The alternative oxidase family of Vitis vinifera reveals an attractive model to study the importance of genomic design

'Genomic design' refers to the structural organization of gene sequences. Recently, the role of intron sequences for gene regulation is being better understood. Further, introns possess high rates of polymorphism that are considered as the major source for speciation. In molecular breeding, the length of gene-specific introns is recognized as a tool to discriminate genotypes with diverse traits of agronomic interest. 'Economy selection' and 'time-economy selection' have been proposed as models for explaining why highly expressed genes typically contain small introns. However, in contrast to these theories, plant-specific selection reveals that highly expressed genes contain introns that are large. In the presented research, 'wet'Aox gene identification from grapevine is advanced by a bioinformatics approach to study the species-specific organization of Aox gene structures in relation to available expressed sequence tag (EST) data. Two Aox1 and one Aox2 gene sequences have been identified in Vitis vinifera using grapevine cultivars from Portugal and Germany. Searching the complete genome sequence data of two grapevine cultivars confirmed that V. vinifera alternative oxidase (Aox) is encoded by a small multigene family composed of Aox1a, Aox1b and Aox2. An analysis of EST distribution revealed high expression of the VvAox2 gene. A relationship between the atypical long primary transcript of VvAox2 (in comparison to other plant Aox genes) and its expression level is suggested. V. vinifera Aox genes contain four exons interrupted by three introns except for Aox1a which contains an additional intron in the 3'-UTR. The lengths of primary Aox transcripts were estimated for each gene in two V. vinifera varieties: PN40024 and Pinot Noir. In both varieties, Aox1a and Aox1b contained small introns that corresponded to primary transcript lengths ranging from 1501 to 1810 bp. The Aox2 of PN40024 (12 329 bp) was longer than that from Pinot Noir (7279 bp) because of selection against a transposable-element insertion that is 5028 bp in size. An EST database basic local alignment search tool (BLAST) search of GenBank revealed the following ESTs percentages for each gene: Aox1a (26.2%), Aox1b (11.9%) and Aox2 (61.9%). Aox1a was expressed in fruits and roots, Aox1b expression was confined to flowers and Aox2 was ubiquitously expressed. These data for V. vinifera show that atypically long Aox intron lengths are related to high levels of gene expression. Furthermore, it is shown for the first time that two grapevine cultivars can be distinguished by Aox intron length polymorphism

Differential expression and co-regulation of carrot AOX genes (Daucus carota)

Alternative oxidase (AOX) is a mitochondrial protein encoded by the nuclear genome. In higher plants AOX genes form a small multigene family mostly consisting of the two subfamilies AOX1 and AOX2. Daucus carota L. is characterized by a unique extension pattern of AOX genes. Different from other plant species studied so far it contains two genes in both subfamilies. Therefore, carrot was recently highlighted as an important model in AOX stress research to understand the evolutionary importance of both AOX subfamilies. Here we report on the expression patterns of DcAOX1a, DcAOX1b and DcAOX2a and DcAOX2b. Our results demonstrate that all of the four carrot AOX genes are expressed. Differential expression was observed in organs, tissues and during de novo induction of secondary root phloem explants to growth and development. DcAOX1a and DcAOX2a indicated a differential transcript accumulation but a similar co-expression pattern. The genes of each carrot AOX sub-family revealed a differential regulation and responsiveness. DcAOX2a indicated high inducibility in contrast to DcAOX2b, which generally revealed low transcript abundance and rather weak responses. In search for withingene sequence differences between both genes as a potential reason for the differential expression patterns, the structural organization of the two genes was compared. DcAOX2a and DcAOX2b showed high sequence similarity in their open reading frames (ORFs). However, length variability was observed in the N-terminal exon1 region. The predicted cleavage site of the mitochondrial targeting sequence in this locus is untypical small for both genes and consists of 35 amino acids for DcAOX2a and of 21 amino acids for DcAOX2b. The importance of structural gene organization and the relevancy of within-gene sequence variations are discussed. Our results strengthen the value of carrot as a model plant for future studies on the importance of AOX sub family evolution

Interaction des 2',3'-dialdéhydes adénine nucléotides avec l'ATPase des mitochondries de coeur de boeuf.

Beef heart mitochondrial F1-ATPase was inactivated by 2',3'-dialdehyde derivatives of ATP, ADP and AMP (dialATP, dialADP, dialAMP). In the absence of Mg2+, analysis of enzyme inactivation kinetics showed that inactivation resulted from the binding of 1 mol nucleotide analog per active unit of F1. The most efficient analog was dialADP, followed by dialAMP and dialATP. Complete inactivation was correlated with the binding of about 11 mol dialADP per mol F1 as measured directly with radioactive nucleotides. After correction for non-specific labeling, the number of specifically bound dialADP was 3 mol per mol F1. By sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, dialADP was found to bind covalently mainly to the alpha and beta subunits.La F1-ATPase des mitochondries de coeur de boeuf est inactivée par les dérivés 2',3'-dialdéhydiques de l'ATP, ADP et AMP (dialATP, dialADP, dialAMP). L'analyse de la cinétique d'inactivation enzymatique montre qu'en l'absence de Mg2+, l'inactivation résulte de la fixation d'une mole d'analogue de nucléotide par unité active de F1-ATPase. L'analogue le plus efficace est le dialADP, suivi par le dialAMP et le dialATP. L'utilisation de nucléotides radioactif montre que l'inactivation complète nécessite la fixation d'environ 11 moles de dialADP par mole de F1. Après correction pour le marquage non-sélectif, le le nombre de dialADP fixés spécifiquement est de 3 par F1. Par électrophorèse sur gel de polyacrylamide en présence de dodécylsulfate de sodium (SDS), le dialADP se fixe de manière covalente principalement sur les sous-unités alpha et beta

Caractérisation de la réponse de deux cultivars de Vigna unguiculata à une contrainte ozone combinée ou non à la sécheresse (Régulation de protéines membranaires (AOX, PTOX et pUCP))

Les interactions entre chloroplastes et mitochondries restent encore mal connues, notamment en réponse à des conditions de stress. Dans ces conditions, il est suggéré que les voies de transfert d'électrons liées à des protéines découplantes AOX ou pUCP (mitochondriales) et PTOX (plastidiale) pourraient limiter la formation de ROS afin d'atténuer les dommages oxydatifs dans ces organites. Dans le cadre de notre travail, nous avons retenu deux cultivars de Vigna unguiculata, cv 1183 et cv EPACE. Les deux cultivars n'ont pas montré de réelles différences de sensibilité à la sécheresse. Par contre le cultivar EPACE s'est montré plus tolérant à l'O3 sur la base du développement des nécroses et plusieurs paramètres physiologiques (Fv/Fm, [phi]PSII) et biochimiques (glutathion). Pour les profils d'expression des gènes codant pour l'AOX, la pUCP et la PTOX deux réponses ont été clairement identifiées chez le cultivar EPACE. Sur un court terme l'expression de ces protéines est généralement stimulée. Sur un plus long terme (14 jours), la réponse diffère en fonction de la contrainte. Sous O3, la plus forte expression des protéines mitochondriales est maintenue alors que le gène codant pour la PTOX est sousrégulé. Sous sécheresse seule la protéine plastidiale (PTOX) reste sur-régulée. Dans des conditions de combinaison de contrainte, la sécheresse a peu d'effet sur l'influx d'O3 dans les feuilles, et les gènes VuPTOX et VuUCP1b sont sur-régulés après 3 et 7 jours de contrainte. Cette sur-régulation, déjà observée en réponse à la sécheresse seule, pourrait jouer un rôle déterminant pour prévenir la formation de ROS à la fois dans la mitochondrie et dans le chloroplastePossible interactions between chloroplast and mitochondria remain poorly known, particularly in response to stress conditions. Under these conditions, it is suggested that the electron transfer pathways linked to AOX or pUCP (mitochondrial uncoupling proteins) and PTOX (plastidial uncoupling protein) could limit the formation of ROS to reduce oxidative damage in cellular organelles. In our work, we selected two cultivars of Vigna unguiculata, cv 1183 and cv EPACE. Under our experimental conditions, both cultivars showed no real differences in sensitivity to drought. However cv EPACE was more tolerant to O3 based on the development of necrosis and several physiological parameters (Fv/Fm, [phi]PSII) and biochemical (glutathione content). For the expression profiles of genes encoding AOX, PUCP and PTOX two responses were clearly identified in the cultivar EPACE. On a shortterm, expression of these proteins is generally stimulated. On a longer term (14 days), the answer differs depending on the treatment. Under O3, the strongest expression of mitochondrial proteins is maintained while the gene encoding the PTOX is down-regulated. Under drought only the plastid protein (PTOX) is upregulated. Under conditions of stress combination, drought has little effect on the influx of O3 in the leaves, and the VuPTOX and VuUCP1b genes are up-regulated after 3 and 7 days of stress. This over-regulation, already observed in response to drought alone could play a role in preventing the formation of ROS in both mitochondria and chloroplastsMETZ-SCD (574632105) / SudocNANCY1-Bib. numérique (543959902) / SudocNANCY2-Bibliotheque electronique (543959901) / SudocNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Étude du métabolisme des lipides de membranes chloroplastiques et des gènes associés chez Vigna unguiculata dans le cadre de la sécheresse et de la reprise après réhydratation

Les membranes cellulaires sont des cibles préférentielles de la dégradation induite par les espèces réactives de l oxygène produites durant la sécheresse et par la stimulation d activités hydrolytiques. La biosynthèse des lipides des chloroplastes peut être importante pour la tolérance à la sécheresse ainsi que pour la reprise après réhydratation. Dans ce travail nous avons étudié le métabolisme des lipides des membranes chloroplastiques, monogalactosyldiacylglycerol (MGDG), digalactosyl-diacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), phosphatidyl-glycerol (PG), dans le cadre de la sécheresse et de la reprise après la fin de la contrainte hydrique. Dans ce but, nous avons mesuré la teneur des lipides des feuilles, suivi l incorporation du précurseur 14C-acétate dans les lipides et analysé l expression des gènes codant les enzymes de biosynthèse des lipides (MGD1, MGD2, DGD1, DGD2, SQD2 et PGP1) durant le stress hydrique et après réhydratation. Afin de mieux comprendre le rapport entre le métabolisme de ces lipides et la tolérance à la sécheresse, nous avons travaillé sur deux cultivars de Vigna unguiculata L. Walp, un tolérant (cv. EPACE) et l autre sensible (cv. 1183) à la sécheresse. Les séquences complètes des ADNc des gènes VuMGD1, VuMGD2, VuDGD1, VuDGD2, VuSQD2 et VuPGP1 ont été obtenues par le criblage d une banque d ADNc de V.unguiculata. Les résultats montrent qu en condition de stress hydrique le cultivar tolérant, en plus de préserver la teneur en lipides, est capable de stimuler la biosynthèse du DGDG augmentant significativement le rapport DGDG:MGDG de ces membranes. Nous suggérons que le DGDG accumulé en sécheresse est exporté vers les membranes extrachloroplastiques et que cela contribue à la tolérance à la contrainte hydrique. Les effets de la perte d eau cellulaire sur les membranes ont des conséquences directes sur la capacité des plantes à reprendre après réhydratation. 48 heures après réarrosage, le cv. sensible 1183 n est pas capable de récupérer en termes de teneurs en galactolipides et incorporation de précurseur. Chez le cv. tolérant, par contre, la teneur en DGDG demeure élevé, même après réhydratation. En conclusion, nos résultats suggèrent l importance des lipides membranaires dans la tolérance/sensibilité des plantes au déficit hydrique, en particulier la balance entre des classes lipidiques de propriétés physico-chimiques différentes (SQDG versus PG et DGDG versus MGDG) qui pourraient affecter la structure et le fonctionnement des membranesMembranes are main targets of degradation by reactive oxygen species and hydrolytic activities induced by drought. Chloroplasts lipid biosynthesis, especially galactolipids monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG) are important for plant tolerance to water deficit and for recovery after rehydration. In this thesis, we studied the metabolism of the chloroplast membrane lipids, MGDG, DGDG, sulphoquinovosyl-diacylglycerol (SQDG), phosphatidyl-glycerol (PG) under drought and during recovery from drought. Aiming this, we measured leaf lipids content, followed 14Cacétate incorporation and expression of genes coding for chloroplast membrane lipid synthases (MGD1, MGD2, DGD1, DGD2, SQD2 and PGP1) during drought and recovery. In order to better understand the relationship between drought tolerance and lipid metabolism, two cultivars of Vigna unguiculata L. Walp, one drought tolerant (cv. EPACE) the other drought susceptible (cv. 1183) were compared. The cDNA complete sequences for VuMGD1, VuMGD2, VuDGD1, VuDGD2, VuSQD2 and VuPGP1 were obtained from screening of a V.unguiculata cDNA library. The results showed that under water stress conditions, the tolerant cultivar, besides its ability to preserve its lipids pool despites drought, is able to strongly stimulate the DGDG biosynthesis, increasing the DGDG:MGDG ratio in its membranes. We suggest that DGDG accumulated under drought condition, when phosphate is deficient, is exported for extrachloroplastic membranes, and thus contributes to plant drought tolerance. Effects of loss of water on cell membranes have direct consequences on plant capacity to recover from stress. 48 hours after rewatering, the susceptible cv. 1183 was not able to fully recover from a moderate stress in terms of leaf galactolipid content and acetate incorporation into MGDG. In EPACE-1, MGDG leaf content remained unchanged after rehydration and DGDG remained higher than in the control plants. In conclusion, our results highlight the importance of membrane lipids in plant adaptation to water deficit and in their capacity to recover from stress. Of particular importance is the balance between lipid classes with various physico-chemical properties (SQDG versus PG, DGDG versus MGDG), since they most likely have a profound influence on membrane structure and functionPARIS-EST-Université (770839901) / SudocSudocFranceF

Expressão diferencial dos genes VuUCP1a e VuUCP1b em caupi sob estresse salino

O estresse salino afeta o crescimento e o desenvolvimento das plantas, induzindo respostas bioquímicas e fisiológicas como mecanismo de adaptação para sua sobrevivência. As proteínas desacopladoras de planta (pUCPs) dissipam o gradiente eletroquímico mitocondrial de prótons como calor e são codificadas por famílias multigênicas. Elas atuam como sistema de defesa evitando a formação de espécies reativas de oxigênio geradas em resposta a estresses ambientais. O objetivo do trabalho foi estudar a expressão de genes das pUCPs (VuUCP1a e VuUCP1b ) em raízes e folhas de plântulas de Vigna unguiculata submetidas ao estresse salino (NaCl 100 mM). Sementes foram germinadas no escuro e após 3 dias, as plântulas foram transferidas para solução de Hoagland, permanecendo 3 dias antes da aplicação do estresse. As raízes e folhas foram coletadas com 0; 6; 12 e 24 horas, após adição de NaCl, para avaliar o perfil de expressão dos genes da pUCP por RT- PCR. A análise de transcritos mostrou que VuUCP1a é expresso em raízes e folhas revelando um perfil constitutivo enquanto que VuUCP1b é dependente do tecido, isto é, expresso nas folhas, sem alteração em resposta ao estresse e induzido pelo estresse salino nas raízes. A peculiaridade da duplicação gênica de pUCP1 em caupi com perfil diferencial de expressão, sugere tanto um papel dessa enzima nos mecanismos de ajustamento ao estresse salino quanto promove essa espécie como um modelo atrativo para compreensão do papel da família multigênica da pUCP em plantas.Salt stress affects growth and development of plants, inducing a variety of physiological and biochemical responses as an adaptation mechanism for survival. The plant uncoupling mitochondrial proteins (pUCPs) are able to dissipate the proton electrochemical gradient as heat and are encoded by a multigene family. They works as defence systems avoiding the formation of reactive oxygen species promoted by environmental stress. The aim of this work was to study gene expression of pUCPs (VuUCP1a and VuUCP1b) in roots and leaves from Vigna unguiculata seedlings under salt stress (100 mM NaCl). Seeds were germinated in the dark and after 3 days, the seedlings were transferred to Hoagland's medium and grown for 3 additional days before being submitted to the stress condition. Roots and leaves were harvested at 0; 6; 12 and 24 hours after addition of NaCl for total RNA isolation and RT-PCR assays. Expression analysis by RT-PCR showed that VuUCP1a is constitutive in leaves and roots while VuUCP1b is expressed as tissue-dependent presenting a constitutive profile in leaves and a differential one in roots from seedlings under salt stress. The uniqueness of pUCP1 gene duplication in cowpea with differential expression suggest a role of this enzyme in the adjustment of salt stress as well as promotes this species as an attractive model to understand the role of pUCP gene members in plants.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Immunogenicity and modulatory effect of the lectins from Artocarpus heterophyllus (Jackfruit) Seeds, artocarpin and jacalin

Se estudió la respuesta inmune de ratones inmunizados subcutáneamiente con dos lectinas de semillas de Artocarpus Heterophyllus, artocarpina y jacalina, y sus posibles efectos moduladores en la síntesis de anticuerpos de ratones inmunizados con un antígeiio no relacionado estructuralmente. Las dos lectinas inducen la síntesis específica de anticuerpos, independientemente de sus dosis inmunizantes. Con respecto al efecto modulador sobre la síntesis de inmunoglobulinas totales antiovoalbúmina, la artocarpina estimuló la síntesis de anticuerpos antiovoalbúmina independientemente de su dosis y la jacalina tuvo tendencia a estimular la síntesis del anticuerpo según su dosis. La discriminación de las síntesis de IgGl y IgE anti-ovoalbumina demostró que la artocarpina modula la IgG1, en tanto que la jacalina modula la IgE.The immune response of mice subcutaneously immunized with two lectins from Artocarpus heterophyllus seeds, artocarpin and jacalin, and their possible modulatory effect on antibody synthesis of mice immunized with an unrelated antigen, were studied. Both lectins induced specific synthesis of antibodies, irrespective of their immunizing dose. Concerning the modulatory effect on the synthesis of antiovalbumin total immunoglobulins, artocarpin stimulated the synthesis of anti-ovalbumin antibodies irrespective of its dose and jacalin had a tendency to stimulate such antibody synthesis according to its dose. Discrimination of anti-ovalbumin synthesis of IgGl and IgE showed that the artocarpin modulated IgGl whilst jacalin modulated IgE.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Immunogenicity and modulatory effect of the lectins from Artocarpus heterophyllus (Jackfruit) Seeds, artocarpin and jacalin

Se estudió la respuesta inmune de ratones inmunizados subcutáneamiente con dos lectinas de semillas de Artocarpus Heterophyllus, artocarpina y jacalina, y sus posibles efectos moduladores en la síntesis de anticuerpos de ratones inmunizados con un antígeiio no relacionado estructuralmente. Las dos lectinas inducen la síntesis específica de anticuerpos, independientemente de sus dosis inmunizantes. Con respecto al efecto modulador sobre la síntesis de inmunoglobulinas totales antiovoalbúmina, la artocarpina estimuló la síntesis de anticuerpos antiovoalbúmina independientemente de su dosis y la jacalina tuvo tendencia a estimular la síntesis del anticuerpo según su dosis. La discriminación de las síntesis de IgGl y IgE anti-ovoalbumina demostró que la artocarpina modula la IgG1, en tanto que la jacalina modula la IgE.The immune response of mice subcutaneously immunized with two lectins from Artocarpus heterophyllus seeds, artocarpin and jacalin, and their possible modulatory effect on antibody synthesis of mice immunized with an unrelated antigen, were studied. Both lectins induced specific synthesis of antibodies, irrespective of their immunizing dose. Concerning the modulatory effect on the synthesis of antiovalbumin total immunoglobulins, artocarpin stimulated the synthesis of anti-ovalbumin antibodies irrespective of its dose and jacalin had a tendency to stimulate such antibody synthesis according to its dose. Discrimination of anti-ovalbumin synthesis of IgGl and IgE showed that the artocarpin modulated IgGl whilst jacalin modulated IgE.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Identification of duplicated and stress-inducible Aox2b gene co-expressed with Aox1 in species of the Medicago genus reveals a regulation linked to gene rearrangement in leguminous genomes

In flowering plants, alternative oxidase (Aox) is encoded by 3-5 genes distributed in 2 subfamilies (Aox1 and Aox2). In several species only Aox1 is reported as a stress-responsive gene, but in the leguminous Vigna unguiculata Aox2b is also induced by stress. In this work we investigated the Aox genes from two leguminous species of the Medicago genus (Medicago sativa and Medicago truncatula) which present one Aox1, one Aox2a and an Aox2b duplication (named here Aox2b1 and Aox2b2). Expression analyses by semi-quantitative RT-PCR in M. sativa revealed that Aox1, Aox2b1 and Aox2b2 transcripts increased during seed germination. Similar analyses in leaves and roots under different treatments (SA, PEG, H2O2 and cysteine) revealed that these genes are also induced by stress, but with peculiar spatio-temporal differences. Aox1 and Aox2b1 showed basal levels of expression under control conditions and were induced by stress in leaves and roots. Aox2b2 presented a dual behavior, i.e., it was expressed only under stress conditions in leaves, and showed basal expression levels in roots that were induced by stress. Moreover, Aox2a was expressed at higher levels in leaves and during seed germination than in roots and appeared to be not responsive to stress. The Aox expression profiles obtained from a M. truncatula microarray dataset also revealed a stress-induced co-expression of Aox1, Aox2b1 and Aox2b2 in leaves and roots. These results reinforce the stress-inducible co-expression of Aox1/Aox2b in some leguminous plants. Comparative genomic analysis indicates that this regulation is linked to Aox1/Aox2b proximity in the genome as a result of the gene rearrangement that occurred in some leguminous plants during evolution. The differential expression of Aox2b1/2b2 suggests that a second gene has been originated by recent gene duplication with neofunctionalization. © 2013 Elsevier GmbH. All rights reserved

Isolation and partial characterization of heterophyllin, a new lectin from Artocarpus heterophyllus seeds

Fueron aisladas cuatro (4) lectinas presentes en las semillas de Artocarpus heterophyllus Lamk. ("jaca") mediante el uso de criterios de solubilidad y varias cromatografias de afinidad: goma de guar, quitina y agarosa-D-manosa. De las dos lectinas que tuvieron afinidad a la goma de guar, una se comportó como albúmina (AII) y fue identificada como la bien conocida lectina "jacalina" por su especificidad al azúcar, movilidad electroforética y secuencia de aminoácidos en el extremo N-terminal. La otra lectina (GII) que se une a la goma de guar tiene las mismas propiedades anteriormente mencionadas, a excepción de su naturaleza globulínica. La lectina albumínica (AIMII), la cual no se unió a la goma de guar, fue aislada gracias a su asociación con agarosa-D-manosa y fue identificada como la lectina artocarpina previamente descripta. Una nueva lectina fue aislada de la fracción globulínica a través de su asociación con quitina. La unión de esta lectina com a quitina fue inhibida por N-acetil-D-glucosamina. La nueva lectina, designada heterofilina, con un pI de 6.5, contiene tres subunidades de pesos moleculares 31,4, 18.7 y 16,3 kDa, según se ha determinado por SDS-PAGE.Four lectins present in the seeds of jackfruit (Artocarpus heterophyllus, Lamk.) were isolated by employing solubility criteria and by guar gum, chitin and agarose-D-mannose affinity chromatography. One of the two lectins that bound to guar gum behaved as an albumin (AII) and was identified as the well known jacalin lectin by its sugar specificity, electrophoretic mobility and N-terminal amino acid sequence. The other guar gum binding lectin (GII) had the same above properties but was distinguished by its globulin nature. An albumin lectin (AIMII) which did not bind to guar gum was isolated due to its association with agarose-D-mannose and was identified as the lectin artocarpin. A newly identified lectin was isolated from the globulin fraction through its association with chitin. Chitin binding of the lectin was competed by N-acetyl-D-glucosamine. The new lectin designated heterophyllin, with a pI value of 6,5 , contained three subunits of molecular weight 31.4,18.7 and 16.3 kDa as estimated by SDS-PAGE.Colegio de Farmacéuticos de la Provincia de Buenos Aire