Function of antioxidant enzymes and metabolites during maturation of pea fruits

Panel (S2-P29) presentado en la Reunión de la Sociedad Española de Fisiología Vegetal (SEFV) (18ª. Zaragoza. 8-11 septiembre 2009).Leguminous plants such as pea (Fisum sativum), bean (Fhaseolus vulgaris) or alfalfa (Medicago sativa) are crops of major economical value as protein source for human and animal consumption. They are also essential to sustainable agricultural systems because of their ability to establish nitrogen-fixing symbioses with soil bacteria, thus providing a biological alternative to chemical fertilization. The concentration of antioxidants in fruits is important agronomically, as it is well documented that antioxidants may protect fruit tissues from potentially toxic reactive oxygen species (ROS) and thus contribute to the stress tolerance of crops. Furthermore, following harvest, fruits have a relatively short shelf-life during which they undergo changes in texture, colour and flavour. Because ROS may be involved in the oxidative processes contributing to fruit deterioration, the antioxidant content is important in terms of both the nutritional value and the post-harvest storage of the fruits. Although the antioxidants are believed to play a crucial role in the ripening process of climacteric fruits, their role in the development, maturation and post-harvest storage of legume fruits is poorly defined. We determined the antioxidant capacity (ascorbate-glutathione pathway, superoxide dismutases, catalases, glutathione peroxidases and peroxiredoxins) of mature fruits from nodulated plants and from plants supplied with combined nitrogen, and concluded that pea fruits from plants dependent on nitrogen fixation show similar antioxidant levels to those dependent on chemical fertilization. Furthermore, oir results showed that fruit maturation causes a decline in the antioxidants of pea fruits. However, the concentration of lipid peroxides and oxidized proteins remained unaltered, suggesting that, contrary to the situation described in climacteric fruits, a decrease in the antioxidant capacity does not necessarily lead to oxidative stress in maturing pea fruits. Similarly, oir data showed that despite the decreases in several antioxidants, oxidative processes are probably not involved in legume fruit deterioration during storage at room temperature. Finally, our results underscore the importance of ascorbate, one of the key components of the antioxidant network, in pea fruit growth and development.This work was funded by Ministerio de Ciencia e Innovación (grant AGL2008-01298) and Gobierno de Aragón (PIP137/2005 and group A53).Peer reviewe

Natural variation in FLOWERING LOCUS T, HvFT1

1 .pdf copy (A3) of the original poster presented by the Authors.The barley ortholog of FLOWERING LOCUS T, HvFT1, also called VrnH3, is the main integrator of the photoperiod and vernalization signals leading to the transition from the vegetative to the reproductive stage. Results gathered by us and other groups for the last years have repeatedly identified variation in this gene related with flowering time QTL in mapping populations and also in genome wide association studies. Differences in the promoter, SNPs in the first intron and also copy number variation have all being associated with phenotypic and expression differences, resulting in earlier or later heading. The first reports found that mutations in the HvFT1 first intron differentiated plants with dominant and recessive alleles, with large phenotypic effect on time to flowering. The catalog of polymorphisms at this gene with potential phenotypic effect has been enlarged with copy number variation and sequence variation at the promoter. There is variation in the number of copies of the HvFT1 gene, apparently related to growth habit. A large set of winter genotypes, with a functional VrnH2 allele, has one copy of VrnH3, whereas variable number (1-5), was found in also a large set of spring or facultative barleys (without VrnH2). The dominant VrnH3 allele, which overrides the vernalization requirement of winter VrnH1 and VrnH2 alleles, is found only in Nordic barleys and carries a particular structure of the gene, with one promoter and variable number of transcribed regions. Using two indels from the promoter region and allele-specific markers for two SNPs in the first intron, we were able to classify four VrnH3 haplotypes, which showed differences in heading time among Spanish landraces. We will present results from several mapping populations and association analyses to contribute to describe the different polymorphisms that should be taken into consideration when analyzing this gene and its phenotypic effects.Peer reviewe

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24–48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses

Metabolismo de ascorbato y tioles en leguminosas

El objetivo principal de la tesis fue el estudio del metabolismo en leguminosas de ascorbato (vitamina C) y tioles, los antioxidantes solubles más abundantes en plantas, en condiciones de estrés abiótico y oxidativo, tras el tratamiento de una hormona relacionada con la señalización del estrés (ácido jasmónico), y también durante la senescencia natural de los nódulos. Las conclusiones más importantes son las siguientes: (1) Los nódulos de leguminosas pueden sintetizar ascorbato de novo. (2) Los estreses tienen un efecto minoritario en el metabolismo del ascorbato en el nódulo. La enzima L-galactono-1,4-lactona deshidrogenasa no es la determinante del contenido de ascorbato. (3) El ácido jasmónico provoca un aumento transcripcional de la actividad ascorbato oxidasa y una inhibición postraduccional de la actividad deshidroascorbato reductasa, resultando en la disminución del estado redox del ascorbato en el nódulo. (4) Los estreses tienen un efecto pequeño en el metabolismo del (homo)glutatión en los nódulos. La actividad γ-glutamilcisteína sintetasa está regulada a nivel traduccional en estas condiciones. (5) La vida del nódulo está regulada, al menos en parte, por factores endógenos. El ascorbato desempeña un papel fundamental en la senescencia nodular. (6) La existencia de una homofitoquelatina sintasa específica de leguminosas resulta improbable. Algunos metales fisiológicamente importantes, como Cu, Zn o Fe, activan a la enzima fitoquelatina sintasa purificada in vitro. (7) Un exceso de los metales anteriores no induce la síntesis de (homo)fitoquelatinas in vivo.Tesis posible con la financiación recibida de los proyectos AGL2002-02876 y AGL2005-01404 del Ministerio de Educación y Ciencia - Fondos Europeos de Desarrollo REgional, y E33 (Grupo de Investigación Consolidado) del Gobierno de Aragón - Fondo Social Europeo. También del Proyecto PIP137/2005 del Gobierno de Aragón.Peer reviewe

Changes in the antioxidants of mitochondria during natural senescence of legume nodules

2 Pag., 1 Fig.Legume nodules have elevated respiratory rates in mitochondria and bacteroids to meet the energy requirements for N2 fixation. However, respiration inevitably produces reactive oxygen species (ROS) such as superoxide radicals and hydrogen peroxide (H2O2). We have studied the changes associated with natural senescence (aging) in the antioxidant system of mitochondria purified from common bean (Phaseolus vulgaris) nodules. During nodule aging, the concentrations of lipid peroxides and oxidized proteins increased in mitochondria, but not in the cytosolic fraction. Also, the activities of three enzymes involved in ascorbate synthesis and regeneration were down-regulated. By contrast, qRT-PCR analysis, immunoblots, and enzyme activity assays reveal a significant up-regulation of manganese superoxide dismutase (MnSOD) activity during aging, whereas the protein level of peroxiredoxin IIF (PrxIIF) remained unchanged. Overall, our results suggest that, in legume nodules, natural senescence is associated with a decline in the capacity of mitochondria to synthesize and regenerate ascorbate, and that this organelle is a preferential target of oxidative stress.This work was supported by the MICINN (AGL2008-01298) and Gobierno de Aragón (group A53).Peer reviewe

Ascorbate and Homoglutathione Metabolism in Common Bean Nodules under Stress Conditions and during Natural Senescence

11 pages, 9 figures.-- Full-text version available Open Access at the journal site.Ascorbate and glutathione are major antioxidants and redox buffers in plant cells but also play key functions in growth, development, and stress responses. We have studied the regulation of ascorbate and homoglutathione biosynthesis in common bean (Phaseolus vulgaris) nodules under stress conditions and during aging. The expression of five genes of the major ascorbate biosynthetic pathway was analyzed in nodules, and evidence was found that L-galactono-1,4-lactone dehydrogenase, the last committed step of the pathway, is posttranscriptionally regulated. Also, in nodules under stress conditions, {gamma}-glutamylcysteine synthetase was translationally regulated, but homoglutathione synthetase (mRNA and activity) and homoglutathione (content and redox state) were not affected. Most interestingly, in nodules exposed to jasmonic acid, dehydroascorbate reductase activity was posttranslationally suppressed, ascorbate oxidase showed strong transcriptional up-regulation, and dehydroascorbate content increased moderately. These changes were not due to a direct effect of jasmonic acid on the enzyme activities but might be part of the signaling pathway in the response of nodules to stress. We determined ascorbate, homoglutathione, and ascorbate-glutathione pathway enzyme activities in two senescing stages of nodules undergoing oxidative stress. When all parameters were expressed on a nodule fresh weight basis, we found that in the first stage ascorbate decreased by 60% and homoglutathione and antioxidant activities remained fairly constant, whereas in the second stage ascorbate and homoglutathione, their redox states, and their associated enzyme activities significantly decreased. The coexistence in the same plants of nodules at different senescence stages, with different ascorbate concentrations and redox states, indicates that the life span of nodules is in part controlled by endogenous factors and points to ascorbate as one of the key players.This work was supported by Ministerio de Educación y Ciencia-Fondos Europeos de Desarrollo Regional (grant no. AGL2005–01404) and by Gobierno de Aragón (group E33 and grant no. PIP137/2005; predoctoral fellowship [B041/2004] to J.L.)Peer reviewe

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

1 copia .pdf (a3) de póster original presentado por los autores. 4 Figs.The thiol tripeptide GSH (yGIu-Cys-GIy) is a major antioxidant and redox buffer in plants, where it also performs critical functions in ccli cycle regulation, development, sulfur transport and storage, stress response, and heavy metal detoxification. In legumes, hGSH (yGlu-Cys-bAla) may partially or completely replace GSH with presumably the same functions. The synthesis of GSH is accomplished in two sequential reactions catalyzed by yECS and GSHS, whereas the synthesis of hGSH shares the same first enzyme and then requires a specific hGSHS. A better understanding of the regulation of GSH and hGSH biosynthesis in legumes during the stress response requires a precise determination of the subcellular localization of the enzymes and a quantitative expression analysis of the genes involved. For this purpose, two types of experiments were performed. First, these proteins were immunolocalized in legumes using electron microscopy. Second, the expression pattern of the three genes was determined in the model legume Lotus japonicus following treatment with several hormones that are crucial for plant development and stress signaling.This work was funded by grant AGL2008-01298 from MICINN-FEDER.Peer reviewe

¿Hasta cuándo dura el invierno para la cebada? Expresión de genes de floración de cebada afectada por el fotoperiodo natural

2 Pags. Contribución de los autores originalmente presentada, como comunicación,en el VIII Congreso de Mejora Genética de Plantas (Vitoria-Gasteiz, 2016).Se sabe que la expresión de VRNH2 se produce en condiciones de día Largo. Sin embargo, se desconocen las horas de luz necesarias para inducirlo en condiciones naturales y su efecto sobre otros genes o caracteres de desarrollo. Este trabajo persigue conocer la longitud del día a la que se induce VRNH2. Este umbral fisiológico es importante para definir los modelos de variedad que se persiguen, pues supone el momento del año en el que una variedad de invierno deberá tener cumplida su vernalización. En caso contrario, VRNH2 retrasaría la floración más allá de lo agronómicamcnte óptimo, como ocurre (de modo exagerado) cuando se siembra una variedad de invierno en primavera.Trabajo financiado por el MINECO, dentro del proyecto AGL20I 3-48756-R.Peer reviewe

Polimorfismo y expresión de HvFT1, el gen integrador de las rutas del fotoperíodo y la vernalización en cebada

2 Pags.Este estudio tiene por objeto i) examinar la distribución de los polimorfismos de secuencia y CNV de HvFTI en una muestra amplia de germoplasma de cebada y ii) confirmar si el polimorfismo CNV se relaciona con el fenotipo (Loscos et al., 2014).Peer reviewe