Lignificación en cultivos celulares de gimnospermas basales

[Resumen] La lignificación de la pared celular es el proceso de sellado de las paredes vegetales a través de la deposición de lignina, que proporciona fuerza mecánica a los tallos. Las células en suspensión tan sólo presentan paredes celulares primarias y muchas veces no lignifican. Se llevó al cabo la caracterización del crecimiento de callos y suspensiones celulares de Betula pendula y Ginkgo biloba, así como la caracterización de la actividad peroxidasa. La mayor actividad peroxidasa se presentó usando alcohol coniferílico como sustrato, aunque estas peroxidasas fueron capaces de oxidar grupos siringilo como lo demostró el hecho que de tanto el alcohol sinapílico como la siringaldazina fueron sustratos de las enzimas. Se comparó la lignificación en plantas y suspensiones celulares de B. pendula, G. biloba y Cycas revoluta, revelando que éstas presentaron lignificación en sus paredes celulares. Se observaron diferencias en la presencia de grupos siringilo en las gimnospermas estudiadas, indicando que tienen el potencial de sintetizar estos grupos pero que su síntesis está reprimida, indicando una fuerte regulación de la lignificación. Los datos obtenidos (mayor cantidad de unidades H y mayor ramificación) sugieren que las ligninas de los cultivos celulares son similares a las de las paredes celulares primarias, y validan los cultivos celulares como herramienta de estudio de la lignificación de la pared celular primaria. Se estudió el proteoma extracelular de B. pendula, G. biloba y C. revoluta, mostrando la presencia universal de peroxidasas capaces de oxidar los alcoholes cinamílicos a ligninas, junto con otras proteínas que intervienen en la formación de la pared celular secundaria y la xilogénesis, y que se expresan diferencialmente durante el proceso de lignificación de las paredes celulares, tanto primarias como secundarias. Se purificó y caracterizó la proteína más abundante en el proteoma extracelular de G. biloba, resultando ser una peroxidasa de clase III con propiedades moleculares y catalíticas distintivas, como su espectro de absorción con un máximo de absorción a 414nm, pero con una funcionalidad clara en la lignificación, como se deduce de su capacidad de oxidar tanto el alcohol coniferílico como el alcohol sinapílico. Se purificaron y caracterizaron peroxidasas del proteoma extracelular de C. revoluta. Todas estas proteínas fueron peroxidasas de alto espín capaces de oxidar los alcoholes cinamílicos, lo que les asigna un papel en la lignificación de las paredes celulares, especialmente a las peroxidasas básicas, que muestran mayor afinidad que las ácidas por el alcohol sinapílico

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

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

Digestibility of silages in relation to their hydroxycinnamic acid content and lignin composition

BACKGROUND: The effectiveness of the analysis of cell wall-bound hydroxycinnamic acids and the composition of lignin to evaluate the in vivo digestibility of a silage collection with unknown botanical composition was evaluated.RESULTS: Syringyl units content and total etherified phenols showed the highest correlation coefficients with in vivo dry matter digestibility (IVDMD) (r = − 0.792 and r = − 0.703, respectively), while guaiacyl units and total phenols showed the highest correlation coefficients with in vivo organic matter digestibility (IVOMD) (r = − 0.871 and r = − 0.817, respectively). Using the above-mentioned chemical parameters, 10 equations were also developed to predict in vivo digestibility. The prediction of IVDMD produced a high adjusted R2 value (0.710) using syringyl, total lignin, etherified total phenols, esterified ferulic acid and total phenol content as predictors. The prediction of IVOMD produced a higher adjusted R2 value (0.821) using guaiacyl, total phenols, total ferulic acid and etherified p-coumaric acid content as predictors.CONCLUSION: Cell wall digestibility depends on a multiplicity of factors and it is not possible to attribute a causal effect on in vivo digestibility to any single factor. However, syringyl and guaiacyl content and etherified phenols emerge as good predictors of digestibility.Xunta de Galicia (PGIDIT04RAG503018PR); INIA–European Social FundPeer Reviewe

Low concentrations of the toxin ophiobolin A lead to an arrest of the cell cycle and alter the intracellular partitioning of glutathione between the nuclei and cytoplasm

Ophiobolin A, a tetracyclic sesterpenoid produced by phytopathogenic fungi, is responsible for catastrophic losses in crop yield but its mechanism of action is not understood. The effects of ophiobolin A were therefore investigated on the growth and redox metabolism of Tobacco Bright Yellow-2 (TBY-2) cell cultures by applying concentrations of the toxin that did not promote cell death. At concentrations between 2 and 5 μM, ophiobolin A inhibited growth and proliferation of the TBY-2 cells, which remained viable. Microscopic and cytofluorimetric analyses showed that ophiobolin A treatment caused a rapid decrease in mitotic index, with a lower percentage of the cells at G1 and increased numbers of cells at the S/G2 phases. Cell size was not changed following treatment suggesting that the arrest of cell cycle progression was not the result of a block on cell growth. The characteristic glutathione redox state and the localization of glutathione in the nucleus during cell proliferation were not changed by ophiobolin A. However, subsequent decreases in glutathione and the re-distribution of glutathione between the cytoplasm and nuclei after mitosis occurring in control cells, as well as the profile of glutathionylated proteins, were changed in the presence of the toxin. The profile of poly ADP-ribosylated proteins were also modified by ophiobolin A. Taken together, these data provide evidence of the mechanism of ophiobolin A action as a cell cycle inhibitor and further demonstrate the link between nuclear glutathione and the cell cycle regulation, suggesting that glutathione-dependent redox controls in the nuclei prior to cell division are of pivotal importance

Nitric oxide is required for determining root architecture and lignin composition in sunflower. Supporting evidence from microarray analyses

Nitric oxide (NO) is a signal molecule involved in several physiological processes in plants, including root development. Despite the importance of NO as a root growth regulator, the knowledge about the genes and metabolic pathways modulated by NO in this process is still limited. A constraint to unravel these pathways has been the use of exogenous applications of NO donors that may produce toxic effects. We have analyzed the role of NO in root architecture through the depletion of endogenous NO using the scavenger cPTIO. Sunflower seedlings growing in liquid medium supplemented with cPTIO showed unaltered primary root length while the number of lateral roots was deeply reduced; indicating that endogenous NO participates in determining root branching in sunflower. The transcriptional changes induced by NO depletion have been analyzed using a large-scale approach. A microarray analysis showed 330 genes regulated in the roots (p ⩽ 0.001) upon endogenous NO depletion. A general cPTIO-induced up-regulation of genes involved in the lignin biosynthetic pathway was observed. Even if no detectable changes in total lignin content could be detected, cell walls analyses revealed that the ratio G/S lignin increased in roots treated with cPTIO. This means that endogenous NO may control lignin composition in planta. Our results suggest that a fine tuning regulation of NO levels could be used by plants to regulate root architecture and lignin composition. The functional implications of these findings are discussed.Fil: Corti Monzón, Georgina de la Paz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Pinedo, Marcela Lilian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Di Rienzo, Julio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Córdoba. Facultad de Cs.exactas Físicas y Naturales. Departamento de Matemáticas. Cátedra de Estadística y Biometria; ArgentinaFil: Novo Uzal, Esther. Universidad de Murcia; EspañaFil: Pomar, Federico. Universidad da Coruña; EspañaFil: Lamattina, Lorenzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: de la Canal, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin