Virulence factors of the genus Fusarium with targets in plants.

Fusarium spp. comprise various species of filamentous fungi that cause severe diseases in plant crops of both agricultural and forestry interest. These plant pathogens produce a wide range of molecules with diverse chemical structures and biological activities. Genetic functional analyses of some of these compounds have shown their role as virulence factors (VF). However, their mode of action and contributions to the infection process for many of these molecules are still unknown. This review aims to analyze the state of the art in Fusarium VF, emphasizing their biological targets on the plant hosts. It also addresses the current experimental approaches to improve our understanding of their role in virulence and suggests relevant research questions that remain to be answered with a greater focus on species of agroeconomic importance. In this review, a total of 37 confirmed VF are described, including 22 proteinaceous and 15 non-proteinaceous molecules, mainly from Fusarium oxysporum and Fusarium graminearum and, to a lesser extent, in Fusarium verticillioides and Fusarium solani.

Gracilis Muscle-Free Flap Objective Improvement of Oral Commissure Excursion in Facial Palsy Reanimation Surgery: Systematic Review and Meta-Analysis.

Background: Facial palsy patients face significant challenges. Gracilis free flap transfer is a key procedure in facial reanimation. Objective: This study aims to analyze oral commissure excursion improvement after gracilis free flap transfer and the differences regarding donor nerve: cross-facial nerve graft (CFNG), hypoglossal or spinal accessory nerves, motor nerve to masseteric (MNTM), and most recently, double anastomosis using both the MNTM and CFNG. Methods: A systematic review and meta-analysis were conducted of studies reporting oral commissure excursion improvement after free gracilis muscle transfer. Pooled proportions were calculated using a random-effects model. Results: Eighteen studies, 453 patients, and 488 free gracilis flaps were included. The mean change in perioperative oral commissure excursion was 7.0 mm, for CFNG 7.2 mm, for MNTM 7.7, and for double anastomoses 5.5 mm. Conclusions: There is a significant improvement in oral commissure excursion after gracilis muscle-free flap. Unfortunately, we could not make definitive conclusions regarding the optimal choice of donor nerve.

Use of practice tests with immediate feedback in an undergraduate molecular biology course.

Multiple studies have shown that testing contributes to learning at all educational levels. In this observational classroom study, we report the use of a learning tool developed for a Genetics and Molecular Biology course at the college level. An interactive set of practice exams that included 136 multiple choice questions (MCQ) or matching queries was developed in the open-source Moodle platform. All MCQ questions contained four answer choices and configured for immediate feedback upon answering. Feedback consisted of providing the right answer and a short explanation of the learning objective examined. The interactive material was tested and refined for several semesters. Usefulness of this tool was assessed in two distinct settings: (1) during a face-to-face semester (Fall 2019) by comparing the grades in a final departmental exam between students who used the tool and those who did not, and (2) during an online semester (Fall 2020) by analyzing the grades in the first and last attempts on study sessions and students' performance in monthly exams. We found that when solving practice tests, students obtained a significantly higher scores in the last attempt compared with their first attempt, and that students who used the material performed better than those who did not. In all cases, answering the practice exams was optional, but students made full use of them preferentially during the online semester. This classroom research exemplifies the documented effectiveness of practice tests enhanced with feedback in biological sciences education through an open-source learning platform.

An online course on applied biochemistry and molecular biology through case-based learning.

An undergraduate online course on Applied Biochemistry and Molecular Biology was developed through different formats of case study that included lecture, class discussion, small-groups discussion, and individual work. Cases covering health, biotechnology, agriculture, and other issues were developed or adapted from the literature to reach the desired learning goals. Multiple web resources were employed for information integration that were presented and discussed in the synchronous sessions and assignments. Formative and summative assessment was achieved through multiple-choice questionnaires, exams, and homework assignments.

In Search of Resistance Against Fusarium Ear Rot: Ferulic Acid Contents in Maize Pericarp Are Associated With Antifungal Activity and Inhibition of Fumonisin Production.

Fusarium verticillioides is a fungal pathogen of maize that causes seedling blight, stem rot, and Fusarium ear rot. Fungal infestation of maize kernels and ears affects grain quality from the ensuing mycotoxin buildup. Among the mycotoxins produced by F. verticillioides, fumonisins accumulate to high levels in Fusarium-infected maize kernels, fumonisin B1 (FB1) being the most abundant in naturally infected maize. Achieving resistance to Fusarium ear rot has been challenging, as various environmental factors facilitate fungal infection. Among the maize grain components that contribute to resistance to F. verticillioides infection, the pericarp is the first barrier faced by the fungus and thus plays a key role. Phenolic acids are major constituents of maize pericarp, of which ferulic acid (FA) is the predominant molecular species. In this work, we explored the relationship between FA levels, fungal infection, and FB1 production in 51 maize genotypes and whether the antioxidant activity of FA might play a role. We confirmed that FA is a major component of the seed pericarp, whose levels as bound FA varied between 4.5 and 26.3 mg/g across maize genotypes. We selected two pools of five maize varieties, with contrasting FA contents: low FA (LFA; 6.14 ± 0.40 mg/g) and high FA (HFA; 15.49 ± 1.31 mg/g). In vitro, HFA extracts inhibited fungal growth with effects comparable to FA concentrations in the 0.25-0.50 mM range. We also established a kernel assay to study F. verticillioides colonization and FB1 production in the LFA and HFA genotypes. Fungal colonization was significantly lower in HFA genotypes relative to LFA genotypes, based on ergosterol levels. Moreover, FB1 production was also inhibited in the HFA genotypes. Importantly, the antioxidant activity of maize pericarp extracts was associated with FA contents, with HFA extracts exhibiting a greater antioxidant activity than LFA extracts. Overall, our results highlight the role of FA and its antioxidant activity on resistance to Fusarium ear rot and provide the basis of a phenotypic trait that can be deployed for breeding selection.

Effect of Tinnitus in Distortion Products Otoacoustic Emissions (DPOAEs) in Normal Hearing Patients.

Tinnitus is a symptom whose pathophysiology remains still unclear. Its diagnosis and treatment is complicated, due to its subjectivity. The generation of tinnitus is commonly linked with the impaired functioning of the outer hair cells (OHC) inside the cochlea. Distortion product otoacoustic emissions (DPOAEs) are the objective test used to assess their activity. This study investigates the cochlear outer hair cell function in patients with tinnitus and normal hearing using DPOAEs. We performed a prospective study of the cochlear function in normal hearing patients complaining of tinnitus by analysing DPOAEs amplitude and signal/noise (S/N) ratio. We gathered a sample of 21 ears from adults that attended to the ENT Department complaining of tinnitus with normal hearing. We compared their results with a control group of 21 ears, with the same demographic characteristics, presenting normal hearing but without tinnitus in order to exclude the influence of age in DPOAEs results. A decreased mean of S/N levels in DPOAEs was found in tinnitus and normal hearing group comparing with control group, although these differences were not statistically significant (p > 0.05). Based on the results, OHC dysfunction is not necessary to experience tinnitus. The majority of the patients that present OHC dysfunction do not present a tinnitus at the moment. Other mechanisms in auditory pathway may be evaluated in the tinnitus development.

Characterization of the Exo-Metabolome of the Emergent Phytopathogen Fusarium kuroshium sp. nov., a Causal Agent of Fusarium Dieback.

Fusarium kuroshium is the fungal symbiont associated with the ambrosia beetle Euwallacea kuroshio, a plague complex that attacks avocado, among other hosts, causing a disease named Fusarium dieback (FD). However, the contribution of F. kuroshium to the establishment of this disease remains unknown. To advance the understanding of F. kuroshium pathogenicity, we profiled its exo-metabolome through metabolomics tools based on accurate mass spectrometry. We found that F. kuroshium can produce several key metabolites with phytotoxicity properties and other compounds with unknown functions. Among the metabolites identified in the fungal exo-metabolome, fusaric acid (FA) was further studied due to its phytotoxicity and relevance as a virulence factor. We tested both FA and organic extracts from F. kuroshium at various dilutions in avocado foliar tissue and found that they caused necrosis and chlorosis, resembling symptoms similar to those observed in FD. This study reports for first-time insights regarding F. kuroshium associated with its virulence, which could lead to the potential development of diagnostic and management tools of FD disease and provides a basis for understanding the interaction of F. kuroshium with its host plants.

Head and neck squamous cell carcinoma in solid organ transplant recipients.

PURPOSE: Characterize head and neck squamous cell carcinomas in solid transplant recipients and compare outcomes with non-transplanted population. METHODS: We carried a retrospective cohort analysis in a tertiary care center in Madrid, Spain. The study reviews 26 cases of non-cutaneous HNSCC diagnosed in solid organ transplant recipients between 2000 and 2017. We select a cohort of 130 (1:5) non-transplanted patients among all non-transplanted patients diagnosed during the same period for comparison purposes, through hierarchical clustering analysis. Univariate, overall and specific survival analysis and multivariate Cox proportional hazards regression were used to evaluate our objectives. RESULTS: The overall risk of non-cutaneous HNSCC in solid transplant recipients was 7.2 cases per 1000. Alcohol abuse (p = 0.021) and upfront surgery (p = 0.019) were more likely to occur in transplanted patients. Age > 60 was an independent predictor of worse outcomes both for overall (HR = 2.4, p < 0.001) and specific (HR = 2.1, p = 0.012) survival. Transplant significantly worse overall survival (HR = 2.1, p = 0.012) but no statistical significant differences were observed on specific survival (p = 0.392). CONCLUSIONS: Solid organ transplant recipients have a higher risk of suffering non-cutaneous HNSCC. The higher mortality rate of these patients does not appear to be directly related to suffering from head and neck cancer, although it seems to contribute to developing other fatal complications in these fragile patients.

Maize Thymidine Kinase Activity Is Present throughout Plant Development and Its Heterologous Expression Confers Tolerance to an Organellar DNA-Damaging Agent.

Thymidine kinase 1 (TK1) phosphorylates thymidine nucleosides to generate thymidine monophosphate. This reaction belongs to the pyrimidine salvage route that is phylogenetically conserved. In the model plant Arabidopsis thaliana, TK activity contributes to maintain nuclear and organellar genome integrity by providing deoxythymidine-triphosphate (dTTP) for DNA synthesis. Arabidopsis has two TK1 genes (TK1a and TK1b) and double mutants show an albino phenotype and develop poorly. In contrast, maize (Zea mays L.) has a single TK1 (ZmTK1) gene and mutant plants are albino and display reduced genome copy number in chloroplasts. We studied the role of ZmTK1 during development and genotoxic stress response by assessing its activity at different developmental stages and by complementing Arabidopsis tk1 mutants. We found that ZmTK1 transcripts and activity are present during germination and throughout maize development. We show that ZmTK1 translocation to chloroplasts depends on a 72-amino-acid N-signal and its plastid localization is consistent with its ability to complement Arabidopsis tk1b mutants which are hypersensitive to ciprofloxacin (CIP), a genotoxic agent to organellar DNA. Also, ZmTK1 partly complemented the Arabidopsis double mutant plants during development. Our results contribute to the understanding of TK1 function in monocot species as an organellar enzyme for genome replication and repair.

Sphingolipid Effects on the Plasma Membrane Produced by Addition of Fumonisin B1 to Maize Embryos.

Fumonisin B1 is a mycotoxin produced by Fusarium verticillioides that modifies the membrane properties from animal cells and inhibits complex sphingolipids synthesis through the inhibition of ceramide synthase. The aim of this work was to determine the effect of Fumonisin B1 on the plant plasma membrane when the mycotoxin was added to germinating maize embryos. Fumonisin B1 addition to the embryos diminished plasma membrane fluidity, increased electrolyte leakage, caused a 7-fold increase of sphinganine and a small decrease in glucosylceramide in the plasma membrane, without affecting phytosphingosine levels or fatty acid composition. A 20%-30% inhibition of the plasma membrane H+-ATPase activity was observed when embryos were germinated in the presence of the mycotoxin. Such inhibition was only associated to the decrease in glucosylceramide and the addition of exogenous ceramide to the embryos relieved the inhibition of Fumonisin B1. These results indicate that exposure of the maize embryos for 24 h to Fumonisin B1 allowed the mycotoxin to target ceramide synthase at the endoplasmic reticulum, eliciting an imbalance of endogenous sphingolipids. The latter disrupted membrane properties and inhibited the plasma membrane H+-ATPase activity. Altogether, these results illustrate the mode of action of the pathogen and a plant defense strategy.

The Protective Effect of Trichoderma asperellum on Tomato Plants against Fusarium oxysporum and Botrytis cinerea Diseases Involves Inhibition of Reactive Oxygen Species Production.

Trichoderma species are fungi widely employed as plant-growth-promoting agents and for biological control. Several commercial and laboratory-made solid formulations for mass production of Trichoderma have been reported. In this study, we evaluated a solid kaolin-based formulation to promote the absortion/retention of Trichoderma asperellum in the substrate for growing tomato plants. The unique implementation of this solid formulation resulted in an increased growth of the tomato plants, both in roots and shoots after 40 days of its application. Plants were challenged with two fungal pathogens, Fusarium oxysporum and Botrytis cinerea, and pretreatment with T. asperellum resulted in less severe wilting and stunting symptoms than non-treated plants. Treatment with T. asperellum formulation inhibited Reactive Oxygen Species (ROS) production in response to the pathogens in comparison to plants that were only challenged with both pathogens. These results suggest that decrease in ROS levels contribute to the protective effects exerted by T. asperellum in tomato.

Role of pyrimidine salvage pathway in the maintenance of organellar and nuclear genome integrity.

Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes -TK1a and TK1b- that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress.

La azatioprina reduce el riesgo de recaída audiométrica en hipoacusia inmunomediada / Azathioprine reduces the risk of audiometric relapse in immune-mediated hearing loss

INTRODUCCIÓN: Los esquemas actuales de tratamiento de la hipoacusia inmunomediada con corticoides, dosis baja y pauta corta, son insuficientes. MÉTODOS: Para determinar el papel de la azatioprina en el control del deterioro auditivo se ha llevado a cabo un estudio observacional descriptivo longitudinal con 20 pacientes tratados con azatioprina por vía oral (1,5-2,5 mg/kg/día en dos dosis) durante 1año. Se consideró recaída la pérdida de 10 dB en dos frecuencias consecutivas o de 15 dB en una frecuencia aislada. RESULTADOS: La edad media de los pacientes fue de 52,50 años (IC 95%: 46,91-58,17), y la mitad fueron mujeres. La afectación bilateral fue del 65%. Un 75% presentaban enfermedad organoespecífica y un 25%, enfermedad autoinmune sistémica. La diferencia entre la PTA basal (46,49 dB; DE18,90) y la PTA a los 12meses (45,47 dB; DE 18,88) no alcanzó significación estadística (p = 0,799). Existía una correlación positiva moderada entre sexo femenino y presencia de enfermedad sistémica (R = 0,577). Aplicando t de Student para datos apareados se obtuvo una diferencia significativa (p = 0,042) entre el descenso de la PTA en frecuencias hasta 1.000Hz (PTA125-1.000Hz). La tasa relativa de incidencia de recaída por año fue de 0,52 recaídas/año (IC 95%: 0,19-1,14). El tiempo medio de supervivencia libre de recaída audiométrica fue de 9,70 meses (DE 1,03). CONCLUSIONES: La azatioprina mantiene el umbral de audición, disminuye el riesgo de recaída y frena la velocidad con la que los pacientes recaen, alterando el curso de la enfermedad inmunomediada del oído interno

INTRODUCTION: Current schemes for treatment of immune-mediated hearing loss with sporadic short-course, low-dose corticosteroids, are insufficient. METHODS: To determine the role of azathioprine in the control of auditory impairment, a longitudinal, observational, descriptive study was performed with 20 patients treated with azathioprine (1.5-2.5 mg/kg/day into two doses) for 1year. The loss of 10 dB on two consecutive frequencies or 15 dB on an isolated frequency was considered as relapse. RESULTS: The mean age of the patients was 52.50 years (95% CI: 46.91-58.17), half were women. Bilateral affectation was 65%. 75% had organ specific disease and 25% had systemic autoimmune disease. The difference between baseline PTA (46.49 dB; DS 18.90) and PTA at 12 months (45.47dB; DS 18.88) did not reach statistical significance (P = .799). There was a moderate positive correlation between female sex and the presence of systemic disease (R = .577). By applying Student's t for paired data, a significant difference (P = .042) was obtained between the PTA in frequencies up to 1000 Hz (PTA125-1000Hz). The relative incidence rate of relapse per year was .52 relapses/year (95% CI: .19-1.14]). The median time to audiometric relapse-free was 9.70 months (DS 1.03). CONCLUSIONS: Azathioprine maintains the hearing threshold, decreases the risk of relapse, and slows down the rate at which patients relapse, altering the course of immune-mediated inner ear disease

Análisis de supervivencia libre de recaída audiométrica en pacientes con hipoacusia inmunomediada tratados exclusivamente con corticoides / Analysis of audiometric relapse-free survival in patients with immune-mediated hearing loss exclusively treated with corticosteroids

OBJETIVO: Describir los resultados en términos de supervivencia libre de recaída audiométrica y el ritmo de recaída en pacientes con hipoacusia inmunomediada tratados exclusivamente con corticoides. MÉTODO: Estudio retrospectivo de pacientes, con recaídas audiométricas, en seguimiento desde 1995 hasta 2014, en 2 centros de la Comunidad de Madrid. RESULTADOS: Se evaluaron 31 pacientes con una media de edad de 48,52 años (14,67 DE) de los cuales el 61,3% fueron mujeres. La mayoría de las hipoacusias fueron fluctuantes (48,4%). Solo el 16,1% de los pacientes presentaban enfermedad autoinmune sistémica. Existe una correlación positiva moderada entre ser mujer y presentar afectación sistémica (coeficiente de correlación de Spearman = 0,356). La tasa relativa de incidencia de recaída en el primer año en nuestra serie fue de 2,01 recaídas/año con un IC95% (1,32-2,92). El tiempo de supervivencia medio del evento (recaída audiométrica) fue de 5,25 meses (DE 0,756). Con el análisis multivariante, la única variable que consiguió significación estadística fue la edad, con una hazard ratio de 1,032 (IC95%; 1,001-1,063, p = 0,043). CONCLUSIONES: La enfermedad inmunomediada del oído interno es una enfermedad crónica con recaídas. La mitad de los pacientes tratados exclusivamente con corticoides recaen antes de los 6 meses de seguimiento. Además, si un paciente no ha presentado recaída, tiene más riesgo de recaer cada año que pasa. El análisis de la supervivencia libre de recaída audiométrica permitirá comparar el efecto de tratamientos futuros y su capacidad para reducir el ritmo de recaídas

OBJECTIVE: To describe the results in terms of audiometric relapse-free survival and relapse rate in immunomediated hearing loss patients treated exclusively with corticosteroids. METHOD: Retrospective study of patients with audiometric relapses, monitored from 1995 to 2014, in two centres of the Community of Madrid. RESULTS: We evaluated 31 patients with a mean age of 48.52 years (14.67 SD), of which 61.3% were women. Most hearing loss was fluctuating (48.4%). Only 16.1% of patients had systemic autoimmune disease. There is a moderate positive correlation between the sex variable and the systemic involvement variable (Spearman's correlation coefficient = 0.356): specifically, between being female and systemic disease. The relative incidence rate of relapse in the first year was 2.01 relapses/year with a 95% CI (1.32 to 2.92). The mean survival time of the event (audiometric relapse) was 5.25 months (SD 0.756). With multivariate analysis, the only variable that achieved statistical significance was age, with a hazard ratio of 1.032 (95% CI; 1.001-1.063, P = .043). CONCLUSIONS: Immune-mediated disease of the inner ear is a chronic disease with relapses. Half of the patients with immunomediated hearing loss treated exclusively with corticosteroids relapse before 6 months of follow-up. In addition, if a patient has not relapsed, they are more likely to relapse as each year passes. Analysis of the of audiometric relapse- free survival will enable the effect of future treatments to be compared and their capacity to reduce the rhythm of relapses

Azathioprine reduces the risk of audiometric relapse in immune-mediated hearing loss. / La azatioprina reduce el riesgo de recaída audiométrica en hipoacusia inmunomediada.

INTRODUCTION: Current schemes for treatment of immune-mediated hearing loss with sporadic short-course, low-dose corticosteroids, are insufficient. METHODS: To determine the role of azathioprine in the control of auditory impairment, a longitudinal, observational, descriptive study was performed with 20 patients treated with azathioprine (1.5-2.5mg/kg/day into two doses) for 1year. The loss of 10dB on two consecutive frequencies or 15dB on an isolated frequency was considered as relapse. RESULTS: The mean age of the patients was 52.50years (95%CI: 46.91-58.17), half were women. Bilateral affectation was 65%. 75% had organ specific disease and 25% had systemic autoimmune disease. The difference between baseline PTA (46.49dB; DS18.90) and PTA at 12months (45.47dB; DS18.88) did not reach statistical significance (P=.799). There was a moderate positive correlation between female sex and the presence of systemic disease (R=.577). By applying Student's t for paired data, a significant difference (P=.042) was obtained between the PTA in frequencies up to 1000 Hz (PTA125-1000Hz). The relative incidence rate of relapse per year was .52 relapses/year (95%CI: .19-1.14]). The median time to audiometric relapse-free was 9.70months (DS1.03). CONCLUSIONS: Azathioprine maintains the hearing threshold, decreases the risk of relapse, and slows down the rate at which patients relapse, altering the course of immune-mediated inner ear disease.

Analysis of audiometric relapse-free survival in patients with immune-mediated hearing loss exclusively treated with corticosteroids. / Análisis de supervivencia libre de recaída audiométrica en pacientes con hipoacusia inmunomediada tratados exclusivamente con corticoides.

OBJECTIVE: To describe the results in terms of audiometric relapse-free survival and relapse rate in immunomediated hearing loss patients treated exclusively with corticosteroids. METHOD: Retrospective study of patients with audiometric relapses, monitored from 1995 to 2014, in two centres of the Community of Madrid. RESULTS: We evaluated 31 patients with a mean age of 48.52 years (14.67 SD), of which 61.3% were women. Most hearing loss was fluctuating (48.4%). Only 16.1% of patients had systemic autoimmune disease. There is a moderate positive correlation between the sex variable and the systemic involvement variable (Spearman's correlation coefficient=0.356): specifically, between being female and systemic disease. The relative incidence rate of relapse in the first year was 2.01 relapses/year with a 95% CI (1.32 to 2.92). The mean survival time of the event (audiometric relapse) was 5.25 months (SD 0.756). With multivariate analysis, the only variable that achieved statistical significance was age, with a hazard ratio of 1.032 (95% CI; 1.001-1.063, P=.043). CONCLUSIONS: Immune-mediated disease of the inner ear is a chronic disease with relapses. Half of the patients with immunomediated hearing loss treated exclusively with corticosteroids relapse before 6 months of follow-up. In addition, if a patient has not relapsed, they are more likely to relapse as each year passes. Analysis of the of audiometric relapse- free survival will enable the effect of future treatments to be compared and their capacity to reduce the rhythm of relapses.

Long-term response on growth, antioxidant enzymes, and secondary metabolites in salicylic acid pre-treated Uncaria tomentosa microplants.

OBJECTIVE: To obtain micro propagated Uncaria tomentosa plantlets with enhanced secondary metabolites production, long-term responses to salicylic acid (SA) pre-treatments at 1 and 100 µM were evaluated after propagation of the plantlets in a SA-free medium. RESULTS: SA pre-treatments of single node cuttings OF U. tomentosa produced long-term responses in microplants grown for 75 days in a SA-free medium. Reduction in survival rate, root formation, and stem elongation were observed only with 100 µM SA pre-treatments with respect to the control (0 + DMSO).Both pre-treatments enhanced H2O2 and inhibited superoxide dismutase and catalase activities, while guaiacol peroxidase was increased only with 1 µM SA. Also, both pre-treatments increased total monoterpenoid oxindole alkaloids by ca. 55 % (16.5 mg g(-1) DW), including isopteropodine, speciophylline, mitraphylline, isomitraphylline, rhynchopylline, and isorhynchopylline; and flavonoids by ca. 21 % (914 µg g(-1) DW), whereas phenolic compounds were increased 80 % (599 µg g(-1) DW) at 1 µM and 8.2 % (359 µg g(-1) DW) at 100 µM SA. CONCLUSION: Pre-treatment with 1 µM SA of U.tomentosa microplants preserved the survival rate and increased oxindole alkaloids, flavonoids, and phenolic compounds in correlation with H2O2 and peroxidase activity enhancements, offering biotechnological advantages over non-treated microplants.

Deciphering the link between salicylic acid signaling and sphingolipid metabolism.

The field of plant sphingolipid biology has evolved in recent years. Sphingolipids are abundant in cell membranes, and genetic analyses revealed essential roles for these lipids in plant growth, development, and responses to abiotic and biotic stress. Salicylic acid (SA) is a key signaling molecule that is required for induction of defense-related genes and rapid and localized cell death at the site of pathogen infection (hypersensitive response) during incompatible host-pathogen interactions. Conceivably, while levels of SA rapidly increase upon pathogen infection for defense activation, they must be tightly regulated during plant growth and development in the absence of pathogens. Genetic and biochemical evidence suggest that the sphingolipid intermediates, long-chain sphingoid bases, and ceramides, play a role in regulating SA accumulation in plant cells. However, how signals generated from the perturbation of these key sphingolipid intermediates are transduced into the activation of the SA pathway has long remained to be an interesting open question. At least four types of molecules - MAP kinase 6, reactive oxygen species, free calcium, and nitric oxide - could constitute a mechanistic link between sphingolipid metabolism and SA accumulation and signaling.

Arabidopsis thaliana thymidine kinase 1a is ubiquitously expressed during development and contributes to confer tolerance to genotoxic stress.

Thymidine kinase catalyzes the first step in the nucleotide salvage pathway by transferring a phosphate group to a thymidine molecule. In mammals thymidine kinase supplies deoxyribonucleotides for DNA replication and DNA repair, and the expression of the gene is tightly regulated during the cell cycle. Although this gene is phylogenetically conserved in many taxa, its physiological function in plants remains unknown. The genome of the model plant Arabidopsis thaliana has two thymidine kinase genes (AtTK1a and AtTK1b) and microarray data suggest they might have redundant roles. In this study we analyzed the TK1a function by evaluating its expression pattern during development and in response to genotoxic stress. We also studied its role in DNA repair by the characterization of a mutant that contained the T-DNA insertion in the promoter region of the TK1a gene. We found that TK1a is expressed in most tissues during plant development and it was differentially induced by ultraviolet-C radiation because TK1b expression was unaffected. In the mutant, the T-DNA insertion caused a 40 % rise in transcript levels and enzyme activity in Arabidopsis seedlings compared to wild-type plants. This elevation was enough to confer tolerance to ultraviolet-C irradiation in dark conditions, as determined by root growth, and meristem length and structure. TK1a overexpression also provided tolerance to genotoxins that induce double-strand break. Our results suggest that thymidine kinase contributes to several DNA repair pathways by providing deoxythymidine triphosphate that serve as precursors for DNA repair and to balance deoxyribonucleotides pools.

Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f. sp. lycopersici.

Sphingolipids play an important role in signal transduction pathways that regulate physiological functions and stress responses in eukaryotes. In plants, recent evidence suggests that their metabolic precursors, the long-chain bases (LCBs) act as bioactive molecules in the immune response. Interestingly, the virulence of two unrelated necrotrophic fungi, Fusarium verticillioides and Alternaria alternata, which are pathogens of maize and tomato plants, respectively, depends on the production of sphinganine-analog mycotoxins (SAMs). These metabolites inhibit de novo synthesis of sphingolipids in their hosts causing accumulation of LCBs, which are key regulators of programmed cell death. Therefore, to gain more insight into the role of sphingolipids in plant immunity against SAM-producing necrotrophic fungi, we disrupted sphingolipid metabolism in Nicotiana benthamiana through virus-induced gene silencing (VIGS) of the serine palmitoyltransfersase (SPT). This enzyme catalyzes the first reaction in LCB synthesis. VIGS of SPT profoundly affected N. benthamiana development as well as LCB composition of sphingolipids. While total levels of phytosphingosine decreased, sphinganine and sphingosine levels increased in SPT-silenced plants, compared with control plants. Plant immunity was also affected as silenced plants accumulated salicylic acid (SA), constitutively expressed the SA-inducible NbPR-1 gene and showed increased susceptibility to the necrotroph A. alternata f. sp. lycopersici. In contrast, expression of NbPR-2 and NbPR-3 genes was delayed in silenced plants upon fungal infection. Our results strongly suggest that LCBs modulate the SA-dependent responses and provide a working model of the potential role of SAMs from necrotrophic fungi to disrupt the plant host response to foster colonization.