EIN2 and COI1 control the antagonism between ethylene and jasmonate in adventitious rooting of Arabidopsis thaliana thin cell layers

Auxins induce adventitious roots (ARs) in numerous culture-systems, and indole-3-butyric acid (IBA) is frequently the best AR-inducer. Vitamin requirements vary according to species, explant, and culture-conditions. Arabidopsis thaliana thin cell layers (AtTCLs) are uncapable of AR-formation on hormone-free medium containing thiamine and myo-inositol, whereas ARs are induced when IBA (10 μM), with/without kinetin (Kin, 0.1 μM), is added. The research frst aim was to determine whether a synergism between IBA and myo-inositol and thiamine was necessary for AR-formation. Results showed that IBA induced AR-formation without myo-inositol and thiamine, but better when both vitamins were also present. Deciphering hormonal action on AR formation under optimal vitamin content would be essential for improving the AR process. Ethylene (ET)/jasmonic acid (JA) signaling cross-talk has been demonstrated as being involved in AR-formation in IBA+Kincultured AtTCLs, by using ein3eil1 and coi1-16 mutants. ETHYLENE INSENSITIVE3 (EIN3)/EIN3-LIKE1 (EIL1) are positive regulators of ethylene (ET)-signaling, whereas CORONATINE INSENSITIVE1 (COI1) is involved in JA-signaling. The ETHYLENE INSENSITIVE2 (EIN2) protein activates EIN3/EIL1 in ET-presence. To understand whether EIN2 was also involved, the AR-response of ein2-1 and coi1-16 TCLs was evaluated adding the ET-precursor 1-aminocyclopropane1-carboxylic acid (ACC, 0.1 μM) and/or the JA-donor methyl jasmonate (JAMe, 0.01 μM) to IBA+vitamins-containing medium. AR-formation was enhanced by JAMe, reduced by ACC, but unchanged by JAMe+ACC in the wild type TCLs, whereas remained similarly low in ein2-1 and coi1-16 under all treatments. Collectively, these results demonstrate that the antagonism between JA and ET in AR-formation from AtTCLs involves a cross-talk by EIN2 and COI1

Indole-3-butyric acid induces ectopic formation of metaxylem in the hypocotyl of Arabidopsis thaliana without conversion into indole-3-acetic acid and with a positive interaction with ethylene

The role of the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) and of the auxin-interacting phytohormone ethylene, on the ectopic formation of primary xylem (xylogenesis in planta) is still little known. In particular, auxin/ethylene-target tissue(s), modality of the xylary process (trans-differentiation vs. de novo formation), and the kind of ectopic elements formed (metaxylem vs. protoxylem) are currently unknown. It is also unclear whether IBA may act on the process independently of conversion into IAA. To investigate these topics, histological analyses were carried out in the hypocotyls of Arabidopsis wild type seedlings and ech2ibr10 and ein3eil1 mutants, which are blocked in IBA-to-IAA conversion and ethylene signalling, respectively. The seedlings were grown under darkness with either IAA or IBA, combined or not with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Adventitious root formation was also investigated because this process may compete with xylogenesis. Our results show that ectopic formation of protoxylem and metaxylem occurred as an indirect process starting from the pericycle periclinal derivatives of the hypocotyl basal part. IAA favoured protoxylem formation, whereas IBA induced ectopic metaxylem with ethylene cooperation through the EIN3EIL1 network. Ectopic metaxylem differentiation occurred independently of IBA-to-IAA conversion as mediated by ECH2 and IBR10, and in the place of IBA-induced adventitious root formation

Jasmonate and nitric oxide roles in the control of xylary cell formation and identity in Arabidopsis seedlings

In basal hypocotyls of dark-grown Arabidopsis seedlings, xylary cells may form from the pericycle as an alternative to another developmental program, i.e. adventitious roots. It is known that several hormones may induce xylogenesis, as jasmonic acid (JA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), which also affect xylary cell identity. Recent studies with the ethylene (ET)-perception mutant ein3eil1 and the ET-precursor 1-aminocyclopropane-1-carboxylic acid (ACC) have shown ET involvement in IBA induced ectopic metaxylem. Nitric oxide (NO) is a reactive free radical molecule, which acts as a messenger in several cell differentiation events, including programmed cell death, moreover it can be produced after IBA/IAA-treatments influencing JA signalling and interacting positively/negatively with ET. To date, NO involvement in ET/JA-mediated xylogenesis has never been investigated.The aim of the present research was to determine the involvement of JA, ET and NO in the control of endogenous/exogenous auxin-induced xylogenesis through a possible crosstalk mediated by EIN3/EIL1. To this aim, ectopic xylem formation was investigated in the hypocotyl of dark-grown Arabidopsis seedlings exposed to various concentrations of JA methyl-ester (JAMe) with/without ACC, IBA or IAA. The xylogenic response in the wild-type (wt) was compared with that of the ein3eil1 mutant, the NO signal was quantified and the its role evaluated by measuring the effects of treatments with a NO donor/scavenger (SNP/cPTIO). Results show that the ectopic formation of protoxylem was enhanced in the wt by JAMe when applied alone at a specific concentration (i.e. 10μM), whereas in ein3eil1 mutant it occurred with any JAMe concentration (i.e. 0.01, 1 and 10 μM). This stimulation of xylary elements mediated by JAMe suggests that a negative interaction between JA and ET-signalling is involved in this developmental program. The negative interaction was confirmed by the reduction in xylogenesis observed in the wt after the combined application of JAMe with ACC, in comparison with JAMe alone. Nitric oxide was detected at early stages of both xylogenesis and adventitious rooting in the hypocotyl pericycle cells and its production was highly enhanced by JAMe at the highest concentration, combined or not with IBA (10 μM). Histological analyses showed that the xylary identity changed when JAMe was applied with each auxin in comparison with treatments with auxin alone. In addition, the IBA/IAA-induced adventitious rooting was increased by the same JAMe concentration enhancing xylogenesis when applied alone. This suggests a role for JA in modulating both developmental programs (adventitious rooting and xylogenesis) in the same target cells (hypocotyl pericycle cells), through an interaction with NO, as summarized in the model proposed (Fig. 1)

Jasmonates, ethylene and brassinosteroids control adventitious and lateral rooting as stress avoidance responses to heavy metals and metalloids

Developmental and environmental signaling networks often converge during plant growth in response to changing conditions. Stress-induced hormones, such as jasmonates (JAs), can influence growth by crosstalk with other signals like brassinosteroids (BRs) and ethylene (ET). Nevertheless, it is unclear how avoidance of an abiotic stress triggers local changes in development as a response. It is known that stress hormones like JAs/ET and BRs can regulate the division rate of cells from the first asymmetric cell divisions (ACDs) in meristems, suggesting that stem cell activation may take part in developmental changes as a stress-avoidance-induced response. The root system is a prime responder to stress conditions in soil. Together with the primary root and lateral roots (LRs), adventitious roots (ARs) are necessary for survival in numerous plant species. AR and LR formation is affected by soil pollution, causing substantial root architecture changes by either depressing or enhancing rooting as a stress avoidance/survival response. Here, a detailed overview of the crosstalk between JAs, ET, BRs, and the stress mediator nitric oxide (NO) in auxin-induced AR and LR formation, with/without cadmium and arsenic, is presented. Interactions essential in achieving a balance between growth and adaptation to Cd and As soil pollution to ensure survival are reviewed here in the model species Arabidopsis and rice

Nitric oxide cooperates with auxin to mitigate the alterations in the root system caused by cadmium and arsenic

Oryza sativa L. is a worldwide food-crop frequently growing in cadmium (Cd)/arsenic (As) polluted soils, with its root-system as the first target of the pollutants. Root-system development involves the establishment of optimal indole-3-acetic acid (IAA) levels, also requiring the conversion of the IAA natural precursor indole-3-butyric acid (IBA) into IAA, causing nitric oxide (NO) formation. Nitric oxide is a stress-signaling molecule. In rice, a negative interaction of Cd or As with endogenous auxin has been demonstrated, as some NO protective effects. However, a synergism between the natural auxins (IAA and/or IBA) and NO was not yet determined and might be important for ameliorating rice metal(oid)-tolerance. With this aim, the stress caused by Cd/As toxicity in the root cells and the possible recovery by either NO or auxins (IAA/IBA) were evaluated after Cd or As (arsenate) exposure, combined or not with the NO-donor compound sodium-nitroprusside (SNP). Root fresh weight, membrane electrolyte leakage, and H2O2 production were also measured. Moreover, endogenous IAA/IBA contents, transcription-levels of OsYUCCA1 and OsASA2 IAA-biosynthetic-genes, and expression of the IAA-influx-carrier OsAUX1 and the IAA-responsive DR5::GUS construct were analyzed, and NO-epifluorescence levels were measured. Results showed that membrane injury by enhanced electrolyte leakage occurred under both pollutants and was reduced by the treatment with SNP only in Cd-presence. By contrast, no membrane injury was caused by either exogenous NO or IAA or IBA. Cd- and As-toxicity also resulted into a decreased root fresh weight, mitigated by the combination of each pollutant with either IAA or IBA. Cd and As decreased the endogenous NO-content, increased H2O2 formation, and altered auxin biosynthesis, levels and distribution in both adventitious (ARs) and mainly lateral roots (LRs). The SNP-formed NO counteracted the pollutants’ effects on auxin distribution/levels, reduced H2O2 formation in Cd-presence, and enhanced AUX1-expression, mainly in As-presence. Each exogenous auxin, but mainly IBA, combined with Cd or As at 10 µM, mitigated the pollutants’ effects by increasing LR-production and by increasing NO-content in the case of Cd. Altogether, results demonstrate that NO and auxin(s) work together in the rice root system to counteract the specific toxic-effects of each pollutant

Brassinosteroids interact with nitric oxide in the response of rice root systems to arsenic stress

Brassinosteroids (BRs), an emerging class of phytohormones, affect numerous plant physiological and metabolic processes and can improve plant defense systems to counteract metalloid phytotoxicity. Nitric oxide (NO), a reactive nitrogen species (RNS), behaves as a signalling molecule activating plant cellular responses to various environmental conditions. Brassinosteroids induce NO synthesis through nitrate reductase (NR) and NO synthase (NOS) activities. Arsenite and arsenate, inorganic forms of the metalloid arsenic (As), cause both soil pollution and many disorders in numerous plants, including important crops like rice, due to the oxidative stress generated by the imbalance between RNS and reactive oxygen species (ROS). Rice is very susceptible to As toxicity because both As availability and solubility are high in flooded paddy fields in many cultivated areas. The research aims to investigate the effects of BRs on the rice root systems exposed to 10-4 M Na2HAsO40.7 H2O [As(V)] or 2.5 × 10-5 M NaAsO2 [As(III)], highlighting the induced cyto-histological events and dissecting the NO role in the root response. A specific concentration (10-7 M) of 24-epibrassinolide (24-eBL), an exogenously applied BR, increases lateral root (LR) formation of more than 50% in the presence of As(III) or As(V). In addition, eBL attenuates the thickening of the cell walls induced by As in the outermost root cortical layers of LRs and in the adventitious roots (ARs) by reducing of ⁓ 50% the lignin deposition, while it restores the As(v)-altered NO levels by increasing OsNOS1 expression and the cellular NO distribution

Jasmonate promotes auxin-induced adventitious rooting in dark-grown Arabidopsis thaliana seedlings and stem thin cell layers by a cross-talk with ethylene signalling and a modulation of xylogenesis

Background: Adventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA + Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA + Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored. Results: MeJA, at 0.01 μM, enhances AR-formation, when combined with IBA + Kin, and the response of the early-JA-biosynthesis mutant dde2–2 and the JA-signalling mutant coi1–16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression. Conclusions: The JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species

High Risk of Secondary Infections Following Thrombotic Complications in Patients With COVID-19

Background. This study’s primary aim was to evaluate the impact of thrombotic complications on the development of secondary infections. The secondary aim was to compare the etiology of secondary infections in patients with and without thrombotic complications. Methods. This was a cohort study (NCT04318366) of coronavirus disease 2019 (COVID-19) patients hospitalized at IRCCS San Raffaele Hospital between February 25 and June 30, 2020. Incidence rates (IRs) were calculated by univariable Poisson regression as the number of cases per 1000 person-days of follow-up (PDFU) with 95% confidence intervals. The cumulative incidence functions of secondary infections according to thrombotic complications were compared with Gray’s method accounting for competing risk of death. A multivariable Fine-Gray model was applied to assess factors associated with risk of secondary infections. Results. Overall, 109/904 patients had 176 secondary infections (IR, 10.0; 95% CI, 8.8–11.5; per 1000-PDFU). The IRs of secondary infections among patients with or without thrombotic complications were 15.0 (95% CI, 10.7–21.0) and 9.3 (95% CI, 7.9–11.0) per 1000-PDFU, respectively (P = .017). At multivariable analysis, thrombotic complications were associated with the development of secondary infections (subdistribution hazard ratio, 1.788; 95% CI, 1.018–3.140; P = .043). The etiology of secondary infections was similar in patients with and without thrombotic complications. Conclusions. In patients with COVID-19, thrombotic complications were associated with a high risk of secondary infections

An explainable model of host genetic interactions linked to COVID-19 severity

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as "Respiratory or thoracic disease", supporting their link with COVID-19 severity outcome.A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19