Transcriptome sequencing of three Pseudo-nitzschia species reveals comparable gene sets and the presence of Nitric Oxide Synthase genes in diatoms

Diatoms are among the most diverse eukaryotic microorganisms on Earth, they are responsible for a large fraction of primary production in the oceans and can be found in different habitats. Pseudo-nitzschia are marine planktonic diatoms responsible for blooms in coastal and oceanic waters. We analyzed the transcriptome of three species, Pseudo-nitzschia arenysensis, Pseudo-nitzschia delicatissima and Pseudo-nitzschia multistriata, with different levels of genetic relatedness. These species have a worldwide distribution and the last one produces the neurotoxin domoic acid. We were able to annotate about 80% of the sequences in each transcriptome and the analysis of the relative functional annotations allowed comparison of the main metabolic pathways, pathways involved in the biosynthesis of isoprenoids (MAV and MEP pathways), and pathways putatively involved in domoic acid synthesis. The search for homologous transcripts among the target species and other congeneric species resulted in the discovery of a sequence annotated as Nitric Oxide Synthase (NOS), found uniquely in Pseudo-nitzschia multistriata. The predicted protein product contained all the domains of the canonical metazoan sequence. Putative NOS sequences were found in other available diatom datasets, supporting a role for nitric oxide as signaling molecule in this group of microalgae

Redox activities and ROS, NO and phenylpropanoids production by axenically cultured intact olive seedling roots after interaction with a mycorrhizal or a pathogenic fungus

Las raíces de las plántulas de olivo, en cultivo axénico, fueron colocadas alternativamente en contacto con Rhizophagus irregulares (micorrícicos) o con hongos Verticillim dahliae (patógenos). También se incluyeron tratamientos MeJA. Las raíces intactas (generación de anión superóxido, superóxido dismutasa y actividades de peroxidasa) se midieron en las actividades in vivo del apoplasto. Todos nuestros resultados mostraron que las actividades redox apoplásticas de raíces de las plántulas intactas en contacto con el hongo micorriza compatible fueron claramente atenuados en comparación con el hongo patógeno o tratado con MeJA, incluso en las primeras etapas usadas en el tratamiento. Los fenoles totales, flavonoides y glucósidos fenilpropanoides, también fueron cuantificados. Las raíces en contacto con el hongo micorriza no mejoraron la biosíntesis de compuestos fenólicos con respecto a los controles, mientras que los de contacto con el patógeno mejoraron de forma significativa la biosíntesis de todas las fracciones fenólicas medidas. Las especies reactivas del oxígeno y la acumulación de óxido nítrico en las raíces fueron examinadas por microscopía de fluorescencia. Todos ellas presentaron una acumulación mucho mayor en las raíces en contacto con el patógeno que con el hongo micorriza. En total, estos resultados indican que las raíces de las plántulas intactas de olivo, claramente diferenciadas entre micorrizas y hongos patógenos, atenuan las reacciones de defensa contra la primera para facilitar su creación, mientras que induce una reacción de defensa fuerte y sostenida contra el segundo. Ambas especies reactivas de oxígeno y nitrógeno parecían estar involucrados en estas respuestas desde los primeros momentos de contacto. Sin embargo, se necesitan más investigaciones para aclarar la diafonía propuesta entre ellos y sus respectivas funciones en estas respuestas ya que las imágenes de fluorescencia de las raíces revelaron que las especies reactivas del oxígeno se acumulan principalmente en el apoplasto (congruente con las actividades redox medidas en este compartimento), mientras el óxido nítrico se almacena principalmente en el citosol.Roots of intact olive seedlings, axenically cultured, were alternatively placed in contact with Rhizophagus irregularis (mycorrhizal) or Verticillim dahliae (pathogenic) fungi. MeJA treatments were also included. In vivo redox activities in the apoplast of the intact roots (anion superoxide generation, superoxide dismutase and peroxidase activities) were measured. All our results showed that apoplastic redox activities of intact seedling roots in contact with the compatible mycorrhizal fungus were clearly attenuated in comparison with the pathogenic fungus or treated with MeJA, even at the early stages of treatment used. Total phenolics, flavonoids and phenylpropanoid glycosides were also quantified. Roots in contact with the mycorrhizal fungus did not enhance the biosynthesis of phenolic compounds with respect to controls, while those in contact with the pathogenic one significantly enhanced the biosynthesis of all phenolic fractions measured. Reactive oxygen species and nitric oxid accumulation in roots were examined by fluorescence microscopy. All of them presented much higher accumulation in roots in contact with the pathogenic than with the mycorrhizal fungus. Altogether these results indicate that intact olive seedling roots clearly differentiated between mycorrhizal and pathogenic fungi, attenuating defense reactions against the first to facilitate its establishment, while inducing a strong and sustained defense reaction against the second. Both reactive oxygen and nitrogen species seemed to be involved in these responses from the first moments of contact. However, further investigations are required to clarify the proposed crosstalk between them and their respective roles in these responses since fluorescence images of roots revealed that reactive oxygen species were mainly accumulated in the apoplast (congruently with the measured redox activities in this compartment) while nitric oxid was mainly stored in the cytosol.-- Ministerio de Ciencia e Innovación. Proyecto CGL2009-12406 -- Junta de Extremadura. Proyecto PRI09A023peerReviewe

Nitric Oxide Controls Constitutive Freezing Tolerance in Arabidopsis by Attenuating the Levels of Osmoprotectants, Stress-Related Hormones and Anthocyanins

[EN] Plant tolerance to freezing temperatures is governed by endogenous constitutive components and environmental inducing factors. Nitric oxide (NO) is one of the endogenous components that participate in freezing tolerance regulation. A combined metabolomic and transcriptomic characterization of NO-deficient nia1,2noa1¿2 mutant plants suggests that NO acts attenuating the production and accumulation of osmoprotective and regulatory metabolites, such as sugars and polyamines, stress-related hormones, such as ABA and jasmonates, and antioxidants, such as anthocyanins and flavonoids. Accordingly, NO-deficient plants are constitutively more freezing tolerant than wild type plants.This work was supported by grants BIO2011-27526 and BIO2014-56067-P from the Spanish Ministry of Economy and Competitiveness and FEDER funds. We thank support and comments from Janice Jones and Danny Alexander (Metabolon Inc., USA) on metabolomic analyses.Costa-Broseta, Á.; Perea-Resa, C.; Castillo López Del Toro, MC.; Ruíz, MF.; Salinas, J.; Leon Ramos, J. (2018). Nitric Oxide Controls Constitutive Freezing Tolerance in Arabidopsis by Attenuating the Levels of Osmoprotectants, Stress-Related Hormones and Anthocyanins. Scientific Reports. 8. https://doi.org/10.1038/s41598-018-27668-8S8Janská, A., Marsík, P., Zelenková, S. & Ovesná, J. Cold stress and acclimation - what is important for metabolic adjustment? Plant Biol (Stuttg) 12, 395–405 (2010).Eremina, M., Rozhon, W. & Poppenberger, B. Hormonal control of cold stress responses in plants. Cell Mol Life Sci 73, 797–810 (2016).Winkel-Shirley, B. Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5, 218–223 (2002).Cuevas, J. C. et al. 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Nitric oxide triggers a transient metabolic reprogramming in Arabidopsis

[EN] Nitric oxide (NO) regulates plant growth and development as well as responses to stress that enhanced its endogenous production. Arabidopsis plants exposed to a pulse of exogenous NO gas were used for untargeted global metabolomic analyses thus allowing the identification of metabolic processes affected by NO. At early time points after treatment, NO scavenged superoxide anion and induced the nitration and the S-nitrosylation of proteins. These events preceded an extensive though transient metabolic reprogramming at 6 h after NO treatment, which included enhanced levels of polyamines, lipid catabolism and accumulation of phospholipids, chlorophyll breakdown, protein and nucleic acid turnover and increased content of sugars. Accordingly, lipid-related structures such as root cell membranes and leaf cuticle altered their permeability upon NO treatment. Besides, NO-treated plants displayed degradation of starch granules, which is consistent with the increased sugar content observed in the metabolomic survey. The metabolic profile was restored to baseline levels at 24 h post-treatment, thus pointing up the plasticity of plant metabolism in response to nitroxidative stress conditions.This work was supported by grants BIO2011-27526 and BIO2014-56067-P from the Spanish Ministry of Economy and Competitiveness and FEDER funds. We thank support and comments from Danny Alexander (Metabolon Inc., USA) on metabolomic analyses.Leon Ramos, J.; Costa-Broseta, Á.; Castillo López Del Toro, MC. (2016). Nitric oxide triggers a transient metabolic reprogramming in Arabidopsis. Scientific Reports. 6:1-14. doi:10.1038/srep37945S1146Arc, E., Galland, M., Godin, B., Cueff, G. & Rajjou, L. Nitric oxide implication in the control of seed dormancy and germination. Front. Plant Sci. 4, 346 (2013).Beligni, M. V. & Lamattina, L. 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Cyanobacterial NOS expression improves nitrogen use efficiency, nitrogen-deficiency tolerance and yield in Arabidopsis.

Developing strategies to improve nitrogen (N) use efficiency (NUE) in plants is a challenge to reduce environmental problems linked to over-fertilization. The nitric oxide synthase (NOS) enzyme from the cyanobacteria Synechococcus PCC 7335 (SyNOS) has been recently identified and characterized. SyNOS catalyzes the conversion of arginine to citrulline and nitric oxide (NO), and then approximately 75 % of the produced NO is rapidly oxidized to nitrate by an unusual globin domain in the N-terminus of the enzyme. In this study, we assessed whether SyNOS expression in plants affects N metabolism, NUE and yield. Our results showed that SyNOS-expressing transgenic Arabidopsis plants have greater primary shoot length and shoot branching when grown under N-deficient conditions and higher seed production both under N-sufficient and N-deficient conditions. Moreover, transgenic plants showed significantly increased NUE in both N conditions. Although the uptake of N was not modified in the SyNOS lines, they showed an increase in the assimilation/remobilization of N under conditions of low N availability. In addition, SyNOS lines have greater N-deficiency tolerance compared to control plants. Our results support that SyNOS expression generates a positive effect on N metabolism and seed production in Arabidopsis, and it might be envisaged as a strategy to improve productivity in crops under adverse N environments