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OBJECTIVE Patient-reported outcome measures (PROMs) are currently the gold standard to evaluate patient physical performance and ability to recover after spine surgery. However, PROMs have significant limitations due to the qualitative and subjective nature of the information reported as well as the impossibility of using this method in a continuous manner. The smartphone global positioning system (GPS) can be used to provide continuous, quantitative, and objective information on patient mobility. The aim of this study was to use daily mobility features derived from the smartphone GPS to characterize the perioperative period of patients undergoing spine surgery and to compare these objective measurements to PROMs, the current gold standard. METHODS Eight daily mobility features were derived from smartphone GPS data in a population of 39 patients undergoing spine surgery for a period of 2 months starting 3weeks before surgery. In parallel, three different PROMs for pain (visual analog scale [VAS]), disability (Oswestry Disability Index [ODI]) and functional status (Patient-Reported Outcomes Measurement Information System [PROMIS]) were serially measured. Segmented linear regression analysis was used to assess trends before and after surgery. The Student paired t-test was used to compare pre- and postoperative PROM scores. Pearson’s correlation was calculated between the daily average of each GPS-based mobility feature and the daily average of each PROM score during the recovery period. RESULTS Smartphone GPS features provided data documenting a reduction in mobility during the immediate postoperative period, followed by a progressive and steady increase with a return to baseline mobility values 1 month after surgery. PROMs measuring pain, physical performance, and disability were significantly different 1 month after surgery compared to the 2 immediate preoperative weeks. The GPS-based features presented moderate to strong linear correlation with pain VAS and PROMIS physical score during the recovery period (Pearson r > 0.7), whereas the ODI and PROMIS mental scores presented a weak correlation (Pearson r approximately 0.4). CONCLUSIONS Smartphone-derived GPS features were shown to accurately characterize perioperative mobility trends in patients undergoing surgery for spine-related diseases. Features related to time (rather than distance) were better at describing patient physical and performance status. Smartphone GPS has the potential to be used for the development of accurate, noninvasive and personalized tools for patient mobility monitoring after surgery.

Background Clinical practice guidelines aim to support clinicians in providing clinical care and should be supported by evidence. There is currently no information on whether clinical practice guidelines in laparoscopic surgery are supported by evidence. Methods We performed a systematic review and identified clinical practice guidelines of laparoscopic surgery published in PubMed and Embase between March 2016 and February 2019. We performed an independent assessment of the strength of recommendation based on the evidence provided by the guideline authors. We used the ‘Appraisal of Guidelines for Research & Evaluation II’ (AGREE-II) Tool’s ‘rigour of development’, ‘clarity of presentation’, and ‘editorial independence’ domains to assess the quality of the guidelines. We performed a mixed-effects generalised linear regression modelling. Results We retrieved 63 guidelines containing 1905 guideline statements. The median proportion of ‘difference in rating’ of strength of recommendation between the guideline authors and independent assessment was 33.3% (quartiles: 18.3%, 55.8%). The ‘rigour of development’ domain score (odds ratio 0.06; 95% confidence intervals 0.01–0.48 per unit increase in rigour score; P value = 0.0071) and whether the strength of recommendation was ‘strong’ by independent evaluation (odds ratio 0.09 (95% confidence intervals 0.06–0.13; P value Conclusion A considerable proportion of guideline statements in clinical practice guidelines in laparoscopic surgery are not supported by evidence. Guideline authors systematically overrated the strength of the recommendation (i.e., even when the evidence points to weak recommendation, guideline authors made strong recommendations).

Background: Clinical practice guidelines (CPGs) contain recommendations intended to aid clinicians in providing optimal care for patients and should be derived from the best quality evidence available. The highest quality evidence is obtained through systematic reviews using Cochrane methodology. This exploratory study aimed to determine the proportion of guideline statements from major liver organisations in USA and Europe that were consistent with the conclusions of recent systematic reviews using Cochrane methodology. Methods: We selected four topics related to decompensated cirrhosis on which our group has recently published Cochrane systematic reviews (variceal bleeding; spontaneous bacterial peritonitis; ascites; hepatorenal syndrome). We performed a purposive sample of CPGs from leading organisations covering these topics: American Association for the Study of Liver Diseases (AASLD); British Society of Gastroenterology (BSG); European Association for the Study of the Liver (EASL); and National Institute for Health and Care Excellence (NICE) until January 2022. For each guideline recommendation statement, two researchers independently assessed whether there was agreement in the statement and the strength of recommendation given by guideline authors when compared to those derived from the Cochrane reviews. We meta-analysed the proportion of agreement using ‘meta’ package for R utilising the ‘Generalised Linear Mixed Model’ method for meta-analysis of proportions. Results: We identified 9 CPGs containing 62 guideline statements that were also covered in 7 Cochrane reviews. Agreement between the guideline authors and independent assessment was 0.145 (95% CI: 0.077 to 0.256), i.e., there was disagreement in 85.5% of recommendation statements. There was disagreement on four of the 62 guideline statements about the use or consideration of an intervention when there was no evidence to support their use or consideration. The remaining disagreements related to the disagreement in the strength of recommendation: in each of these disagreements, the guideline author had provided a strong recommendation for or against an intervention when there was only weak evidence for or against the intervention. For each disagreement in the strength of recommendation, the strength of recommendation was overstated rather than understated by the guideline authors. The disagreement could not be explained by the difference in the search dates between the guidelines and Cochrane reviews. Conclusion: The strength of recommendation given in the majority of CPGs from leading liver organisations on decompensated liver cirrhosis did not match those derived from Cochrane reviews. Our findings suggest a systematic issue with guideline developers overstating the strength of recommendations, which has the potential for direct impact on patient care.

Stomata optimize land plants’ photosynthetic requirements and limit water vapor loss. So far, all of the molecular and electrical components identified as regulating stomatal aperture are produced, and operate, directly within the guard cells. However, a completely autonomous function of guard cells is inconsistent with anatomical and biophysical observations hinting at mechanical contributions of epidermal origins. Here, potassium (K+) assays, membrane potential measurements, microindentation, and plasmolysis experiments provide evidence that disruption of the Arabidopsis thaliana K+ channel subunit gene AtKC1 reduces pavement cell turgor, due to decreased K+ accumulation, without affecting guard cell turgor. This results in an impaired back pressure of pavement cells onto guard cells, leading to larger stomatal apertures. Poorly rectifying membrane conductances to K+ were consistently observed in pavement cells. This plasmalemma property is likely to play an essential role in K+ shuttling within the epidermis. Functional complementation reveals that restoration of the wild-type stomatal functioning requires the expression of the transgenic AtKC1 at least in the pavement cells and trichomes. Altogether, the data suggest that AtKC1 activity contributes to the building of the back pressure that pavement cells exert onto guard cells by tuning K+ distribution throughout the leaf epidermis.

Summary Background Guidelines are essential for safe and effective treatment. They usually have multiple statements. Since the supporting information for the guidelines varies widely, the degree to which these statements are recommended also differ. We rely on recommendation systems for grading the recommendations for different statements. All recommendation systems have different grading criteria and they could potentially cause confusion and affect the quality of recommendations. Therefore, there is a need to determine the extent of variation and explore the potential reasons behind it. Methods A purposive sampling on PubMed was conducted to find four different laparoscopic guidelines using different methods to grade the recommendations. Each statement was then re-evaluated using the GRADE recommendation system. Results The guidelines used GRADE, Oxford Methodology, SIGN, and ‘bespoke’ systems. The number of statements with similar strength for the different statements as the re-evaluated strengths in the four guidelines were 24.1, 62.2, 35.8 and 50.0% respectively. Conclusion There were a wide variety of recommendation systems for laparoscopic guidelines and there were differences between the recommendations from the guidelines using GRADE, Oxford Methodology, SIGN and the ‘Bespoke’ system when re-evaluated by GRADE. A systematic review of recent laparoscopic guidelines might provide the extent and the main reasons of the problem.

Clinical practice guidelines aim to support clinicians in providing clinical care and should be supported by evidence. There is currently no information on whether clinical practice guidelines in laparoscopic surgery are supported by evidence.We performed a systematic review and identified clinical practice guidelines of laparoscopic surgery published in PubMed and Embase between March 2016 and February 2019. We performed an independent assessment of the strength of recommendation based on the evidence provided by the guideline authors. We used the 'Appraisal of Guidelines for ResearchEvaluation II' (AGREE-II) Tool's 'rigour of development', 'clarity of presentation', and 'editorial independence' domains to assess the quality of the guidelines. We performed a mixed-effects generalised linear regression modelling.We retrieved 63 guidelines containing 1905 guideline statements. The median proportion of 'difference in rating' of strength of recommendation between the guideline authors and independent assessment was 33.3% (quartiles: 18.3%, 55.8%). The 'rigour of development' domain score (odds ratio 0.06; 95% confidence intervals 0.01-0.48 per unit increase in rigour score; P value = 0.0071) and whether the strength of recommendation was 'strong' by independent evaluation (odds ratio 0.09 (95% confidence intervals 0.06-0.13; P value 0.001) were the only determinants of difference in rating between the guideline authors and independent evaluation.A considerable proportion of guideline statements in clinical practice guidelines in laparoscopic surgery are not supported by evidence. Guideline authors systematically overrated the strength of the recommendation (i.e., even when the evidence points to weak recommendation, guideline authors made strong recommendations).

BackgroundAcute calculus cholecystitis (ACC) has a high incidence in the general population. The presence of several areas of uncertainty, along with the availability of new evidence, prompted the current update of the 2016 WSES (World Society of Emergency Surgery) Guidelines on ACC.Materials and methodsThe WSES president appointed four members as a scientific secretariat, four members as an organization committee and four members as a scientific committee, choosing them from the expert affiliates of WSES. Relevant key questions were constructed, and the task force produced drafts of each section based on the best scientific evidence from PubMed and EMBASE Library; recommendations were developed in order to answer these key questions. The quality of evidence and strength of recommendations were reviewed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria (seehttps://www.gradeworkinggroup.org/). All the statements were presented, discussed and voted upon during the Consensus Conference at the 6th World Congress of the World Society of Emergency Surgery held in Nijmegen (NL) in May 2019. A revised version of the statements was voted upon via an online questionnaire until consensus was reached.ResultsThe pivotal role of surgery is confirmed, including in high-risk patients. When compared with the WSES 2016 guidelines, the role of gallbladder drainage is reduced, despite the considerable technical improvements available. Early laparoscopic cholecystectomy (ELC) should be the standard of care whenever possible, even in subgroups of patients who are considered fragile, such as the elderly; those with cardiac disease, renal disease and cirrhosis; or those who are generally at high risk for surgery. Subtotal cholecystectomy is safe and represents a valuable option in cases of difficult gallbladder removal.Conclusions, knowledge gaps and research recommendationsELC has a central role in the management of patients with ACC. The value of surgical treatment for high-risk patients should lead to a distinction between high-risk patients and patients who are not suitable for surgery. Further evidence on the role of clinical judgement and the use of clinical scores as adjunctive tools to guide treatment of high-risk patients and patients who are not suitable for surgery is required. The development of local policies for safe laparoscopic cholecystectomy is recommended.

Background Liver transplantation is considered the definitive treatment for people with liver failure. As part of post-liver transplantation management, immunosuppression (suppressing the host immunity) is given to prevent graft rejections. Immunosuppressive drugs can be classified into those that are used for a short period during the beginning phase of immunosuppression (induction immunosuppression) and those that are used over the entire lifetime of the individual (maintenance immunosuppression), because it is widely believed that graft rejections are more common during the first few months after liver transplantation. Some drugs such as glucocorticosteroids may be used for both induction and maintenance immunosuppression because of their multiple modalities of action. There is considerable uncertainty as to whether induction immunosuppression is necessary and if so, the relative efficacy of different immunosuppressive agents. Objectives To assess the comparative benefits and harms of different induction immunosuppressive regimens in adults undergoing liver transplantation through a network meta-analysis and to generate rankings of the different induction immunosuppressive regimens according to their safety and efficacy. Search methods We searched CENTRAL, MEDLINE, Embase, Science Citation Index Expanded, World Health Organization International Clinical Trials Registry Platform, and trials registers until July 2019 to identify randomised clinical trials in adults undergoing liver transplantation. Selection criteria We included only randomised clinical trials (irrespective of language, blinding, or status) in adults undergoing liver transplantation. We excluded randomised clinical trials in which participants had multivisceral transplantation and those who already had graft rejections. Data collection and analysis We performed a network meta-analysis with OpenBUGS using Bayesian methods and calculated the odds ratio (OR), rate ratio, and hazard ratio (HR) with 95% credible intervals (CrIs) based on an available case analysis, according to National Institute of Health and Care Excellence Decision Support Unit guidance. Main results We included a total of 25 trials (3271 participants; 8 treatments) in the review. Twenty-three trials (3017 participants) were included in one or more outcomes in the review. The trials that provided the information included people undergoing primary liver transplantation for various indications and excluded those with HIV and those with renal impairment. The follow-up in the trials ranged from three to 76 months, with a median follow-up of 12 months among trials. All except one trial were at high risk of bias, and the overall certainty of evidence was very low. Overall, approximately 7.4% of people who received the standard regimen of glucocorticosteroid induction died and 12.2% developed graft failure. All-cause mortality and graft failure was lower with basiliximab compared with glucocorticosteroid induction: all-cause mortality (HR 0.53, 95% CrI 0.31 to 0.93; network estimate, based on 2 direct comparison trials (131 participants; low-certainty evidence)); and graft failure (HR 0.44, 95% CrI 0.28 to 0.70; direct estimate, based on 1 trial (47 participants; low-certainty evidence)). There was no evidence of differences in all-cause mortality and graft failure between other induction immunosuppressants and glucocorticosteroids in either the direct comparison or the network meta-analysis (very low-certainty evidence). There was also no evidence of differences in serious adverse events (proportion), serious adverse events (number), renal failure, any adverse events (proportion), any adverse events (number), liver retransplantation, graft rejections (any), or graft rejections (requiring treatment) between other induction immunosuppressants and glucocorticosteroids in either the direct comparison or the network meta-analysis (very low-certainty evidence). However, because of the wide CrIs, clinically important differences in these outcomes cannot be ruled out. None of the studies reported health-related quality of life. Funding the source of funding for 14 trials was drug companies who would benefit from the results of the study; two trials were funded by neutral organisations who have no vested interests in the results of the study; and the source of funding for the remaining nine trials was unclear. Authors' conclusions Based on low-certainty evidence, basiliximab induction may decrease mortality and graft failure compared to glucocorticosteroids induction in people undergoing liver transplantation. However, there is considerable uncertainty about this finding because this information is based on small trials at high risk of bias. The evidence is uncertain about the effects of different induction immunosuppressants on other clinical outcomes, including graft rejections. Future randomised clinical trials should be adequately powered, employ blinding, avoid post-randomisation dropouts (or perform intention-to-treat analysis), and use clinically important outcomes such as mortality, graft failure, and health-related quality of life.

SnRK2 kinases, PP2C phosphatases and the PYR/PYL/RCAR receptors constitute the core abscisic acid (ABA) signaling module that is thought to contain all of the intrinsic properties to self-regulate the hormone signal output. Here we identify Casein Kinase (CK)2 as a novel negative regulator of SnRK2. CK2 phosphorylates a cluster of conserved serines at the ABA box of SnRK2, increasing its binding to PP2C and triggering protein degradation. Consequently, CK2 action has implications on SnRK2 protein levels, as well as kinase activity and its response to abiotic stimuli., This work was supported by MCYT, Spain (Consolider-Ingenio 2010CSD2007-00036, BIO2009-13044); Comissionat per Universitats i Recerca de la Generalitat de Catalunya (CIRIT2009SGR626); Fundação para a Ciência e Tecnologia (PhD grant SFRH/BD/62070/2009); and European Union Marie-Curie (Early Stage Training Fellowships MEST-CT-2005-020232-2 ADONIS).

In the opening salvos of his engaging article, How Stomata Resolve the Dilemma of Opposing Priorities, Raschke (1976) wrote that “Land plants are in a dilemma throughout their lives: assimilation of CO2 from the atmosphere requires intensive gas exchange; the prevention of excessive water loss demands that gas exchange be kept low.” What seemed a rarefied musing of a plant academic nearly 40 years ago has since broadened into global environmental concerns.

This is a commentary on an accepted manuscript by Ye et al., requested by Plant Physiology.

The combined daily consumption of fresh water ranges from 200 to 700 liters per capita per day in most developed countries, with about 70% being used for agricultural needs. Unlike other resources such as the different forms of energy, water has no other alternatives. With the looming prospect of global water crisis, the recent laudable success in deciphering the early steps in the signal transduction of the "stress hormone" abscisic acid (ABA) has ignited hopes that crops can be engineered with the capacity to maintain productivity while requiring less water input. Although ABA was first discovered in plants, it has resurfaced in the human brain (and many other non-plant organisms : sea sponge, some parasites, hydra to name a few), suggesting that its existence may be widespread. In humans, more amazingly, ABA has shown anti-inflammatory and antiviral properties. Even its receptors and key signaling intermediates have homologs in the human genome suggesting that evolution has re-fashioned these same proteins into new functional contexts. Thus, learning about the molecular mechanisms of ABA in action using the more flexible plant model will be likely beneficial to other organisms, and especially in human diseases, which is topical in the medical circle. ABA can accumulate up to 10 to 30-fold in plants under drought stress relative to unstressed conditions. The built up of the hormone then triggers diverse adaptive pathways permitting plants to withstand temporary bouts of water shortage. One favorite experimental model to unravel ABA signaling mechanisms in all of its intimate detail is based on the hormone's ability to elicit stomatal closure - a rapid cellular response of land plants to limit water loss through transpiration. Each microscopic stoma, or pore, is contoured by two specialized kidney-shaped cells called the guard cells. Because land plants are protected by a waxy cuticle impermeable to gas exchange, the stomatal pores are thus the primary portals for photosynthetic CO(2) uptake. Drought, by biasing pathways that lead to rapid closure of these pores, has therefore a negative impact on photosynthesis, and consequently, biomass as well. The stomatal aperture widens and narrows by expansion and contraction, respectively, of these flanking guard cells caused by changes in the intracellular concentrations of ion fluxes. These transport mechanisms most likely share fundamental principles with any excitable cell. These events require coordination of channels, vacuolar and membrane transporters that generate a specific pattern of electrical signals that relay the ABA stimulus. Research on ABA begun in the 1960's has now been crowned by the achievement of having identified the soluble ABA receptor that turns on and off the activities of a kinase/phosphatase pair, as the heart of the signaling complex. Results distilled from the latest structural studies on these ABA receptors, characterized by the so-called START domain, are beginning to tender the most exciting promise for rational design of agonists and antagonists towards modulating stress adaptive ability in plants. This review will chart the recent extraordinary progress that has enlightened us on how ABA controls membrane transport mechanisms that evoke the fast stomatal closing pathway.

The year 2009 marked a real turnaround in our understanding of the mode of abscisic acid (ABA) action. Nearly 25 years had elapsed since the first biochemical detection of ABA-binding proteins in the plasmalemma of Vicia guard cells was reported. This recent--and laudable--achievement is owed largely to the discovery of the soluble ABA receptors whose major interacting proteins happen to be some of the most well-established components of earliest steps in ABA signaling. These soluble receptors, with the double name of PYRABACTIN RESISTANCE (PYR) or REGULATORY COMPONENT OF ABA RECEPTOR (RCAR), are a family of Arabidopsis proteins of about 150-200 amino acids that share a conserved START domain. The ABA signal transduction circuitry under non-stress conditions is muted by the clade A protein phosphatases 2C (PP2C) (notably HAB1, ABI1, and ABI2). During the initial steps of ABA signaling, the binding of the hormone to the receptor induces a conformational change in the latter that allows it to sequester the PP2Cs. This excludes them from the negative regulation of the downstream ABA-activated kinases (OST1/SnRK2.6/SRK2E, SnRK2.2, and SnRK2.3), thus unleashing the pathway by freeing them to phosphorylate downstream targets that now include several b-ZIP transcription factors, ion channels (SLAC1, KAT1), and a NADPH oxidase (AtrbohF). The discovery of this family of soluble receptors and the rich insight already gained from structural studies of their complexes with different isoforms of ABA, PP2C, and the synthetic agonist pyrabactin lay the foundation towards rational design of chemical switches that can bolster drought hardiness in plants.

Stomatal guard cells are functionally specialized epidermal cells usually arranged in pairs surrounding a pore. Changes in ion fluxes, and more specifically osmolytes, within the guard cells drive opening/closing of the pore, allowing gas exchange while limiting water loss through evapo-transpiration. Adjustments of the pore aperture to optimize these conflicting needs are thus centrally important for land plants to survive, especially with the rise in CO(2) associated with global warming and increasing water scarcity this century. The basic biophysical events in modulating membrane transport have been gradually delineated over two decades. Genetics and molecular approaches in recent years have complemented and extended these earlier studies to identify major regulatory nodes. In Arabidopsis, the reference for guard cell genetics, stomatal opening driven by K(+) entry is mainly through KAT1 and KAT2, two voltage-gated K(+) inward-rectifying channels that are activated on hyperpolarization of the plasma membrane principally by the OST2 H(+)-ATPase (proton pump coupled to ATP hydrolysis). By contrast, stomatal closing is caused by K(+) efflux mainly through GORK, the outward-rectifying channel activated by membrane depolarization. The depolarization is most likely initiated by SLAC1, an anion channel distantly related to the dicarboxylate/malic acid transport protein found in fungi and bacteria. Beyond this established framework, there is also burgeoning evidence for the involvement of additional transporters, such as homologues to the multi-drug resistance proteins (or ABC transporters) as intimated by several pharmacological and reverse genetics studies. General inhibitors of protein kinases and protein phosphatases have been shown to profoundly affect guard cell membrane transport properties. Indeed, the first regulatory enzymes underpinning these transport processes revealed genetically were several protein phosphatases of the 2C class and the OST1 kinase, a member of the SnRK2 family. Taken together, these results are providing the first glimpses of an emerging signalling complex critical for modulating the stomatal aperture in response to environmental stimuli.

The mode of abscisic acid (ABA) action, and its relations to drought adaptive responses in particular, has been a captivating area of plant hormone research for much over a decade. The hormone triggers stomatal closure to limit water loss through transpiration, as well as mobilizes a battery of genes that presumably serve to protect the cells from the ensuing oxidative damage in prolonged stress. The signaling network orchestrating these various responses is, however, highly complex. This review summarizes several significant advances made within the last few years. The biosynthetic pathway of the hormone is now almost completely elucidated, with the latest identification of the ABA4 gene encoding a neoxanthin synthase, which seems essential for de novo ABA biosynthesis during water stress. This leads to the interesting question on how ABA is then delivered to perception sites. In this respect, regulated transport has attracted renewed focus by the unexpected finding of a shoot-to-root translocation of ABA during drought response, and at the cellular level, by the identification of a s-galactosidase that releases biologically active ABA from inactive ABA-glucose ester. Surprising candidate ABA receptors were also identified in the form of the Flowering Time Control Protein A (FCA) and the Chloroplastic Magnesium Protoporphyrin-IX Chelatase H subunit (CHLH) in chloroplast-nucleus communication, both of which have been shown to bind ABA in vitro. On the other hand, the protein(s) corresponding to the physiologically detectable cell-surface ABA receptor(s) is (are) still not known with certainty. Genetic and physiological studies based on the guard cell have reinforced the central importance of reversible phosphorylation in modulating rapid ABA responses. Sucrose Non-Fermenting Related Kinases (SnRK), Calcium-Dependent Protein Kinases (CDPK), Protein Phosphatases (PP) of the 2C and 2A classes figure as prominent regulators in this single-cell model. Identifying their direct in vivo targets of regulation, which may include H + -ATPases, ion channels, 14-3-3 proteins and transcription factors, will logically be the next major challenge. Emerging evidence also implicates ABA as a repressor of innate immune response, as hinted by the highly similar roster of genes elicited by certain pathogens and ABA. Undoubtedly, the most astonishing revelation is that ABA is not restricted to plants and mosses, but overwhelming evidence now indicates that it also exists in metazoans ranging from the most primitive to the most advance on the evolution scale (sponges to humans). In metazoans, ABA has healing properties, and plays protective roles against both environmental and pathogen related injuries. These cross-kingdom comparisons have shed light on the surprising ancient origin of ABA and its attendant mechanisms of signal transduction.

Plants rely on a diverse set of small-molecule hormones to regulate every aspect of their biological processes including development, growth, and adaptation. Since the discovery of the first plant hormone, auxin, hormones have always been at the frontier of plant biology. Although the physiological functions of most plant hormones have been studied for decades, the last 15–20 years have seen dramatic progress in our understanding of the molecular mechanisms of hormone actions. The publication of the whole-genome sequences of the model systems of Arabidopsis and rice, together with the advent of multidisciplinary approaches, has opened the door to successful experimentation on plant hormone actions.

Light activates proton (H(+))-ATPases in guard cells, to drive hyperpolarization of the plasma membrane to initiate stomatal opening, allowing diffusion of ambient CO(2) to photosynthetic tissues. Light to darkness transition, high CO(2) levels and the stress hormone abscisic acid (ABA) promote stomatal closing. The overall H(+)-ATPase activity is diminished by ABA treatments, but the significance of this phenomenon in relationship to stomatal closure is still debated. We report two dominant mutations in the OPEN STOMATA2 (OST2) locus of Arabidopsis that completely abolish stomatal response to ABA, but importantly, to a much lesser extent the responses to CO(2) and darkness. The OST2 gene encodes the major plasma membrane H(+)-ATPase AHA1, and both mutations cause constitutive activity of this pump, leading to necrotic lesions. H(+)-ATPases have been traditionally assumed to be general endpoints of all signaling pathways affecting membrane polarization and transport. Our results provide evidence that AHA1 is a distinct component of an ABA-directed signaling pathway, and that dynamic downregulation of this pump during drought is an essential step in membrane depolarization to initiate stomatal closure.

The Arabidopsis mitogen-activated kinase MPK6 transmits a diversity of stress signals. However, much less is known about resetting mechanisms subsequent to the stress response. We show that MPK6 is a potential target of abscisic acid-insensitive 1 (ABI1), a protein phosphatase 2C that acts as a key element in attenuating abscisic acid (ABA)-dependent stress signaling. MPK6 can bind to ABI1 in vitro and in yeast, and that a complex containing these two proteins can be co-precipitated from transfected Arabidopsis cells. In whole plants, MPK6 is hyper-reactive to osmotic stress in mutants compromised in ABI1 activity, but not in its closest functional homolog ABI2. Moreover, conditional expression of a dominant-negative form of mpk6 renders plants hypersensitive to ABA. MPK6 enhances the synthesis of ethylene [1]. ABI1 may therefore down modulate ethylene as part of the resetting mechanism after stress.

International audience; Faced with declining soil-water potential, plants synthesize abscisic acid (ABA), which then triggers stomatal closure to conserve tissue moisture. Closed stomates, however, also create several physiological dilemmas. Among these, the large CO2 influx required for net photosynthesis will be disrupted. Depleting CO2 in the plant will in turn bias stomatal opening by suppressing ABA sensitivity, which then aggravates transpiration further. We have investigated the molecular basis of how C3 plants resolve this H2 O-CO2 conflicting priority created by stomatal closure. Here, we have identified in Arabidopsis thaliana an early drought-induced spermidine spermine-N(1) -acetyltransferase homolog, which can slow ABA-mediated stomatal closure. Evidence from genetic, biochemical and physiological analyses has revealed that this protein does so by acetylating the metabolite 1,3-diaminopropane (DAP), thereby turning on the latter's intrinsic activity. Acetylated DAP triggers plasma membrane electrical and ion transport properties in an opposite way to those by ABA. Thus in adapting to low soil-water availability, acetyl-DAP could refrain stomates from complete closure to sustain CO2 diffusion to photosynthetic tissues.

The influence of the plant water-stress hormone abscisic acid (ABA) on anion channel activity and its interaction with protein kinase and phosphatase antagonists was examined in stomatal guard cells of wild-type Nicotiana benthamiana L. and of transgenic plants expressing the dominant-negative (mutant) Arabidopsis abi1-1 protein phosphatase. Intact guard cells were impaled with double-barrelled micro-electrodes and membrane current was recorded under voltage clamp in the presence of 15 mM CsCl and 15 mM tetraethylammonium chloride (TEA-Cl) to eliminate K+ channel currents. Under these conditions, the free-running voltage was situated close to 0 mV (+9 +/- 6 mV, n = 18) and the membrane under voltage clamp was dominated by anion channel current (ICl) as indicated from tall current reversal near the expected chloride equilibrium potential, current sensitivity to the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, and by its voltage-dependent kinetics. Pronounced activation of ICl was recorded on stepping from a conditioning voltage of -250 mV to voltages between -30 and +50 mV, and the current deactivated with a voltage-dependent halftime at more negative voltages (tau approximately equal to 0.3 sec at -150 mV). Challenge with 20 microM ABA increased the steady-state current conductance, gCl, near 0 mV by 1.2- to 2.6-fold and at -150 mV by 4.5- to sixfold with a time constant of 40 +/- 4 sec, and it slowed ICl deactivation as much as fourfold at voltages near -50 mV, introducing two additional voltage-sensitive kinetic components to these current relaxations. Neither the steady-state and kinetic characteristics of ICl nor its sensitivity to ABA were influenced by H7 or staurosporine, both broad-range protein kinase antagonists. However, the protein phosphatase 1/2A antagonist calyculin A mimicked the effects of ABA on gCl and current relaxations on its own and exhibited a synergistic interaction with ABA, enhancing ICl sensitivity to ABA three- to four-fold. Quantitatively similar current characteristics were recorded from guard cells of abi1-1 transgenic N. benthamiana, indicating that the abi1-1 protein phosphatase does not influence the anion current or its response to ABA directly. These results demonstrate that ABA stimulates ICl and modulates its voltage sensitivity. Furthermore, they show that ABA promotes ICl, either by introducing additional long-lived states of the channel or by activating a second anion channel with similar permeation characteristics but with a very long dwell time in the open state. Overall, the data are broadly consistent with the view that ABA action engenders coordinate control of ICl together with guard cell K+ channels to effect solute loss and stomatal closure.

BV was financed by Fundação para a Ciência e Tecnologia PhD grant SFRH/BD/62070/2009 and European Union Marie-Curie Early Stage Training Fellowships MEST-CT-2005-020232-2 ADONIS. This work was supported by Consolider-Ingenio 2010CSD2007-00036 from MCYT (Spain) and grants BIO2009-13044 from MCYT (Spain) and CIRIT2009SGR626 from Comissionat per Universitats i Recerca de la Generalitat de Catalunya to MP.

International audience; Plant mitogen-activated protein kinases (MAPKs) are involved in important processes, including stress signaling and development. In a functional yeast screen, we identified mutations that render Arabidopsis thaliana MAPKs constitutively active (CA). Importantly, CA-MAPKs maintain their specificity toward known activators and substrates. As a proof-of-concept, Arabidopsis MAPK4 (MPK4) function in plant immunity was investigated. In agreement with the phenotype of mpk4 mutants, CA-MPK4 plants were compromised in pathogen-induced salicylic acid accumulation and disease resistance. MPK4 activity was found to negatively regulate pathogen-associated molecular pattern-induced reactive oxygen species production but had no impact on callose deposition, indicating that CA-MPK4 allows discriminating between processes regulated by MPK4 activity from processes indirectly affected by mpk4 mutation. Finally, MPK4 activity was also found to compromise effector-triggered immunity conditioned by the Toll Interleukin-1 Receptor-nucleotide binding (NB)-Leu-rich repeat (LRR) receptors RPS4 and RPP4 but not by the coiled coil-NB-LRR receptors RPM1 and RPS2. Overall, these data reveal important insights on how MPK4 regulates plant defenses and establishes that CA-MAPKs offer a powerful tool to analyze the function of plant MAPK pathways.

The soluble receptors of abscisic acid (ABA) have been identified in Arabidopsis thaliana. The 14 proteins in this family, bearing the double name of PYRABACTIN RESISTANCE/PYRABACTIN-LIKE (PYR/PYL) or REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR) (collectively referred to as PYR/PYL/RCAR), contain between 150 and 200 amino acids with homology to the steroidogenic acute regulatory-related lipid transfer (START) protein. Structural studies of these receptors have provided rich insights into the early mechanisms of ABA signaling. The binding of ABA to PYR/PYL/RCAR triggers the pathway by inducing structural changes in the receptors that allows them to sequester members of the clade A negative regulating protein phosphatase 2Cs (PP2Cs). This liberates the class III ABA-activated Snf1-related kinases (SnRK2s) to phosphorylate various targets. In guard cells, a specific SnRK2, OPEN STOMATA 1 (OST), stimulates H(2)O(2) production by NADPH oxidase respiratory burst oxidase protein F and inhibits potassium ion influx by the inward-rectifying channel KAT1. OST1, the kinase CPK23, the calcium-dependent kinase CPK21, and the counteracting PP2Cs modulate the slow anion channel SLAC1, a pathway that contributes to stomatal responses to diverse stimuli, including ABA and carbon dioxide. A minimal ABA response pathway that leads to activation of the SLAC1 homolog, SLAH3, and presumably stomatal closure has been reconstituted in vitro. The identification of the soluble receptors and core components of the ABA signaling pathway provides promising targets for crop design with higher resilience to water deficit while maintaining biomass.

On water deficit, abscisic acid (ABA) induces stomata closure to reduce water loss by transpiration. To identify Arabidopsis thaliana mutants which transpire less on drought, infrared thermal imaging of leaf temperature has been used to screen for suppressors of an ABA-deficient mutant (aba3-1) cold-leaf phenotype. Three novel mutants, called hot ABA-deficiency suppressor (has), have been identified with hot-leaf phenotypes in the absence of the aba3 mutation. The defective genes imparted no apparent modification to ABA production on water deficit, were inherited recessively and enhanced ABA responses indicating that the proteins encoded are negative regulators of ABA signalling. All three mutants showed ABA-hypersensitive stomata closure and inhibition of root elongation with little modification of growth and development in non-stressed conditions. The has2 mutant also exhibited increased germination inhibition by ABA, while ABA-inducible gene expression was not modified on dehydration, indicating the mutated gene affects early ABA-signalling responses that do not modify transcript levels. In contrast, weak ABA-hypersensitivity relative to mutant developmental phenotypes suggests that HAS3 regulates drought responses by both ABA-dependent and independent pathways. has1 mutant phenotypes were only apparent on stress or ABA treatments, and included reduced water loss on rapid dehydration. The HAS1 locus thus has the required characteristics for a targeted approach to improving resistance to water deficit. In contrast to has2, has1 exhibited only minor changes in susceptibility to Dickeya dadantii despite similar ABA-hypersensitivity, indicating that crosstalk between ABA responses to this pathogen and drought stress can occur through more than one point in the signalling pathway.

Drought tolerance actually embodies several protective mechanisms deployed by plants commensurate with the stress severity and duration. Against mild drought, one of the most rapid defensive measures is the closing of the stomatal pore, caused by shrinking of the flanking guard cells, in order to reduce transpiration that accounts for ~ 90% of the water loss from plants. In contrast, the widening of the pore caused by expansion and bowing out of the guard cells is stimulated by light, permitting CO 2 entry for carbon fixation. It is clear, therefore, that the two most decisive factors in plant growth—photosynthesis and water consumption—are directly influenced by guard cell regulation. Drought induces the synthesis of the “stress” hormone abscisic acid (ABA). Work within the past two decades has outlined the signalling pathway consisting of phospho-relay and ion transport across membranes that link ABA reception to stomatal closure. The threshold of the stomatal response to stress may also be set by more long-term processes that include gene expression and epigenetic regulation (“stress memory”), implying a feedback integration between rapid and slower protective mechanisms. In this chapter, some of the most recent, insightful, and exciting findings in the signalling network that orchestrate these ABA-dependent adaptive processes will be related. We will also extend our discussion in applying ABA research—not on the more obvious agricultural benefits—but as a novel and potent modulator of the immune system in human.

A powerful means to learn about gene functions in a developmental or physiological context in an organism is to isolate the corresponding mutants with altered phenotypes. Diverse mutagenic agents, including chemical and biological, have been widely employed, and each comes with its own advantages and inconveniences. For Arabidopsis thaliana, whose genome sequence is publicly available, the reliance of reverse genetics to understand the relevant roles of genes particularly those coding for proteins in growth and development is now a common practice. Identifying multiple alleles at each locus is important because they can potentially reveal epistatic relationship in a signaling pathway or components belonging to a common signaling complex by their synergistic or even allele-specific enhancement of the phenotypic severity. In this article, we describe mutagenesis by using ethyl methanesulfonate (EMS) and transfer (T)-DNA-mediated insertion or activation tagging as applied to the most widely used genetic plant model A. thaliana. Also, we demonstrate the utility of several genetic screening approaches to dissect adaptive responses to various abiotic stresses.

A powerful means to learn about gene functions in a developmental or physiological context in an organism is to isolate the corresponding mutants with altered phenotypes. Diverse mutagenic agents, including chemical and biological, have been widely employed, and each comes with its own advantages and inconveniences. For Arabidopsis thaliana, whose genome sequence is publicly available, the reliance of reverse genetics to understand the relevant roles of genes particularly those coding for proteins in growth and development is now a common practice. Identifying multiple alleles at each locus is important because they can potentially reveal epistatic relationship in a signaling pathway or components belonging to a common signaling complex by their synergistic or even allele-specific enhancement of the phenotypic severity. In this article, we describe mutagenesis by using ethyl methanesulfonate (EMS) and transfer (T)-DNA-mediated insertion or activation tagging as applied to the most widely used genetic plant model A. thaliana. Also, we demonstrate the utility of several genetic screening approaches to dissect adaptive responses to various abiotic stresses.

The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.

The plant hormone abscisic acid (ABA) triggers production of reactive oxygen species (ROS) in guard cells via the AtrbohD and AtrbohF NADPH oxidases, leading to stomatal closure. The ABA-activated SnRK2 protein kinase open stomata 1 (OST1) (SRK2E/SnRK2.6) acts upstream of ROS in guard cell ABA signaling. Here, we report that OST1 phosphorylates Ser13 and Ser174 on AtrbohF. In addition, substitution of Ser174 to Ala results in a ∼40% reduction in the phosphorylation of AtrbohF by OST1. We also show that OST1 physically interacts with AtrbohF. These results provide biochemical evidence suggesting that OST1 regulates AtrbohF activity.Structured summaryMINT-7260179, MINT-7260147, MINT-7260165: OST1 (uniprotkb:Q940H6) phosphorylates (MI:0217) ATRBOHF (uniprotkb:O48538) by protein kinase assay (MI:0424)MINT-7260208: OST1 (uniprotkb:Q940H6) and ATRBOHF (uniprotkb:O48538) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)

The dual MAS1'-2' promoter regulating two divergently transcribed mannopine synthase genes has been widely employed in plant expression vectors. As part of an effort towards its rational design as a genetic engineering tool, we have undertaken a functional analysis of the promoter by deletion mutagenesis and by the use of hybrid promoter constructs. Our results indicate that the central region of the intergenic promoter is composed of at least four domains. Three of these contain complementary sequences, which can potentially hybridize to form alternative palindromic structures. These three domains can function cooperatively, and in an orientation-independent manner, in imparting a sevenfold higher expression level at the 2' end relative to the corresponding 1'. The remaining domain is characterized by tracts of repeated A/T-rich elements, and appears to confer the weak activity at the MAS1' promoter end. However, even though this A/T-rich DNA segment is functional, our deletion analysis provided strong evidence that it is completely dispensable for wild-type promoter activity. In addition, the relative distances between these enhancer domains and the 1'-2' TATA-proximal regions can have a pronounced influence on the level of expression in both directions. In young tobacco seedlings, the two promoter ends are expressed in similar, if not identical, tissues in the aerial parts of the plants, but major differences can be observed in roots. Transient expression assays using hybrid promoter constructs showed that cis-elements that can respond to auxin induction signals are redundant in nature, in that they are dispersed throughout the promoter and showed no obvious consensus sequence.

Most signaling networks are regulated by reversible protein phosphorylation. The specificity of this regulation depends in part on the capacity of protein kinases to recognize and efficiently phosphorylate particular sequence motifs in their substrates. Sequenced plant genomes potentially encode over than 1000 protein kinases, representing 4% of the proteins, twice the proportion found in humans. This plethora of plant kinases requires the development of high-throughput strategies to identify their substrates. In this study, we have implemented a semi-degenerate peptide array screen to define the phosphorylation preferences of four kinases from Arabidopsis thaliana that are representative of the plant calcium-dependent protein kinase and Snf1-related kinase superfamily. We converted these quantitative data into position-specific scoring matrices to identify putative substrates of these kinases in silico in protein sequence databases. Our data show that these kinases display related but nevertheless distinct phosphorylation motif preferences, suggesting that they might share common targets but are likely to have specific substrates. Our analysis also reveals that a conserved motif found in the stress-related dehydrin protein family may be targeted by the SnRK2-10 kinase. Our results indicate that semi-degenerate peptide array screening is a versatile strategy that can be used on numerous plant kinases to facilitate identification of their substrates, and therefore represents a valuable tool to decipher phosphorylation-regulated signaling networks in plants.

The Arabidopsis thaliana RNA binding protein UBA2a is the closest homologue of the Vicia faba AKIP1 (56% identity). Like AKIP1, UBA2a is a constitutively-expressed nuclear protein and in response to ABA it is also reorganized within the nucleus in “speckles” suggesting a possible role of this protein in the regulation of mRNA metabolism during ABA signaling. AKIP1 interacts with, and is phosphorylated by, the upstream ABA-activated protein kinase AAPK. We have investigated if a pathway similar to that described in Vicia faba also exists in Arabidopsis. Our results showed that despite the resemblance between the corresponding Vicia and Arabidopsis proteins, it appears that the function of UBA2a is independent of OST1 phosphorylation.

Drought stress is the major limitation to crop productivity. However, crops are genetically complex with many loci contributing quantitatively to a given physiological trait. Nonetheless, significant in-roads into the molecular mechanisms of drought-adaptive responses have been made from the use of Arabidopsis thaliana. In this special review, we will discuss results gleaned from reverse and forward genetic studies that revealed the involvement of both ABA-dependent and ABA-independent components. In particular, mutant analyses have highlighted the surprising prevalence of RNA metabolism in many key steps. We will also discuss our recent use of infrared thermography to visualize stomatal closure in response to dehydration as a means to identify novel regulatory genes. This has allowed us to recover mutations belonging to at least eight complementation groups. Analysis of six of these loci revealed that all of their corresponding mutations affect either abscisic acid (ABA) biosynthesis or perception. Hence, in contrast to molecular studies on gene networks which pointed to the clear existence of multiple ABA-independent pathways in the control of dehydration tolerance, our results reinforce ABA-based signalling pathways as the predominant factor in primary or rapid responses. Finally, we will provide some details learned from the molecular analysis of OPEN STOMATA1 (OST1), a gene that encodes an ABA-activated kinase issued from this targeted genetic approach.

▪ Abstract The plant hormone abscisic acid (ABA) plays a major role in seed maturation and germination, as well as in adaptation to abiotic environmental stresses. ABA promotes stomatal closure by rapidly altering ion fluxes in guard cells. Other ABA actions involve modifications of gene expression, and the analysis of ABA-responsive promoters has revealed a diversity of potential cis-acting regulatory elements. The nature of the ABA receptor(s) remains unknown. In contrast, combined biophysical, genetic, and molecular approaches have led to considerable progress in the characterization of more downstream signaling elements. In particular, substantial evidence points to the importance of reversible protein phosphorylation and modifications of cytosolic calcium levels and pH as intermediates in ABA signal transduction. Exciting advances are being made in reassembling individual components into minimal ABA signaling cascades at the single-cell level.

The Nobel chemist Roald Hoffmann described molecular mimicry as a mechanism founded on subversion and deception. On page 85 of this issue, Soon et al. ( 1 ) report intriguing evidence for such mimicry in the transmission of signals elicited by the plant hormone abscisic acid (ABA). Using the model plant Arabidopsis thaliana , the authors show that the ABA-bound receptor and kinase alternately bind to a phosphatase, turning on and off this stress-responsive pathway, respectively. Not surprisingly, ABA is behind this “subversive” dealing in partner swapping.

The understanding of how leguminous plants establish symbiosis with rhizobia is limited by the lack of genetic system in most legume plants. Here we propose a Medicago truncatula line suitable for genetic analysis of Medicago-Rhizobium meliloti symbiosis because of its high regeneration capacity and broad R. meliloti strain specificity. This line has been isolated by extensive in vitro screening of explants from several ecotypes of M. truncatula that are known to be autogamous and have small diploid genomes. One such derivative identified from ecotype 108-1 has gained the capacity to be readily regenerated in vitro. The derivative, called R108-1, like its parent, can establish effective symbiosis with several widely studied R. meliloti strains (Rm41, Rm2011, Rm1021, F51, and GR4). Importantly, the nodulation characteristics of the R108-1 line were not compromised by the in vitro selection procedure. We have further established the conditions for efficient transformation of R108-1 by co-cultivation with Agrobacterium tumefaciens. Thus, this in vitro-derived plant line has most of the favorable attributes, otherwise rare among legumes, of a complementary model plant system for investigating symbiosis at the genetic and the molecular levels.