The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants.

The transient elevation of cytosolic free calcium concentration ([Ca2+ ]cyt ) induced by cold stress is a well-established phenomenon; however, the underlying mechanism remains elusive. Here, we report that the Ca2+ -permeable transporter ANNEXIN1 (AtANN1) mediates cold-triggered Ca2+ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold-induced [Ca2+ ]cyt increase and upregulation of the cold-responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca2+ transport activity, thereby potentiating Ca2+ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold-induced [Ca2+ ]cyt elevation in the ost1-3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1-AtANN1 to cold-induced Ca2+ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.

Indications and Outcomes for Planned Cesarean Hysterectomy in Non-Placenta Accreta Spectrum Disorder Patients: A Systematic Review.

OBJECTIVE: Planned hysterectomy at the time of cesarean delivery may be reasonable in cases other than placenta accreta spectrum disorders. Our objective was to synthesize the published literature on the indications and outcomes for planned cesarean hysterectomy. DATA SOURCES: We performed a systematic review of published literature from the following databases from inception (1946) to June 2021: MEDLINE, PubMed, EMBASE, Cochrane CENTRAL, DARE, and clinicaltrials.gov. STUDY SELECTION: We included all study designs where subjects underwent planned cesarean delivery with simultaneous hysterectomy. Emergency procedures and those performed for placenta accreta spectrum disorders were excluded. DATA EXTRACTION AND SYNTHESIS: The primary outcome was surgical indication, though other surgical outcomes were evaluated when data permitted. Quantitative analysis was limited to studies published in 1990 or later. Risk of bias was assessed using an adaptation of the ROBINS-I tool. CONCLUSION: The most common indication for planned cesarean hysterectomy was malignancy, with cervical cancer being the most frequent. Other indications included permanent contraception, uterine fibroids, menstrual disorders, and chronic pelvic pain. Common complications included bleeding, infection, and ileus. The surgical skill for cesarean hysterectomy continues to be relevant in contemporary obstetrical practice for reproductive malignancy and several benign indications. Although the data indicate relatively safe outcomes, these studies show significant publication bias and, therefore, further systematic study of this procedure is justified. PROSPERO REGISTRATION NUMBER: CRD42021260545, registered June 16, 2021.

Arabidopsis thaliana CYCLIC NUCLEOTIDE-GATED CHANNEL2 mediates extracellular ATP signal transduction in root epidermis.

Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca2+ ]cyt ) increase as a second messenger. The downstream PM Ca2+ channels remain enigmatic. Here, the Arabidopsis thaliana Ca2+ channel subunit CYCLIC NUCLEOTIDE-GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca2+ ]cyt signalling in roots. Extracellular ATP-induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca2+ ]cyt were measured with aequorin, and root transcriptional changes were determined by quantitative real-time PCR. Two cngc2 loss-of-function mutants were used: cngc2-3 and defence not death1 (which expresses cytosolic aequorin). Extracellular ATP-induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca2+ dependent, requiring CNGC2 but not CNGC4 (its channel co-subunit in immunity signalling). Activation of PM Ca2+ influx currents also required CNGC2. The eATP-induced [Ca2+ ]cyt increase and transcriptional response in cngc2 roots were significantly impaired. CYCLIC NUCLEOTIDE-GATED CHANNEL2 is required for eATP-induced epidermal Ca2+ influx, causing depolarization leading to [Ca2+ ]cyt increase and damage-related transcriptional response.

ANNEXIN1 mediates calcium-dependent systemic defense in Arabidopsis plants upon herbivory and wounding.

Cellular calcium (Ca) transients are endogenous signals involved in local and systemic signaling and defense activation upon environmental stress, including wounding and herbivory. Still, not all Ca2+ channels contributing to the signaling have been identified, nor are their modes of action fully known. Plant annexins are proteins capable of binding to anionic phospholipids and can exhibit Ca channel-like activity. Arabidopsis ANNEXIN1 (ANN1) is suggested to contribute to Ca transport. Here, we report that wounding and simulated-herbivory-induced cytosolic free Ca elevation was impaired in systemic leaves in ann1 loss-of-function plants. We provide evidence for a role of ANN1 in local and systemic defense of plants attacked by herbivorous Spodoptera littoralis larvae. Bioassays identified ANN1 as a positive defense regulator. Spodoptera littoralis feeding on ann1 gained significantly more weight than larvae feeding on wild-type, whereas those feeding on ANN1-overexpressing lines gained less weight. Herbivory and wounding both induced defense-related responses on treated leaves, such as jasmonate accumulation and defense gene expression. These responses remained local and were strongly reduced in systemic leaves in ann1 plants. Our results indicate that ANN1 plays an important role in activation of systemic rather than local defense in plants attacked by herbivorous insects.

Annexin 1 Is a Component of eATP-Induced Cytosolic Calcium Elevation in Arabidopsis thaliana Roots.

Extracellular ATP (eATP) has long been established in animals as an important signalling molecule but this is less understood in plants. The identification of Arabidopsis thaliana DORN1 (Does Not Respond to Nucleotides) as the first plant eATP receptor has shown that it is fundamental to the elevation of cytosolic free Ca2+ ([Ca2+]cyt) as a possible second messenger. eATP causes other downstream responses such as increase in reactive oxygen species (ROS) and nitric oxide, plus changes in gene expression. The plasma membrane Ca2+ influx channels involved in eATP-induced [Ca2+]cyt increase remain unknown at the genetic level. Arabidopsis thaliana Annexin 1 has been found to mediate ROS-activated Ca2+ influx in root epidermis, consistent with its operating as a transport pathway. In this study, the loss of function Annexin 1 mutant was found to have impaired [Ca2+]cyt elevation in roots in response to eATP or eADP. Additionally, this annexin was implicated in modulating eATP-induced intracellular ROS accumulation in roots as well as expression of eATP-responsive genes.

Phosphate Starvation Alters Abiotic-Stress-Induced Cytosolic Free Calcium Increases in Roots.

Phosphate (Pi) deficiency strongly limits plant growth, and plant roots foraging the soil for nutrients need to adapt to optimize Pi uptake. Ca2+ is known to signal in root development and adaptation but has to be tightly controlled, as it is highly toxic to Pi metabolism. Under Pi starvation and the resulting decreased cellular Pi pool, the use of cytosolic free Ca2+ ([Ca2+]cyt) as a signal transducer may therefore have to be altered. Employing aequorin-expressing Arabidopsis (Arabidopsis thaliana), we show that Pi starvation, but not nitrogen starvation, strongly dampens the [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The altered root [Ca2+]cyt response to extracellular ATP manifests during seedling development under chronic Pi deprivation but can be reversed by Pi resupply. Employing ratiometric imaging, we delineate that Pi-starved roots have a normal response to extracellular ATP at the apex but show a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species levels induced by Pi starvation. Excluding iron, as well as Pi, rescues this altered [Ca2+]cyt response and restores reactive oxygen species levels to those seen under nutrient-replete conditions. These results indicate that, while Pi availability does not seem to be signaled through [Ca2+]cyt, Pi starvation strongly affects stress-induced [Ca2+]cyt signatures. These data reveal how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer.

DORN1/P2K1 and purino-calcium signalling in plants: making waves with extracellular ATP.

BACKGROUND AND AIMS: Extracellular ATP governs a range of plant functions, including cell viability, adaptation and cross-kingdom interactions. Key functions of extracellular ATP in leaves and roots may involve an increase in cytosolic free calcium as a second messenger ('calcium signature'). The main aim here was to determine to what extent leaf and root calcium responses require the DORN1/P2K1 extracellular ATP receptor in Arabidopsis thaliana. The second aim was to test whether extracellular ATP can generate a calcium wave in the root. METHODS: Leaf and root responses to extracellular ATP were reviewed for their possible links to calcium signalling and DORN1/P2K1. Leaves and roots of wild type and dorn1 plants were tested for cytosolic calcium increase in response to ATP, using aequorin. The spatial abundance of DORN1/P2K1 in the root was estimated using green fluorescent protein. Wild type roots expressing GCaMP3 were used to determine the spatial variation of cytosolic calcium increase in response to extracellular ATP. KEY RESULTS: Leaf and root ATP-induced calcium signatures differed markedly. The leaf signature was only partially dependent on DORN1/P2K1, while the root signature was fully dependent. The distribution of DORN1/P2K1 in the root supports a key role in the generation of the apical calcium signature. Root apical and sub-apical calcium signatures may operate independently of each other but an apical calcium increase can drive a sub-apical increase, consistent with a calcium wave. CONCLUSION: DORN1 could underpin several calcium-related responses but it may not be the only receptor for extracellular ATP in Arabidopsis. The root has the capacity for a calcium wave, triggered by extracellular ATP at the apex.

MAP Kinase PrMPK9-1 Contributes to the Self-Incompatibility Response.

Mitogen-activated protein kinases (MAPKs) form important signaling modules for a variety of cellular responses in eukaryotic cells. In plants, MAPKs play key roles in growth and development as well as in immunity/stress responses. Pollen-pistil interactions are critical early events regulating pollination and fertilization and involve many signaling events. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants and also is known to utilize signaling to achieve incompatible pollen rejection. Although several pollen-expressed MAPKs exist, very little is known about their function. We previously identified a pollen-expressed MAPK (p56) from Papaver rhoeas that was rapidly activated during SI; several studies implicated its role in signaling to SI-induced programmed cell death involving a DEVDase. However, to date, the identity of the MAPK involved has been unknown. Here, we have identified and cloned a pollen-expressed P. rhoeas threonine-aspartate-tyrosine (TDY) MAPK, PrMPK9-1 Rather few data relating to the function of TDY MAPKs in plants currently exist. We provide evidence that PrMPK9-1 has a cell type-specific function, with a distinct role from AtMPK9 To our knowledge, this is the first study implicating a function for a TDY MAPK in pollen. We show that PrMPK9-1 corresponds to p56 and demonstrate that it is functionally involved in mediating SI in P. rhoeas pollen: PrMPK9-1 is a key regulator for SI in pollen and acts upstream of programmed cell death involving actin and activation of a DEVDase. Our study provides an important advance in elucidating functional roles for this class of MAPKs.

Self-incompatibility-induced programmed cell death in field poppy pollen involves dramatic acidification of the incompatible pollen tube cytosol.

Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly specific interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in field poppy (Papaver rhoeas) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidification of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH]cyt); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH]cyt of the pollen tubes in vivo, we show that [pH]cyt acidification is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidification and identify key targets of this major physiological alteration. A small drop in [pH]cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH]cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and Actin-Depolymerizing Factor are identified as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH]cyt and its effect on several components in the context of SI-induced events and PCD.

Highlights from the tenth ISCB Student Council Symposium 2014.

This report summarizes the scientific content and activities of the annual symposium organized by the Student Council of the International Society for Computational Biology (ISCB), held in conjunction with the Intelligent Systems for Molecular Biology (ISMB) conference in Boston, USA, on July 11th, 2014.

The hydroxyl radical in plants: from seed to seed.

The hydroxyl radical (OH(•)) is the most potent yet short-lived of the reactive oxygen species (ROS) radicals. Just as hydrogen peroxide was once considered to be simply a deleterious by-product of oxidative metabolism but is now acknowledged to have signalling roles in plant cells, so evidence is mounting for the hydroxyl radical as being more than merely an agent of destruction. Its oxidative power is harnessed to facilitate germination, growth, stomatal closure, reproduction, the immune response, and adaptation to stress. It features in plant cell death and is a key tool in microbial degradation of plant matter for recycling. Production of the hydroxyl radical in the wall, at the plasma membrane, and intracellularly is facilitated by a range of peroxidases, superoxide dismutases, NADPH oxidases, and transition metal catalysts. The spatio-temporal activity of these must be tightly regulated to target substrates precisely to the site of radical production, both to prevent damage and to accommodate the short half life and diffusive capacity of the hydroxyl radical. Whilst research has focussed mainly on the hydroxyl radical's mode of action in wall loosening, studies now extend to elucidating which proteins are targets in signalling systems. Despite the difficulties in detecting and manipulating this ROS, there is sufficient evidence now to acknowledge the hydroxyl radical as a potent regulator in plant cell biology.

Taking one for the team: self-recognition and cell suicide in pollen.

Self-incompatibility (SI) is an important genetically controlled mechanism used by many angiosperms to prevent self-fertilization and inbreeding. A multiallelic S-locus allows discrimination between 'self' (incompatible) pollen from 'nonself' pollen at the pistil. Interaction of matching pollen and pistil S-determinants allows 'self' recognition and triggers rejection of incompatible pollen. The S-determinants for Papaver rhoeas (poppy) are PrsS and PrpS. PrsS is a small secreted protein that acts as a signalling ligand to interact with its cognate pollen S-determinant PrpS, a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates increases in cytosolic free Ca(2+) and involves influx of Ca(2+) and K(+). Data implicate involvement of reactive oxygen species and nitric oxide signalling in the SI response. Downstream targets include the cytoskeleton, a soluble inorganic pyrophosphatase, Pr-p26.1, and a MAP kinase, PrMPK9-1. A major focus for SI-induced signalling is to initiate programmed cell death (PCD). In this review we provide an overview of our understanding of SI, with focus on how the signals and components are integrated, in particular, how reactive oxygen species, nitric oxide, and the actin cytoskeleton feed into a PCD network. We also discuss our recent functional expression of PrpS in Arabidopsis thaliana pollen in the context of understanding how PCD signalling systems may have evolved.

Vacuolar Ca2+/H+ exchanger and Ca2+-ATPase homologues are differentially regulated in tipburn-resistant and susceptible lettuce (Lactuca sativa) cultivars.

Tipburn is a physiological disorder of lettuce (Lactuca sativa) and other leafy crops that causes external and internal leaf discolouration and results in serious quality issues for the fresh produce industry. Tipburn occurrence is difficult to predict and no completely effective control methods exist. This is compounded by poor knowledge of the underlying physiological and molecular basis of the condition, which appears to be associated with deficiency of calcium and other nutrients. Vacuolar calcium transporters, which are involved in calcium homeostasis in Arabidopsis, show differential expression in tipburn-resistant and susceptible Brassica oleracea lines. We therefore investigated expression of a subset of L. sativa vacuolar calcium transporter homologues, belonging to the Ca2+/H+ exchanger and Ca2+-ATPase classes, in tipburn-resistant and susceptible cultivars. This indicated that some L. sativa vacuolar calcium transporter homologues belonging to these gene classes exhibited higher expression levels in resistant cultivars, whilst others had higher expression in susceptible cultivars or were independent of tipburn phenotype. In addition, some homologues were more highly expressed in symptomatic versus asymptomatic leaves in susceptible cultivars, suggesting that tipburn-induced increases in expression are unsuccessful in conferring resistance and that differential baseline expression of such genes is important for tipburn resistance. Knowledge of individual genes associated with tipburn resistance will improve breeding for such traits and the development of resistant lettuce varieties.

Reactive oxygen species and nitric oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of papaver.

Pollen-pistil interactions are critical early events regulating pollination and fertilization. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants. Although data implicate the involvement of reactive oxygen species (ROS) and nitric oxide (NO) in pollen-pistil interactions and the regulation of pollen tube growth, there has been a lack of studies investigating ROS and NO signaling in pollen tubes in response to defined, physiologically relevant stimuli. We have used live-cell imaging to visualize ROS and NO in growing Papaver rhoeas pollen tubes using chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and demonstrate that SI induces relatively rapid and transient increases in ROS and NO, with each showing a distinctive "signature" within incompatible pollen tubes. Investigating how these signals integrate with the SI responses, we show that Ca(2+) increases are upstream of ROS and NO. As ROS/NO scavengers alleviated both the formation of SI-induced actin punctate foci and also the activation of a DEVDase/caspase-3-like activity, this demonstrates that ROS and NO act upstream of these key SI markers and suggests that they signal to these SI events. These data represent, to our knowledge, the first steps in understanding ROS/NO signaling triggered by this receptor-ligand interaction in pollen tubes.

Reducing Inequities During the COVID-19 Pandemic: A Rapid Review and Synthesis of Public Health Recommendations.

Objectives: Efforts to contain the COVID-19 pandemic should take into account worsening health inequities. While many public health experts have commented on inequities, no analysis has yet synthesized recommendations into a guideline for practitioners. The objective of this rapid review was to identify the areas of greatest concern and synthesize recommendations. Methods: We conducted a rapid systematic review (PROSPERO: CRD42020178131). We searched Ovid MEDLINE, Embase, PsycINFO, CINAHL and Cochrane Central Register of Controlled Trials databases from December 1, 2019 to April 27, 2020. We included English language peer-reviewed commentaries, editorials, and opinion pieces that addressed the social determinants of health in the context of COVID-19. Results: 338 articles met our criteria. Authors represented 81 countries. Income, housing, mental health, age and occupation were the most discussed social determinants of health. We categorized recommendations into primordial, primary, secondary and tertiary prevention that spoke to the social determinants of COVID-19 and equity. Conclusion: These recommendations can assist efforts to contain COVID-19 and reduce health inequities during the pandemic. Using these recommendations, public health practitioners could support a more equitable pandemic response. Systematic Review Registration: PROSPERO, CRD42020178131.

Damage Signaling by Extracellular Nucleotides: A Role for Cyclic Nucleotides in Elevating Cytosolic Free Calcium?

Extracellular ATP (eATP) is now held to be a constitutive damage-associated molecular pattern (DAMP) that is released by wounding, herbivory or pathogen attack. The concentration of eATP must be tightly regulated as either depletion or overload leads to cell death. In Arabidopsis thaliana, sensing of eATP is by two plasma membrane legume-like lectin serine-threonine receptor kinases (P2K1 and P2K2), although other receptors are postulated. The transcriptional response to eATP is dominated by wound- and defense-response genes. Wounding and pathogen attack can involve the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) which, in common with eATP, can increase cytosolic-free Ca2+ as a second messenger. This perspective on DAMP signaling by eATP considers the possibility that the eATP pathway involves production of cyclic nucleotides to promote opening of cyclic nucleotide-gated channels and so elevates cytosolic-free Ca2+. In silico analysis of P2K1 and P2K2 reveals putative adenylyl and guanylyl kinase sequences that are the hallmarks of "moonlighting" receptors capable of cAMP and cGMP production. Further, an Arabidopsis loss of function cngc mutant was found to have an impaired increase in cytosolic-free Ca2+ in response to eATP. A link between eATP, cyclic nucleotides, and Ca2+ signaling therefore appears credible.

Characterization of a legumain/vacuolar processing enzyme and YVADase activity in Papaver pollen.

Legumains, also known as Vacuolar Processing Enzymes (VPEs) have received considerable attention recently, as they share structural properties with mammalian caspase-1 and exhibit YVADase/caspase-1-like cleavage activity. Although many legumains have been cloned, knowledge about their detailed characteristics and intracellular localization is relatively limited. We previously identified several caspase-like activities activated by self-incompatibility (SI) in pollen; a DEVDase was required for programmed cell death (PCD), but YVADase was not (Bosch and Franklin-Tong in Proc Natl Acad Sci USA 104:18327-18332, 2007; Thomas and Franklin-Tong in Nature 429:305-309, 2004). Here we report identification of a legumain/VPE from Papaver rhoeas pollen (PrVPE1) that binds to the DEVD tetrapeptide, a signature substrate for caspase-3. A detailed characterization of the recombinant PrVPE1 cleavage activity revealed that, like other VPEs, it has YVADase activity and requires an acidic pH for activity. Unlike other legumain/VPEs, it also exhibits DEVDase and IETDase activities and apparently does not require processing for activity. The pollen-expressed PrVPE1 localizes to a reticulate compartment resembling the vacuole. Examination of YVADase activity using live-cell imaging of pollen tubes revealed YVADase activity in mitochondria of growing pollen tubes. The unexpected features of PrVPE1, together with evidence for YVADase activity in plant mitochondria, indicate that VPEs, YVADases, their localization and functions in plant cells merit further investigation.

Phosphate Deprivation Can Impair Mechano-Stimulated Cytosolic Free Calcium Elevation in Arabidopsis Roots.

The root tip responds to mechanical stimulation with a transient increase in cytosolic free calcium as a possible second messenger. Although the root tip will grow through a heterogeneous soil nutrient supply, little is known of the consequence of nutrient deprivation for such signalling. Here, the effect of inorganic phosphate deprivation on the root's mechano-stimulated cytosolic free calcium increase is investigated. Arabidopsisthaliana (cytosolically expressing aequorin as a bioluminescent free calcium reporter) is grown in zero or full phosphate conditions, then roots or root tips are mechanically stimulated. Plants also are grown vertically on a solid medium so their root skewing angle (deviation from vertical) can be determined as an output of mechanical stimulation. Phosphate starvation results in significantly impaired cytosolic free calcium elevation in both root tips and whole excised roots. Phosphate-starved roots sustain a significantly lower root skewing angle than phosphate-replete roots. These results suggest that phosphate starvation causes a dampening of the root mechano-signalling system that could have consequences for growth in hardened, compacted soils.

Iron availability modulates the Arabidopsis thaliana root calcium signature evoked by exogenous ATP.

Plants use changes in cytosolic free Ca2+ ("signatures") to encode information from the specific signals generated in development, immunity and stress perception. Phosphate availability has a significant impact on the Arabidopsis thaliana root calcium signatures generated in response to abiotic stress stimuli and exogenous purine nucleotides. In the case of the response to exogenous ATP, the effect of low phosphate availability is linked to abnormal iron and reactive oxygen species accumulation with iron deprivation's restoring normal signature dynamics. Here, the effect of iron deprivation with normal phosphate availability has been examined. Iron deprivation significantly alters the root calcium signature evoked by exogenous ATP and may link to levels of reactive oxygen species and callose deposition.