Caracterización de la actividad peroxidasa implicada en los procesos de lignificación y su estudio en la interacción : Capsicum annuum L. var annuum-Verticillium dahliae Kleb

[Resumen] Se han estudiado los procesos de lignificación, usando como modelo la especie Zinnia elegans. El análisis de las ligninas en el hipocotilo de Zinnia elegans, muestran la presencia de grupos con iferilaldehído terminales, así como la presencia de alcohol coniferílico y alcohol sinapílico formando parte de estructuras aril-glicerol-b-ariléter. La peroxidasa de Zinnia elegans es capaz de oxidar el alcohol coniferílico, el alcohol sinapílico, el coniferilaldehídoy el sinapilaldehído, tanto en presencia, como en ausencia de H2O2, de acuerdo al modelo cinético propuesto como general para las peroxidasas. Asimismo, esta enzima muestra una afinidad por todos los substratos del orden de uM, similar a la que presentan las enzimas inmediatamente anteriores en el proceso de síntesis de las ligninas. Por medio deluso de inhibidores competitivos, se ha podido demostrar a nivel histoquímico, que la peroxidasa de Z. Elegans se encuentra localizada tanto espacial como temporalmente en los sitios de lignificación activa, lo cual nos permite sugerir que es la enzima clave en la polimerización de las lignimas de tiposiringilo. Asimismo, se ha estudiado la interacción Capsicum annuum-Verticillium dahliae, fijándonos en el comportamiento del sistema peroxidasa y de los procesos del lignificación. La infección de plantas de Capsicum annuum con Verticillium dahliae provoca un aumento en la actividad peroxidasa, proceso que está acompañado por un aumento en la expresión de dos isoenzimas ácidas, de pI 3,6 y 4,0. Esta última isoenzima presenta una reactividad especial con la siringaldazina, lo que confirma su participación en los procesos de lignificación oxidando los grupos siringilo. La infecciónde C. Annuum con V. Dahliae provoca un aumento en la cantidad de lignina en las plantas infectas, conjuntamente con un cambio en su composición, que se refleja en una mayor proproción de grupos guaiacilo en las ligninas de las plantas

Evolutionary Implications of a Peroxidase with High Affinity for Cinnamyl Alcohols from Physcomitrium patens, a Non-Vascular Plant

[Abstract] Physcomitrium (Physcomitrella) patens is a bryophyte highly tolerant to different stresses, allowing survival when water supply is a limiting factor. This moss lacks a true vascular system, but it has evolved a primitive water-conducting system that contains lignin-like polyphenols. By means of a three-step protocol, including ammonium sulfate precipitation, adsorption chromatography on phenyl Sepharose and cationic exchange chromatography on SP Sepharose, we were able to purify and further characterize a novel class III peroxidase, PpaPrx19, upregulated upon salt and H2O2 treatments. This peroxidase, of a strongly basic nature, shows surprising homology to angiosperm peroxidases related to lignification, despite the lack of true lignins in P. patens cell walls. Moreover, PpaPrx19 shows catalytic and kinetic properties typical of angiosperm peroxidases involved in oxidation of monolignols, being able to efficiently use hydroxycinnamyl alcohols as substrates. Our results pinpoint the presence in P. patens of peroxidases that fulfill the requirements to be involved in the last step of lignin biosynthesis, predating the appearance of true lignin.Xunta de Galicia; INCITE08PXIB103182PRPortugal. Fundaçao para a Ciência e a Tecnologia; SFRH/BPD/112587/2015This research was funded by Xunta de Galicia (Spain), grant number INCITE 08PXIB103182PR. E.N.-U. holds an FCT postdoctoral fellowship (SFRH/BPD/112587/2015)

Análisis proteómico de dos peroxidasas de tipo siringilo de Selaginella martensii

Comunicaciones a congreso

The Ve-mediated resistance response of the tomato to Verticillium dahliae involves H2O2, peroxidase and lignins and drives PAL gene expression

<p>Abstract</p> <p>Background</p> <p><it>Verticillium dahliae </it>is a fungal pathogen that infects a wide range of hosts. The only known genes for resistance to <it>Verticillium </it>in the Solanaceae are found in the tomato (<it>Solanum lycopersicum</it>) <it>Ve </it>locus, formed by two linked genes, <it>Ve1 </it>and <it>Ve2</it>. To characterize the resistance response mediated by the tomato <it>Ve </it>gene, we inoculated two nearly isogenic tomato lines, LA3030 (<it>ve</it>/<it>ve</it>) and LA3038 (<it>Ve</it>/<it>Ve</it>), with <it>V. dahliae</it>.</p> <p>Results</p> <p>We found induction of H₂O₂production in roots of inoculated plants, followed by an increase in peroxidase activity only in roots of inoculated resistant plants. Phenylalanine-ammonia lyase (PAL) activity was also increased in resistant roots 2 hours after inoculation, while induction of PAL activity in susceptible roots was not seen until 48 hours after inoculation. Phenylpropanoid metabolism was also affected, with increases in ferulic acid, <it>p</it>-coumaric acid, vanillin and <it>p</it>-hydroxybenzaldehyde contents in resistant roots after inoculation. Six tomato <it>PAL </it>cDNA sequences (<it>PAL1 </it>- <it>PAL6</it>) were found in the SolGenes tomato EST database. RT-PCR analysis showed that these genes were expressed in all organs of the plant, albeit at different levels. Real-time RT-PCR indicated distinct patterns of expression of the different <it>PAL </it>genes in <it>V. dahliae</it>-inoculated roots. Phylogenetic analysis of 48 partial <it>PAL </it>cDNAs corresponding to 19 plant species grouped angiosperm <it>PAL </it>sequences into four clusters, suggesting functional differences among the six tomato genes, with <it>PAL2 </it>and <it>PAL6 </it>presumably involved in lignification, and the remaining <it>PAL </it>genes implicated in other biological processes.</p> <p>An increase in the synthesis of lignins was found 16 and 28 days after inoculation in both lines; this increase was greater and faster to develop in the resistant line. In both resistant and susceptible inoculated plants, an increase in the ratio of guaiacyl/syringyl units was detected 16 days after inoculation, resulting from the lowered amount of syringyl units in the lignins of inoculated plants.</p> <p>Conclusions</p> <p>The interaction between the tomato and <it>V. dahliae </it>triggered a number of short- and long-term defensive mechanisms. Differences were found between compatible and incompatible interactions, including onset of H₂O₂production and activities of peroxidase and PAL, and phenylpropanoid metabolism and synthesis of lignins.</p

Adaptations in energy metabolism and gene family expansions revealed by comparative transcriptomics of three Chagas disease triatomine vectors

Background: Chagas disease is a parasitic infection caused by Trypanosoma cruzi. It is an important public health problem affecting around seven to eight million people in the Americas. A large number of hematophagous triatomine insect species, occupying diverse natural and human-modified ecological niches transmit this disease. Triatomines are long-living hemipterans that have evolved to explode different habitats to associate with their vertebrate hosts. Understanding the molecular basis of the extreme physiological conditions including starvation tolerance and longevity could provide insights for developing novel control strategies. We describe the normalized cDNA, full body transcriptome analysis of three main vectors in North, Central and South America, Triatoma pallidipennis, T. dimidiata and T. infestans. Results: Two-thirds of the de novo assembled transcriptomes map to the Rhodnius prolixus genome and proteome. A Triatoma expansion of the calycin family and two types of protease inhibitors, pacifastins and cystatins were identified. A high number of transcriptionally active class I transposable elements was documented in T. infestans, compared with T. dimidiata and T. pallidipennis. Sequence identity in Triatoma-R. prolixus 1:1 orthologs revealed high sequence divergence in four enzymes participating in gluconeogenesis, glycogen synthesis and the pentose phosphate pathway, indicating high evolutionary rates of these genes. Also, molecular evidence suggesting positive selection was found for several genes of the oxidative phosphorylation I, III and V complexes. Conclusions: Protease inhibitors and calycin-coding gene expansions provide insights into rapidly evolving processes of protease regulation and haematophagy. Higher evolutionary rates in enzymes that exert metabolic flux control towards anabolism and evidence for positive selection in oxidative phosphorylation complexes might represent genetic adaptations, possibly related to prolonged starvation, oxidative stress tolerance, longevity, and hematophagy and flight reduction. Overall, this work generated novel hypothesis related to biological adaptations to extreme physiological conditions and diverse ecological niches that sustain Chagas disease transmission.Fil: Martínez Barnetche, Jesús. Instituto Nacional de Salud Pública; MéxicoFil: Lavore, Andres Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Beliera, Melina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Téllez Sosa, Juan. Instituto Nacional de Salud Pública; MéxicoFil: Zumaya Estrada, Federico A.. Instituto Nacional de Salud Pública; MéxicoFil: Palacio, Victorio Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Godoy Lozano, Ernestina. Instituto Nacional de Salud Pública; MéxicoFil: Rivera Pomar, Rolando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Rodríguez, Mario Henry. Instituto Nacional de Salud Pública; Méxic

Genetic characterization and pungency analysis in a collection of onion landraces from NW Spain: abstract

Publishe

Exploring genetic diversity and quality traits in a collection of onion (Allium cepa L) landraces from North-West Spain

Seventeen onion landraces from North-West Spain were evaluated using microsatellites markers. Eleven polymorphic markers identified 32 alleles in the whole collection, with an average of 2.9 alleles per locus. High values of observed (mean of 0.45) and expected heterozigosity (mean of 0.51) were detected for the majority of loci. Wright’s fixation index confirmed an excess of heterozygotes and a low level of inbreeding within the collection. Multivariate analyses revealed that Oimbra was the most distinctive genotype. The remaining 16 onion genotypes were in part assorted according to some morphological traits of bulbs. Pungency and solid soluble content highly varied among landraces and bulbs. Five landraces were classified as sweet, whereas 9 possessed medium pungency and 3 were recorded as pungent. This onion collection represents a useful source of genetic heterogeneity that might be exploited in breeding programs for the generation of new onion varieties that satisfy consumer demandsPublishe

Overexpression of ZePrx in Nicotiana Tabacum Affects Lignin Biosynthesis Without Altering Redox Homeostasis

[Abstract] Class III plant peroxidases (Prxs) are involved in the oxidative polymerization of lignins. Zinnia elegans Jacq. Basic peroxidase (ZePrx) has been previously characterized as capable of catalyzing this reaction in vitro and the role in lignin biosynthesis of several of its Arabidopsis thaliana homologous has been previously confirmed. In the present work, ZePrx was overexpressed in Nicotiana tabacum to further characterize its function in planta with particular attention to its involvement in lignin biosynthesis. Since Prxs are known to alter ROS levels by using them as electron acceptor or producing them in their catalytic activity, the impact of this overexpression in redox homeostasis was studied by analyzing the metabolites and enzymes of the ascorbate-glutathione cycle. In relation to the modification induced by ZePrx overexpression in lignin composition and cellular metabolism, the carbohydrate composition of the cell wall as well as overall gene expression through RNA-Seq were analyzed. The obtained results indicate that the overexpression of ZePrx caused an increase in syringyl lignin in cell wall stems, suggesting that ZePrx is relevant for the oxidation of sinapyl alcohol during lignin biosynthesis, coherently with its S-peroxidase nature. The increase in the glucose content of the cell wall and the reduction of the expression of several genes involved in secondary cell wall biosynthesis suggests the occurrence of a possible compensatory response to maintain cell wall properties. The perturbation of cellular redox homeostasis occurring as a consequence of ZePrx overexpression was kept under control by an increase in APX activity and a reduction in ascorbate redox state. In conclusion, our results confirm the role of ZePrx in lignin biosynthesis and highlight that its activity alters cellular pathways putatively aimed at maintaining redox homeostasis.Xunta de Galicia; ED431C 2018/57AG-U held an FPU grant from MECD (Spain) (FPU13/04835). This research was possible thanks to the funding of Xunta de Galicia (Spain) (ED431C 2018/57

Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures

18 p.Maize (Zea mays L.) suspension-cultured cells with up to 70% less celluloseQ1 were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulosedeficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and pcoumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulosedeficient cell walls showed a fivefold increase in Klasontype lignin. Thioacidolysis/GC-MS analysis of cellulosedeficient cell walls indicated the presence of a ligninlike polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishmentS