Alternative splicing as a driver of natural variation in abscisic acid response.

Abscisic acid (ABA) is a crucial player in plant responses to the environment. It accumulates under stress, activating downstream signaling to implement molecular responses that restore homeostasis. Natural variance in ABA sensitivity remains barely understood, and the ABA pathway has been mainly studied at the transcriptional level, despite evidence that posttranscriptional regulation, namely, via alternative splicing, contributes to plant stress tolerance. Here, we identified the Arabidopsis accession Kn-0 as less sensitive to ABA than the reference Col-0, as shown by reduced effects of the hormone on seedling establishment, root branching, and stomatal closure, as well as by decreased induction of ABA marker genes. An in-depth comparative transcriptome analysis of the ABA response in the two variants revealed lower expression changes and fewer genes affected for the least ABA-sensitive ecotype. Notably, Kn-0 exhibited reduced levels of the ABA-signaling SnRK2 protein kinases and lower basal expression of ABA-reactivation genes, consistent with our finding that Kn-0 contains less endogenous ABA than Col-0. ABA also markedly affected alternative splicing, primarily intron retention, with Kn-0 being less responsive regarding both the number and magnitude of alternative splicing events, particularly exon skipping. We find that alternative splicing introduces a more ecotype-specific layer of ABA regulation and identify ABA-responsive splicing changes in key ABA pathway regulators that provide a functional and mechanistic link to the differential sensitivity of the two ecotypes. Our results offer new insight into the natural variation of ABA responses and corroborate a key role for alternative splicing in implementing ABA-mediated stress responses.

The SCL30a SR protein regulates ABA-dependent seed traits and germination under stress.

SR proteins are conserved RNA-binding proteins best known as splicing regulators that have also been implicated in other steps of gene expression. Despite mounting evidence for a role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain poorly understood. Here we show that the plant-specific SCL30a SR protein negatively regulates ABA signaling to control seed traits and stress responses during germination in Arabidopsis. Transcriptome-wide analyses revealed that loss of SCL30a function barely affects splicing, but largely induces ABA-responsive gene expression and genes repressed during germination. Accordingly, scl30a mutant seeds display delayed germination and hypersensitivity to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA and salt stress sensitivity. An ABA biosynthesis inhibitor rescues the enhanced mutant seed stress sensitivity, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the gene promotes seed germination under stress by reducing sensitivity to the phytohormone. Our results reveal a new player in ABA-mediated control of early development and stress response.

Tailoring photomorphogenic markers to organ growth dynamics.

When a dark-germinated seedling reaches the soil surface and perceives sunlight for the first time, light signaling is activated to adapt the plant's development and transition to autotrophism. During this process, functional chloroplasts assemble in the cotyledons and the seedling's cell expansion pattern is rearranged to enhance light perception. Hypocotyl cells expand rapidly in the dark, while cotyledon cell expansion is suppressed. However, light reverses this pattern by activating cell expansion in cotyledons and repressing it in hypocotyls. The fact that light-regulated developmental responses, as well as the transcriptional mechanisms controlling them, are organ-specific has been largely overlooked in previous studies of seedling de-etiolation. To analyze the expansion pattern of the hypocotyl and cotyledons separately in a given Arabidopsis (Arabidopsis thaliana) seedling, we define an organ ratio, the morphogenic index (MI), which integrates either phenotypic or transcriptomic data for each tissue and provides an important resource for functional analyses. Moreover, based on this index, we identified organ-specific molecular markers to independently quantify cotyledon and hypocotyl growth dynamics in whole-seedling samples. The combination of these marker genes with those of other developmental processes occurring during de-etiolation will allow improved molecular dissection of photomorphogenesis. Along with organ growth markers, this MI contributes a key toolset to unveil and accurately characterize the molecular mechanisms controlling seedling growth.

The Arabidopsis SR45 Splicing Factor, a Negative Regulator of Sugar Signaling, Modulates SNF1-Related Protein Kinase 1 Stability.

The ability to sense and respond to sugar signals allows plants to cope with environmental and metabolic changes by adjusting growth and development accordingly. We previously reported that the SR45 splicing factor negatively regulates glucose signaling during early seedling development in Arabidopsis thaliana Here, we show that under glucose-fed conditions, the Arabidopsis sr45-1 loss-of-function mutant contains higher amounts of the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1) despite unaffected SnRK1 transcript levels. In agreement, marker genes for SnRK1 activity are upregulated in sr45-1 plants, and the glucose hypersensitivity of sr45-1 is attenuated by disruption of the SnRK1 gene. Using a high-resolution RT-PCR panel, we found that the sr45-1 mutation broadly targets alternative splicing in vivo, including that of the SR45 pre-mRNA itself. Importantly, the enhanced SnRK1 levels in sr45-1 are suppressed by a proteasome inhibitor, indicating that SR45 promotes targeting of the SnRK1 protein for proteasomal destruction. Finally, we demonstrate that SR45 regulates alternative splicing of the Arabidopsis 5PTase13 gene, which encodes an inositol polyphosphate 5-phosphatase previously shown to interact with and regulate the stability of SnRK1 in vitro, thus providing a mechanistic link between SR45 function and the modulation of degradation of the SnRK1 energy sensor in response to sugars.

Pre-mRNA splicing repression triggers abiotic stress signaling in plants.

Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants.

Intron retention in the 5'UTR of the novel ZIF2 transporter enhances translation to promote zinc tolerance in arabidopsis.

Root vacuolar sequestration is one of the best-conserved plant strategies to cope with heavy metal toxicity. Here we report that zinc (Zn) tolerance in Arabidopsis requires the action of a novel Major Facilitator Superfamily (MFS) transporter. We show that ZIF2 (Zinc-Induced Facilitator 2) localises primarily at the tonoplast of root cortical cells and is a functional transporter able to mediate Zn efflux when heterologously expressed in yeast. By affecting plant tissue partitioning of the metal ion, loss of ZIF2 function exacerbates plant sensitivity to excess Zn, while its overexpression enhances Zn tolerance. The ZIF2 gene is Zn-induced and an intron retention event in its 5'UTR generates two splice variants (ZIF2.1 and ZIF2.2) encoding the same protein. Importantly, high Zn favours production of the longer ZIF2.2 transcript, which compared to ZIF2.1 confers greater Zn tolerance to transgenic plants by promoting higher root Zn immobilization. We show that the retained intron in the ZIF2 5'UTR enhances translation in a Zn-responsive manner, markedly promoting ZIF2 protein expression under excess Zn. Moreover, Zn regulation of translation driven by the ZIF2.2 5'UTR depends largely on a predicted stable stem loop immediately upstream of the start codon that is lost in the ZIF2.1 5'UTR. Collectively, our findings indicate that alternative splicing controls the levels of a Zn-responsive mRNA variant of the ZIF2 transporter to enhance plant tolerance to the metal ion.

A major facilitator superfamily transporter plays a dual role in polar auxin transport and drought stress tolerance in Arabidopsis.

Many key aspects of plant development are regulated by the polarized transport of the phytohormone auxin. Cellular auxin efflux, the rate-limiting step in this process, has been shown to rely on the coordinated action of PIN-formed (PIN) and B-type ATP binding cassette (ABCB) carriers. Here, we report that polar auxin transport in the Arabidopsis thaliana root also requires the action of a Major Facilitator Superfamily (MFS) transporter, Zinc-Induced Facilitator-Like 1 (ZIFL1). Sequencing, promoter-reporter, and fluorescent protein fusion experiments indicate that the full-length ZIFL1.1 protein and a truncated splice isoform, ZIFL1.3, localize to the tonoplast of root cells and the plasma membrane of leaf stomatal guard cells, respectively. Using reverse genetics, we show that the ZIFL1.1 transporter regulates various root auxin-related processes, while the ZIFL1.3 isoform mediates drought tolerance by regulating stomatal closure. Auxin transport and immunolocalization assays demonstrate that ZIFL1.1 indirectly modulates cellular auxin efflux during shootward auxin transport at the root tip, likely by regulating plasma membrane PIN2 abundance. Finally, heterologous expression in yeast revealed that ZIFL1.1 and ZIFL1.3 share H(+)-coupled K(+) transport activity. Thus, by determining the subcellular and tissue distribution of two isoforms, alternative splicing dictates a dual function for the ZIFL1 transporter. We propose that this MFS carrier regulates stomatal movements and polar auxin transport by modulating potassium and proton fluxes in Arabidopsis cells.

The major facilitator superfamily transporter ZIFL2 modulates cesium and potassium homeostasis in Arabidopsis.

Potassium (K(+)) is an essential mineral nutrient for plant growth and development, with numerous membrane transporters and channels having been implicated in the maintenance and regulation of its homeostasis. The cation cesium (Cs(+)) is toxic for plants but shares similar chemical properties to the K(+) ion and hence competes with its transport. Here, we report that K(+) and Cs(+) homeostasis in Arabidopsis thaliana also requires the action of ZIFL2 (Zinc-Induced Facilitator-Like 2), a member of the Major Facilitator Superfamily (MFS) of membrane transporters. We show that the Arabidopsis ZIFL2 is a functional transporter able to mediate K(+) and Cs(+) influx when heterologously expressed in yeast. Promoter-reporter, reverse transcription-PCR and fluorescent protein fusion experiments indicate that the predominant ZIFL2.1 isoform is targeted to the plasma membrane of endodermal and pericyle root cells. ZIFL2 loss of function and overexpression exacerbate and alleviate plant sensitivity, respectively, upon Cs(+) and excess K(+) supply, also influencing Cs(+) whole-plant partitioning. We propose that the activity of this Arabidopsis MFS carrier promotes cellular K(+) efflux in the root, thereby restricting Cs(+)/K(+) xylem loading and subsequent root to shoot translocation under conditions of Cs(+) or high K(+) external supply.

Low doses of haloperidol combined with ondansetron are not effective for prophylaxis of postoperative nausea and vomiting in susceptible patients.

BACKGROUND: In this observational study we reviewed the efficacy and side effects of different antiemetic combinations used in our hospital for postoperative nausea and vomiting (PONV) prophylaxis in high-risk women undergoing highly emetogenic surgery. METHODS: After reviewing retrospectively the medical records of patients undergoing highly emetogenic elective surgeries under general anaesthesia, we selected 368 women whose Apfel risk score was ≥ 3 and receiving a combination of 2 antiemetics for PONV prophylaxis. We analysed the incidence of PONV at 2, 6, 12 and 24h after surgery, antiemetic rescue requirements, pattern of occurrence of PONV, side effects and level of sedation were also assessed. The main goal was complete response defined as no PONV within 24h after surgery. RESULTS: Ondansetron 4mg i.v. plus dexamethasone 8mg i.v. (O&Dex), haloperidol 1mg i.v. (O&Hal1), haloperidol 2mg i.v. (O&Hal2) or droperidol 1.25mg i.v. (O&Dro) were the combinations most frequently used. The complete response was better in groups O&Dex: 68.5% (CI: 58-78), O&Hal2: 64.1% (CI: 53-74) and O&Dro 63% (CI: 52-73) than in group O&Hal1: 41.3% (CI: 31-52) (p<0,01). Peak incidence of PONV occurred within the 2-6h period. The incidence of side effects was higher in group O&Hal2. CONCLUSION: In high risk patients for PONV who underwent highly emetogenic surgeries, the efficacy of low-dose haloperidol (1mg) in combination is limited. Higher doses (2mg) are more effective but its use is associated with a high incidence of side effects.

Abscisic acid (ABA) regulation of Arabidopsis SR protein gene expression.

Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses.

ABA signaling prevents phosphodegradation of the SR45 splicing factor to alleviate inhibition of early seedling development in Arabidopsis.

Serine/arginine-rich (SR) proteins are conserved splicing regulators that play important roles in plant stress responses, namely those mediated by the abscisic acid (ABA) hormone. The Arabidopsis thaliana SR-like protein SR45 is a described negative regulator of the ABA pathway during early seedling development. How the inhibition of growth by ABA signaling is counteracted to maintain plant development under stress conditions remains largely unknown. Here, we show that SR45 overexpression reduces Arabidopsis sensitivity to ABA during early seedling development. Biochemical and confocal microscopy analyses of transgenic plants expressing fluorescently tagged SR45 revealed that exposure to ABA dephosphorylates the protein at multiple amino acid residues and leads to its accumulation, due to SR45 stabilization via reduced ubiquitination and proteasomal degradation. Using phosphomutant and phosphomimetic transgenic Arabidopsis lines, we demonstrate the functional relevance of ABA-mediated dephosphorylation of a single SR45 residue, T264, in antagonizing SR45 ubiquitination and degradation to promote its function as a repressor of seedling ABA sensitivity. Our results reveal a mechanism that negatively autoregulates ABA signaling and allows early plant growth under stress via posttranslational control of the SR45 splicing factor.

Marie Poussepin's Influence on Nursing from Her Vocation of Service and Charity.

Objective: This work sought to describe the influence of Marie Poussepin on Nursing from her vocation of service and charity. Methods: Historical-hermeneutic study with participation by 15 Dominican Sisters of Charity in the Presentation of the Blessed Virgin from the city of Manizales and Bogotá, Colombia, who answered semi-structured interviews. The information was gathered and recontextualized via the open and axial coding system through ATLAS.ti9 software. During the interpretation procedure, copying, intensive reading, note taking, analysis, first epigraph of the report, coding, grouping, and determination of categories was made, conducting information triangulation with existing evidence. Results: Three categories emerged: Responding to the call of Jesus through service to the community; Under the legacy of charity, respect for life and the dignity of human beings, and Caring for life as a foundation of nursing. The second category formulated the description that integrates the course of life and objectives of the institutions where the nuns interviewed work. Conclusion: The legacy by Marie Poussepin to nursing care has been manifested since the foundation of the work, influencing the disciplinary work through the vocational commitment of those who make up the congregation, imprinting a character of service and respect for others, in response to the love of God.

Effects of Socio-educational Interventions on the Quality of Life of People with a Digestive Ostomy.

Introduction: The creation of digestive stomata is associated with a change in people's lives. Causing a strong impact that influences all dimensions of life. This paper reports on the effects of socio-educational interventions on the quality of life (QOL) of people with a digestive ostomy. Objective: To describe the effect of a nursing intervention on the QOL of people with a digestive ostomy. Methods: A quasiexperimental study was conducted whose sample consisted of 12 people who were ostomized in a public hospital in Colombia. Participants were selected through convenience sampling and randomly assigned to the intervention group and the control group. For the control group, an educational process was carried out through a theoretical session with virtual mediation (educational video). The QOL was evaluated before and after the interventions through the Montreux questionnaire. Results: The average age was 57(+7) years. No statistical differences were found between the groups in the QOL index or for any of the dimensions that make up QOL. Pretest and post-test analysis for each intervention separately showed improvement in two dimensions of QOL for each group; in the intervention group body image dimension (p = .017) and the positive coping dimension (p = .027). In the control group, the physical well-being dimension (p = .037) and social concerns dimension (p = .034). Conclusions: The personalized educational intervention or carried out through virtual pedagogical mediation, generated a clinically significant increase in the dimensions of QOL, without statistical differences. The study adds knowledge about the impact that digestive stomata have on the QOL, which is why it is necessary to establish specialized interdisciplinary teams to care for the person's new condition at home during the following months.

Etiolated Hypocotyls: A New System to Study the Impact of Abiotic Stress on Cell Expansion.

Abiotic stress impacts a wide range of plant developmental processes. Among them, cell expansion is particularly important given its contribution to plant growth and morphogenesis. Here, we describe a new phenotypic system to quantify accurately the impact of different sources of abiotic stress on the cell's capacity to expand. This approach monitors hypocotyl growth in Arabidopsis thaliana etiolated seedlings, as in the dark this embryonic organ is known to grow solely by expanding its cells, without cell division.

Assessing Abscisic Acid-Mediated Changes in Stomatal Aperture Through High-Quality Leaf Impressions.

Plants live in highly dynamic surroundings and need to cope with constant environmental challenges. In order to do so, they developed quick reactions to stress that allow them to gain time while mounting a major response. This first line of defense includes the stomata, leaf epidermal pores in charge of regulating water loss and photosynthesis. Stomatal movements are controlled by the stress phytohormone abscisic acid (ABA), which induces fast closure of the stomata upon perception of stress conditions. By modulating plasma membrane ion channels, ABA leads to loss of water from the guard cells surrounding the stomatal pore and a consequent reduction of its aperture. Here, we provide a microscopy-based method to assess the plant's response to ABA through measurements of the stomatal aperture. This protocol describes a simple, quick, and unexpensive method to prepare high-quality impressions of leaves from Arabidopsis thaliana seedlings from long-lasting silicone-based casts, allowing detailed imaging and accurate determination of the aperture of stomatal pores.

Assessing Postgermination Development in Arabidopsis thaliana Under Abiotic Stress.

The abscisic acid (ABA) phytohormone is well known to regulate responses to abiotic stress, particularly tolerance to osmotic stress. Screening for phenotypes at the early plant development stages is fundamental to identify new regulators of the ABA pathway, which in turn is extremely relevant for agriculture in a global climate change context. Typically, under experimental conditions, seeds are germinated in hormone-containing plates, and postgermination development is then assessed through scoring of the appearance of green or expanded cotyledons. However, postgermination phenotypes may be either masked or exacerbated by prior defects in seed germination. To circumvent this, we propose a transfer assay to screen specifically and quickly for postgermination phenotypes affected by exogenous ABA. The assay can be applied to different forms of abiotic stress, and we provide tips to score for postgermination phenotypes in genotypes exhibiting differential development.

Biological Anchorage and Canine Orthodontic Movement Rate with a New Technique for Micro-Osteoperforations.

INTRODUCTION: The differential management of anchorage and the acceleration of tooth movement are some of the current greatest challenges for orthodontists. Diverse techniques and devices to reinforce anchorage and increase the rate of tooth movement have been proposed. Whether micro-osteoperforations (MOPs) can be used for both purposes is currently investigated. OBJECTIVES: To propose and describe a new technique for biological anchorage, which involves six MOPs performed every four weeks, and to present its results in a clinical case of upper premolar extraction. Intervention. In a dental class II patient who met the selection criteria, three MOPs both on the buccal and palatine sides on the intervention side were performed on the extraction area following the protocol described. No MOPs were performed on the control side. The allocation of the intervention was randomised. The MOPs were performed three times at an interval of four weeks. A 0.019 × 0.025-inch stainless steel wire was activated with calibrated NiTi springs. The three-dimensional movement of the first molars and upper canines was evaluated. In addition, the comfort, periodontal status, and canine root resorption of the patient were evaluated. RESULTS: Clinical and radiographic results suggest that the MOPs had a positive effect in reducing the loss of biological anchorage of the posterior sector and in the rate of canine tooth movement, without damaging changes in the soft and hard tissues. CONCLUSION: The proposed protocol involving six MOPs every four weeks improved the behaviour of biological anchorage and increased distalization on the intervention side in this clinical case.

The plant-specific SR45 protein negatively regulates glucose and ABA signaling during early seedling development in Arabidopsis.

The plant-specific SR45 belongs to the highly conserved family of serine/arginine-rich (SR) proteins, which play key roles in precursor-mRNA splicing and other aspects of RNA metabolism. An Arabidopsis (Arabidopsis thaliana) loss-of-function mutant, sr45-1, displays pleiotropic phenotypes, such as defects in flower and leaf morphology, root growth, and flowering time. Here, we show that the sr45-1 mutation confers hypersensitivity to glucose (Glc) during early seedling growth in Arabidopsis. Unlike wild-type plants, the sr45-1 mutant displays impaired cotyledon greening and expansion as well as reduced hypocotyl elongation of dark-grown seedlings when grown in the presence of low (3%) Glc concentrations. In addition, SR45 is involved in the control of Glc-responsive gene expression, as the mutant displays enhanced repression of photosynthetic and nitrogen metabolism genes and overinduction of starch and anthocyanin biosynthesis genes. Like many other sugar response mutants, sr45-1 also shows hypersensitivity to abscisic acid (ABA) but appears to be unaffected in ethylene signaling. Importantly, the sr45-1 mutant shows enhanced ability to accumulate ABA in response to Glc, and the ABA biosynthesis inhibitor fluridone partially rescues the sugar-mediated growth arrest. Moreover, three ABA biosynthesis genes and two key ABA signaling genes, ABI3 and ABI5, are markedly overinduced by Glc in sr45-1. These results provide evidence that the SR45 protein defines a novel player in plant sugar response that negatively regulates Glc signaling during early seedling development by down-regulating both Glc-specific ABA accumulation and ABA biosynthesis and signaling gene expression.

Alternative splicing landscapes in Arabidopsis thaliana across tissues and stress conditions highlight major functional differences with animals.

BACKGROUND: Alternative splicing (AS) is a widespread regulatory mechanism in multicellular organisms. Numerous transcriptomic and single-gene studies in plants have investigated AS in response to specific conditions, especially environmental stress, unveiling substantial amounts of intron retention that modulate gene expression. However, a comprehensive study contrasting stress-response and tissue-specific AS patterns and directly comparing them with those of animal models is still missing. RESULTS: We generate a massive resource for Arabidopsis thaliana, PastDB, comprising AS and gene expression quantifications across tissues, development and environmental conditions, including abiotic and biotic stresses. Harmonized analysis of these datasets reveals that A. thaliana shows high levels of AS, similar to fruitflies, and that, compared to animals, disproportionately uses AS for stress responses. We identify core sets of genes regulated specifically by either AS or transcription upon stresses or among tissues, a regulatory specialization that is tightly mirrored by the genomic features of these genes. Unexpectedly, non-intron retention events, including exon skipping, are overrepresented across regulated AS sets in A. thaliana, being also largely involved in modulating gene expression through NMD and uORF inclusion. CONCLUSIONS: Non-intron retention events have likely been functionally underrated in plants. AS constitutes a distinct regulatory layer controlling gene expression upon internal and external stimuli whose target genes and master regulators are hardwired at the genomic level to specifically undergo post-transcriptional regulation. Given the higher relevance of AS in the response to different stresses when compared to animals, this molecular hardwiring is likely required for a proper environmental response in A. thaliana.

Expression of Castanea crenata Allene Oxide Synthase in Arabidopsis Improves the Defense to Phytophthora cinnamomi.

Allene oxide synthase (AOS) is a key enzyme of the jasmonic acid (JA) signaling pathway. The AOS gene was previously found to be upregulated in an Asian chestnut species resistant to infection by the oomycete Phytophthora cinnamomi (Castanea crenata), while lower expression values were detected in the susceptible European chestnut (Castanea sativa). Here, we report a genetic and functional characterization of the C. crenata AOS (CcAOS) upon its heterologous gene expression in a susceptible ecotype of Arabidopsis thaliana, which contains a single AOS gene. It was found that Arabidopsis plants expressing CcAOS delay pathogen progression and exhibit more vigorous growth in its presence. They also show upregulation of jasmonic acid and salicylic acid-related genes. As in its native species, heterologous CcAOS localized to plastids, as revealed by confocal imaging of the CcAOS-eGFP fusion protein in transgenic Arabidopsis roots. This observation was confirmed upon transient expression in Nicotiana benthamiana leaf epidermal cells. To further confirm a specific role of CcAOS in the defense mechanism against the pathogen, we performed crosses between transgenic CcAOS plants and an infertile Arabidopsis AOS knockout mutant line. It was found that plants expressing CcAOS exhibit normal growth, remain infertile but are significantly more tolerant to the pathogen than wild type plants. Together, our results indicate that CcAOS is an important player in plant defense responses against oomycete infection and that its expression in susceptible varieties may be a valuable tool to mitigate biotic stress responses.