated in planta. On the one hand, in situ hybridisation studies showed that CrSTR and CrSGD were coexpressed in the epidermal first barrier of C. roseus aerial organs. However, a combination of GFP-imaging, bimolecular fluorescence complementation and electromobility shift-zymogram experiments revealed that STR from both C. roseus and R. serpentina were localised to the vacuole whereas SGD from both species were shown to accumulate as highly stable supramolecular aggregates within the nucleus. Deletion and fusion studies allowed us to identify and to demonstrate the functionality of CrSTR and CrSGD targeting sequences. CONCLUSIONS: A spatial model was drawn to explain the role of the subcellular sequestration of STR and SGD to control the MIA metabolic flux under normal physiological conditions. The model also illustrates the possible mechanism of massive activation of the strictosidine vacuolar pool upon enzyme-substrate reunion occurring during potential herbivore feeding constituting a so-called "nuclear time bomb" in reference to the "mustard oil bomb" commonly used to describe the myrosinase-glucosinolate defence system in Brassicaceae.

Moderate Fever Cycles as a Potential Mechanism to Protect the Respiratory System in COVID-19 Patients.

Mortality in COVID-19 patients predominantly results from an acute respiratory distress syndrome (ARDS), in which lungs alveolar cells undergo programmed cell death. Mortality in a sepsis-induced ARDS rat model is reduced by adenovirus over-expression of the HSP70 chaperone. A natural rise of body temperature during mild fever can naturally accumulate high cellular levels of HSP70 that can arrest apoptosis and protect alveolar lung cells from inflammatory damages. However, beyond 1-2 h of fever, no HSP70 is being further produced and a decreased in body temperature required to the restore cell's ability to produce more HSP70 in a subsequent fever cycle. We suggest that antipyretics may be beneficial in COVID-19 patients subsequent to several hours of mild (<38.8°C) advantageous fever, allowing lung cells to accumulate protective HSP70 against damages from the inflammatory response to the virus SARS-CoV-2. With age, the ability to develop fever and accumulate HSP70 decreases. This could be ameliorated, when advisable to do so, by thermotherapies and/or physical training.

Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle.

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis.