Browsing by Author "Jordana, Xavier"

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    A Deficiency in the Flavoprotein of Arabidopsis Mitochondrial Complex II Results in Elevated Photosynthesis and Better Growth in Nitrogen-Limiting Conditions
    (AMER SOC PLANT BIOLOGISTS, 2011) Fuentes, Daniela; Meneses, Marco; Nunes Nesi, Adriano; Araujo, Wagner L.; Tapia, Rodrigo; Gomez, Isabel; Holuigue, Loreto; Gutierrez, Rodrigo A.; Fernie, Alisdair R.; Jordana, Xavier
    Mitochondrial complex II (succinate dehydrogenase [SDH]) plays roles both in the tricarboxylic acid cycle and the respiratory electron transport chain. In Arabidopsis (Arabidopsis thaliana), its flavoprotein subunit is encoded by two nuclear genes, SDH1-1 and SDH1-2. Here, we characterize heterozygous SDH1-1/sdh1-1 mutant plants displaying a 30% reduction in SDH activity as well as partially silenced plants obtained by RNA interference. We found that these plants displayed significantly higher CO2 assimilation rates and enhanced growth than wild-type plants. There was a strong correlation between CO2 assimilation and stomatal conductance, and both mutant and silenced plants displayed increased stomatal aperture and density. By contrast, no significant differences were found for dark respiration, chloroplastic electron transport rate, CO2 uptake at saturating concentrations of CO2, or biochemical parameters such as the maximum rates of carboxylation by Rubisco and of photosynthetic electron transport. Thus, photosynthesis is enhanced in SDH-deficient plants by a mechanism involving a specific effect on stomatal function that results in improved CO2 uptake. Metabolic and transcript profiling revealed that mild deficiency in SDH results in limited effects on metabolism and gene expression, and data suggest that decreases observed in the levels of some amino acids were due to a higher flux to proteins and other nitrogen-containing compounds to support increased growth. Strikingly, SDH1-1/sdh1-1 seedlings grew considerably better in nitrogen-limiting conditions. Thus, a subtle metabolic alteration may lead to changes in important functions such as stomatal function and nitrogen assimilation.
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    A Nuclear Gene Encoding the Iron-Sulfur Subunit of Mitochondrial Complex II Is Regulated by B3 Domain Transcription Factors during Seed Development in Arabidopsis
    (AMER SOC PLANT BIOLOGISTS, 2009) Roschzttardtz, Hannetz; Fuentes, Ignacia; Vasquez, Marcos; Corvalan, Claudia; Leon, Gabriel; Gomez, Isabel; Araya, Alejandro; Holuigue, Loreto; Vicente Carbajosa, Jesus; Jordana, Xavier
    Mitochondrial complex II (succinate dehydrogenase) is part of the tricarboxylic acid cycle and the respiratory chain. Three nuclear genes encode its essential iron-sulfur subunit in Arabidopsis (Arabidopsis thaliana). One of them, SUCCINATE DEHYDROGENASE2-3 (SDH2-3), is specifically expressed in the embryo during seed maturation, suggesting that SDH2-3 may have a role as the complex II iron-sulfur subunit during embryo maturation and/or germination. Here, we present data demonstrating that three abscisic acid-responsive elements and one RY-like enhancer element, present in the SDH2-3 promoter, are involved in embryo-specific SDH2-3 transcriptional regulation. Furthermore, we show that ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON2, three key B3 domain transcription factors involved in gene expression during seed maturation, control SDH2-3 expression. Whereas ABI3 and FUS3 interact with the RY element in the SDH2-3 promoter, the abscisic acid-responsive elements are shown to be a target for bZIP53, a member of the basic leucine zipper (bZIP) family of transcription factors. We show that group S1 bZIP53 protein binds the promoter as a heterodimer with group C bZIP10 or bZIP25. To the best of our knowledge, the SDH2-3 promoter is the first embryo-specific promoter characterized for a mitochondrial respiratory complex protein. Characterization of succinate dehydrogenase activity in embryos from two homozygous sdh2-3 mutant lines permits us to conclude that SDH2-3 is the major iron-sulfur subunit of mature embryo complex II. Finally, the absence of SDH2-3 in mutant seeds slows down their germination, pointing to a role of SDH2-3-containing complex II at an early step of germination.
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    A Reverse Transcriptase Activity in Potato Mitochondria
    (1996) Moenne, A.; Jordana, Xavier
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    A Ribosomal Protein S10 Gene Is Found in the Mitochondrial Genome in Solanum Tuberosum
    (1994) Zanlungo Matsuhiro, Silvana; Holuigue Barros, María Loreto; Jordana, Xavier
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    Caracterización de la regulación transcripcional y función del gen SDH2-3 de Arabidopsis thaliana.
    (2013) Restovic Carvajal, Franko; Jordana, Xavier; Pontificia Universidad Católica de Chile. Facultad de Ciencias Biológicas
    Our group has undertaken the study of mitochondrial function in plants focusing on complex II (succinate dehydrogenase, SDH) of Arabidopsis thaliana. This complex plays a pivotal role in the tricarboxylic acid (TCA) cycle and the electron transport chain, two fundamental processes in the energetic plant metabolism. We have described the existence of three nuclear genes coding for iron-sulfur proteins (SDH2), named SDH2-1, SDH2-2 and SDH2-3. Two of them, SDH2-1 and SDH2-2, are almost identical (96%) and share the same exon-intron structure. SDH2-3 on the other hand, has a different exonintron structure and has approximately 70% identity to the other two. Additionally, unlike SDH2-1 and SDH2-2 that are expressed in adult plants, SDH2-3 expression is confined to seed maturation and it decreases during germination, while SDH2-1 and SDH2-2 transcripts are low in seeds and they begin to accumulate after germination and during vegetative growth. We have described seed-specific cis-elements necessary for promoter activity (three ABA-responsive elements or ABRE and one RY element) and determined in vitro binding of seed-specific transcription factors to them (bZIP10, bZIP25 and bZIP53 binding to ABREs; ABI3 and FUS3 binding to RY). Moreover, we have determined that ABI3 and FUS3 are necessary for promoter activity in planta, as mutant lines of these factors showed decreased SDH2-3 levels in seeds. On the other hand, we have determined that SDH2-3 is important for seed germination, indicating a specific role during this developmental stage. The main focus of this thesis work is to study the seed-specific expression and the function of SDH2-3. It is worth noting that no reports of mitochondrial proteins bearing this singular expression pattern have been published. Therefore, we will determine essential regions of the SDH2-3 promoter and additional putative cis-elements controlling transcriptional regulation, transcription factors involved, and the function of this protein during seed maturation and postgerminative growth. Regarding the SDH2-3 promoter, during this thesis we were able to determine a minimal region necessary and sufficient for promoter activity, between -114 and +49 from the transcription start site. Moreover, the 5´UTR region (+1 to +49) is essential for SDH2-3 promoter activity, as determined by loss-of-function experiments. In addition, transient expression assays showed that ABI3 is able to activate SDH2- 3 transcription in vivo alone or in combination with bZIP factors bZIP10, bZIP25 or bZIP53. However, single bZIP factors were unable to activate the promoter, and only transfection with bZIP10 and bZIP53 was able to induce expression. Moreover, SDH2-3 transcript levels are significantly reduced in bzip53 mutant dry seeds. These results indicate the SDH2-3 promoter is activated by bZIP transcription factors and corroborate the importance of ABI3. On the other hand, we determined that SDH2-3 influences seed development and maturation, as lack of this protein resulted in decreased seed weight. Interestingly, protein content also showed a reduction in sdh2-3 mutants while lipid content did not show any biologically significant variation. This is an interesting feature since seed metabolism is directed during maturation towards the formation of seed proteins and lipids. Thus, the decrease in protein content would explain the lower total weight. Although it has been suggested that mitochondria plays a minor role during seed maturation, these results suggest that its role should be reconsidered, as it may carry out important metabolic tasks during this stage. Mitochondrial role during postgerminative growth is well characterized. Here we show that sdh2-3 mutants have impaired hypocotyl growth in the dark. Moreover, TTFA treatment (complex II inhibitor) abolishes hypocotyl growth and seedling establishment. All these results suggest an essential role for complex II during postgerminative growth and establishment. Wild seeds in nature generally germinate underground, in conditions where they lack direct sunlight. A seed with a non-functional SDH2-3 would be in disadvantage over wild-type seeds, which would elongate their hypocotyls further until they reach light in order to promote photoautotrophic growth. SDH2-3 gives an important advantage to the plant in energy-consuming processes such as germination and early stages towards seedling establishment. This work shows the importance that this nonessential gene can have in critical stages of plant development. The existence of a SDH2-3-like gene in the moss Physcomitrella patens has drawn our attention because SDH2-3 has been described as a seed-specific expressed gene in angiosperms, and mosses do not have seeds. We confirmed the SDH2-3-like gene is expressed and increases under osmotic stress, in contrast to saline stress, desiccation and high ABA content. Moreover, we determined that Physcomitrella SDH2-3 promoter lacks significant activity in Arabidopsis, either in seeds or vegetative tissue. These results indicate that the transcriptional regulation of this gene in Physcomitrella evolved in an independent way as compared to Arabidopsis.
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    Caracterización de los genes MIT1 y MIT2 de Arabidopsis Thaliana que codifican para transportadores mitocondriales de hierro
    (2021) Vargas Pérez, Joaquín Ignacio; Roschzttardtz Choucroun, Hannetz France; Jordana, Xavier; Pontificia Universidad Católica de Chile. Facultad de Ciencias Biológicas
    Entre los micronutrientes esenciales para las plantas, el hierro es aquel que se requiere en mayor abundancia ya que es componente de cofactores como hemo y centros hierro azufre, que participan en procesos como la fotosíntesis y la respiración, entre otros. Importantes avances han permitido conocer los mecanismos que rigen la homeostasis del hierro a nivel fisiológico en las plantas, sin embargo a nivel subcelular existen aún muchas interrogantes. En este contexto, las mitocondrias son organelos con un alto requerimiento de hierro ya que albergan parte de la ruta de síntesis de centros hierro azufre y utilizan numerosos cofactores de hierro en la cadena transportadora de electrones, que en total suman al menos 9 hemos y 13 centros hierro azufre, por lo que la función mitocondrial y el metabolismo del hierro se encuentran estrechamente relacionados. Se ha estudiado poco la homeostasis del hierro mitocondrial en plantas y poco se sabe de los transportadores involucrados. Recientemente fue descrito el primer transportador de hierro mitocondrial en plantas, llamado Mitochondrial Iron Transporter (MIT) en Oryza sativa y codificado por un gen esencial. En base a este transportador identificamos dos genes ortólogos de Arabidopsis thaliana, At1g07030 (MIT1) y At2g30160 (MIT2). La expresión de estos genes en levaduras mutantes en los transportadores mitocondriales de hierro de alta afinidad les permite a estas levaduras crecer en medio deficiente en hierro, lo que sugiere fuertemente que MIT1 y MIT2 transportan hierro a la mitocondria en levaduras. Para caracterizar la función de MIT1 y MIT2 se obtuvieron 3 líneas homocigotas mutantes para MIT1 y 2 líneas homocigotas mutantes para MIT2, y ninguna de ellas presentó alteraciones en su fenotipo. Además la segregación del alelo mutado en la descendencia de plantas heterocigotas tampoco mostró alteraciones. Estos resultados sugieren que ambos genes son redundantes, lo que fue confirmado al cruzar líneas mutantes nulas mit1 y mit2 y no poder obtenerse dobles homocigotas mutantes. Este resultado demostró además que la función MIT es esencial en Arabidopsis. Al cruzar una mutante nula de MIT2 y una “knockdown” de MIT1 fue posible obtener plantas dobles homocigotas mutantes que en la primera generación expresaban un 10-20 % de MIT1. Estas plantas resultaron ser viables y presentaron múltiples alteraciones fenotípicas: embriones con tres cotiledones, retraso en la germinación, en el crecimiento post-germinativo y durante todo el desarrollo. Además, se encontraron anormalidades morfológicas en semillas, hojas, flores y tallos. Parte de estas alteraciones podría explicarse directamente por una disfunción mitocondrial generada por deficiencia de hierro en el organelo, lo que se sustenta en la inducción de genes marcadores de respuesta a perturbación/estrés mitocondrial. Otra parte de las alteraciones podría estar relacionada con un efecto indirecto de la perturbación mitocondrial sobre la homeostasis de auxina. El análisis de los datos transcriptómicos revela que estas plantas tienen probablemente reprimida la adquisición de hierro, la síntesis de cumarinas, la formación de la banda de Caspari, la suberización y el desarrollo de los pelos radiculares. Y tienen aumentada la expresión de genes relacionados con desarrollo y mantención de meristemas, formación y límites de órganos, desarrollo de flores. Curiosamente estas plantas dobles homocigotas mutantes recuperaron un fenotipo normal en la siguiente generación, lo que se correlaciona con un aumento de la expresión de MIT1 (70% a nivel de transcrito y 30% a nivel de proteína, respecto de plantas silvestres), probablemente debido al mecanismo descrito de “supresión de T-DNAs intrónicos”. Estas plantas “compensadas” no son diferentes de plantas silvestres en actividad mitocondrial, en el proteoma mitocondrial y en el transcriptoma.
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    cis Recognition Elements in Plant Mitochondion RNA Editing
    (2001) Farre, J.; Jordana, Xavier
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    Cloning and Characterization of the Cdna Coding for the Catalytic a Subunit of Ck2 From Tobacco
    (2001) Salinas, P.; Jordana, Xavier; Holuigue Barros, María Loreto
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    Contiguous RNA editing sites in the mitochondrial nad1 transcript of Arabidopsis thaliana are recognized by different proteins
    (2013) Arenas Miranda, Ana Maribel.; Moreno Ramírez, Sebastián Andrés; Gómez Barrenechea, María Isabel.; Jordana, Xavier
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    Early genomic responses to salicylic acid in Arabidopsis
    (2009) Blanco Herrera, María Francisca; Salinas Salvo, Paula Andrea Ximena; Jordana, Xavier; Holuigue Barros, María Loreto
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    Glutaredoxin GRXS13 plays a key role in protection against photooxidative stress in Arabidopsis
    (OXFORD UNIV PRESS, 2012) Laporte, Daniel; Olate, Ema; Salinas, Paula; Salazar, Marcela; Jordana, Xavier; Holuigue, Loreto
    Glutaredoxins (GRXs) belong to the antioxidant and signalling network involved in the cellular response to oxidative stress in bacterial and eukaryotic cells. In spite of the high number of GRX genes in plant genomes, the biological functions and physiological roles of most of them remain unknown. Here the functional characterization of the Arabidopsis GRXS13 gene (At1g03850), that codes for two CC-type GRX isoforms, is reported. The transcript variant coding for the GRXS13.2 isoform is predominantly expressed under basal conditions and is the isoform that is induced by photooxidative stress. Transgenic lines where the GRXS13 gene has been knocked down show increased basal levels of superoxide radicals and reduced plant growth. These lines also display reduced tolerance to methyl viologen (MeV) and high light (HL) treatments, both conditions of photooxidative stress characterized by increased production of superoxide ions. Consistently, lines overexpressing the GRXS13.2 variant show reduced MeV- and HL-induced damage. Alterations in GRXS13 expression also affect superoxide levels and the ascorbate/dehydroascorbate ratio after HL-induced stress. These results indicate that GRXS13 gene expression is critical for limiting basal and photooxidative stress-induced reactive oxygen species (ROS) production. Together, these results place GRXS13.2 as a member of the ROS-scavenging/antioxidant network that shows a particularly low functional redundancy in the Arabidopsis GRX family.
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    Identificación y caracterización de genes de proteínas con repeticiones de pentatricopéptidos (PPR) necesarias para la edición de transcritos mitocondriales en Arabidopsis thaliana
    (2015) Arenas Miranda, Ana Maribel; Jordana, Xavier; Pontificia Universidad Católica de Chile. Facultad de Ciencias Biológicas
    La edición de RNA es un tipo de procesamieto post-transcripcional que altera la identidad de nucleótidos en las moléculas de RNA. En organelos de plantas con flores, la edición de RNA cambia citosinas a uracilos. Más de 400 sitios en mitocondrias de angiospermas son individualmente identificados por proteínas altamente específicas, pero ninguno de estos factores están codificados en el genoma mitocondrial. Todos estos factores de reconocimiento pertenecen a la familia de proteínas con repeticiones de pentatricopéptido (PPR). El principal objetivo de este trabajo es identificar nuevos Factores de Edición Mitocondrial (MEFs). Para lograr este objetivo, se utilizaron los siguientes criterios: (i) Disponibilidad de secuencias de ESTs o cDNAs de genes PPR en bases de datos, (ii) destinación mitocondrial predicha por distintos algoritmos, (iii) proteínas PPR pertenecientes a la subfamilia PLS con motivos E, E+ o DYW, y (iv) reducida identidad de secuencia con otras proteínas PPR de Arabidopsis. Utilizando estos criterios, cinco genes PPR candidatos fueron seleccionados. Tres de nuestros candidatos, en experimentos de localización subcelular muestran localización en mitocondrias. Plantas mutantes homocigotas en cada uno de los genes candidatos fueron analizadas con la aproximación de SNaPshot. Esta metodología permite evaluar masivamente los niveles de edición en organelos de Arabidopsis, encontrando alteraciones en la edición de diversos transcritos mitocondriales en tres líneas mutantes analizadas. Desde ese momento las proteínas codificados por los genes candidatos fueron nombradas MEF25, MEF26 y MEF31. MEF25 participa en la edición de un sitio en el transcrito nad1. MEF26 es necesaria para la edición completa del sitio cox3-311 y de la edición parcial del sitio nad4-166. MEF31 facilita la edición de un sitio en el transcrito orfX.Los defectos en la edición en las mutantes mef25, mef26 and mef31 fueron confirmados por secuenciación de productos de RT-PCR, análisis de una segunda línea mutante o ensayos de complementación. La edición de los sitios diana de estos factores MEF genera un cambio en la naturaleza del aminoácido que finalmente estará presente en la proteína funcional. Sin embargo, el fenotipo de las plantas mutantes mef25, mef26 and mef31 es indistinguibles de las plantas silvestres, sugiriendo que la edición de sus sitios diana no es esencial al menos bajo condiciones estándar de crecimiento. Por otra parte, recientemente ha sido propuesto un código para el reconocimiento del RNA mediado por las proteínas PPR. Usando este código, los dianas predichos para los factores MEF25, MEF26 y MEF31 son coincidentes con los dianas experimentales identificados en este estudio.