Single-nucleotide and long-patch base excision repair of DNA damage in plants

Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell-free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′- or 3′-blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short-patch BER) or several nucleotides (long-patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways

Homologous Recombination Is Stimulated by a Decrease in dUTPase in Arabidopsis

Deoxyuridine triphosphatase (dUTPase) enzyme is an essential enzyme that protects DNA against uracil incorporation. No organism can tolerate the absence of this activity. In this article, we show that dUTPase function is conserved between E. coli (Escherichia coli), yeast (Saccharomyces cerevisiae) and Arabidopsis (Arabidopsis thaliana) and that it is essential in Arabidopsis as in both micro-organisms. Using a RNA interference strategy, plant lines were generated with a diminished dUTPase activity as compared to the wild-type. These plants are sensitive to 5-fluoro-uracil. As an indication of DNA damage, inactivation of dUTPase results in the induction of AtRAD51 and AtPARP2, which are involved in DNA repair. Nevertheless, RNAi/DUT1 constructs are compatible with a rad51 mutation. Using a TUNEL assay, DNA damage was observed in the RNAi/DUT1 plants. Finally, plants carrying a homologous recombination (HR) exclusive substrate transformed with the RNAi/DUT1 construct exhibit a seven times increase in homologous recombination events. Increased HR was only detected in the plants that were the most sensitive to 5-fluoro-uracils, thus establishing a link between uracil incorporation in the genomic DNA and HR. Our results show for the first time that genetic instability provoked by the presence of uracils in the DNA is poorly tolerated and that this base misincorporation globally stimulates HR in plants

RCD1 and SRO1 are necessary to maintain meristematic fate in Arabidopsis thaliana

The RADICAL-INDUCED CELL DEATH1 and SIMILAR TO RCD ONE1 genes of Arabidopsis thaliana encode members of the poly(ADP-ribose) polymerase (PARP) superfamily and have pleiotropic functions in development and abiotic stress response. In order to begin to understand the developmental and molecular bases of the defects seen in rcd1-3; sro1-1 plants, this study used the root as a model. Double mutant roots are short and display abnormally organized root apical meristems. However, acquisition of most cell fates within the root is not significantly disrupted. The identity of the quiescent centre is compromised, the zone of cell division is smaller than in wild-type roots and abnormal divisions are common, suggesting that RCD1 and SRO1 are necessary to maintain cells in a division-competent state and to regulate division plane placement. In addition, differentiation of several cell types is disrupted in rcd1-3; sro1-1 roots and shoots, demonstrating that RCD1 and SRO1 are also necessary for proper cell differentiation. Based on the data shown in this article and previous work, we hypothesize that RCD1 and SRO1 are involved in redox control and, in their absence, an altered redox balance leads to abnormal development

Xylella fastidiosa gene expression analysis by DNA microarrays

Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM2 and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants

Chemical PARP Inhibition Enhances Growth of Arabidopsis and Reduces Anthocyanin Accumulation and the Activation of Stress Protective Mechanisms

Poly-ADP-ribose polymerase (PARP) post-translationally modifies proteins through the addition of ADP-ribose polymers, yet its role in modulating plant development and stress responses is only poorly understood. The experiments presented here address some of the gaps in our understanding of its role in stress tolerance and thereby provide new insights into tolerance mechanisms and growth. Using a combination of chemical and genetic approaches, this study characterized phenotypes associated with PARP inhibition at the physiological level. Molecular analyses including gene expression analysis, measurement of primary metabolites and redox metabolites were used to understand the underlying processes. The analysis revealed that PARP inhibition represses anthocyanin and ascorbate accumulation under stress conditions. The reduction in defense is correlated with enhanced biomass production. Even in unstressed conditions protective genes and molecules are repressed by PARP inhibition. The reduced anthocyanin production was shown to be based on the repression of transcription of key regulatory and biosynthesis genes. PARP is a key factor for understanding growth and stress responses of plants. PARP inhibition allows plants to reduce protection such as anthocyanin, ascorbate or Non-Photochemical-Quenching whilst maintaining high energy levels likely enabling the observed enhancement of biomass production under stress, opening interesting perspectives for increasing crop productivity

Production de peptides de lieu noir dotés d'une capacité antioxydante par hydrolyse enzymatique en réacteur discontinu et fractionnement sur membrane

Ce travail s inscrit dans le cadre général de la valorisation des co-produits de la pêche. Il porte sur la mise en oeuvre de l hydrolyse enzymatique et des procédés de séparation sur membranes pour produire des peptides dotés d une capacité antioxydante élevée selon un procédé transférable à l échelle industrielle. Le filet de lieu noir a été choisi comme substrat modèle. L hydrolyse enzymatique par l Alcalase® a tout d abord été optimisée à l aide de la méthodologie des plans d expériences. Les conditions d hydrolyse permettant de maximiser l activité antioxydante (test au b-carotène-linoléate) des peptides sont : T = 60 C, pH 8, ratio E/S = 2,23 % (53,8 AU.kg-1), pendant 10,8 min. Le transfert du procédé d hydrolyse à l échelle pilote (réacteur de 20L) a été validé. Le fractionnement sur chromatographie basse pression montre que la fraction inférieure à 1 kDa est majoritairement active. Le fractionnement sur membrane a été utilisé comme un deuxième levier pour agir (i) sur la distribution de la taille des peptides et (ii) sur le niveau d activité des hydrolysats. Le rôle des paramètres opératoires (pression, teneur en peptides, facteur de réduction volumique (FRV)) a été étudié. Tout d abord, on a observé que pour une teneur en peptides fixée, lorsque la pression augmente, le taux de rétention (TR) des peptides augmente et le seuil de coupure apparent (SC) de la membrane augmente. A une pression constante, lorsque la teneur en peptides (ou le FRV) augmente, le TR diminue et le SC augmente. La durée de la concentration renforce cependant le colmatage. Ensuite, on constate qu en mode recyclage total, le fractionnement améliore la capacité antioxydante du perméat. En mode concentration, le fractionnement améliore l activité du perméat tant que la concentration reste modérée (FRV <= 2). L originalité de ce travail de thèse réside dans (i) la maximisation de la capacité antioxydante de l hydrolysat, (ii) l utilisation des paramètres opératoires pour modifier la sélectivité des membranes et obtenir des fractions peptidiques de poids moléculaires désirés et (iii) le développement d outils analytiques adaptés à la quantification des peptides et au suivi de leur rétention par une membrane d UF.This work is conducted in the framework of the fishery by-product up-grading. It concerns the implementation of the enzymatic hydrolysis and the membrane separation processes to produce peptides with a high antioxidant capacity according to a process transferable to the industrial scale. Saithe fillet was chosen as a model substrate. The enzymatic hydrolysis by Alcalase® was, first, optimized by means of the design of experiment methodology. Hydrolysis conditions maximising the antioxidant activity of peptides (b-carotene-linoleate model system assay) are: T = 60 C, pH 8, E/S ratio = 2.23 % (53.8 AU.kg-1) during 10.8 min. The transposition of the hydrolysis process at pilot scale (20 L batch reactor) was validated. The fractionation on a low pressure chromatographic column shows that the fraction lower than 1 kDa are the most active. Membrane fractionation was then used as a second lever to act (i) on size distribution of peptides and (ii) on the activity level of hydrolysates. The role of the operating parameters (pressure, peptide content, volume reduction factor (VRF)) was studied. It was observed that, for a given peptide content, when the pressure increases, the retention rate (RR) of peptides increases and the apparent molecular weight cut-off (MWCO) of the membrane decreases. For a constant pressure, when the peptide content (or of the FRV) increases, the RR decreases and the apparent MWCO increase. However, the duration of concentration step strengthens the membrane fouling. In recirculation mode, the fractionation improve the antioxidant capacity of permeates. In concentration mode, the fractionation improved the activity as long as the concentration remains moderate (FRV <= 2). The originality of this PhD work is (i) the maximisation of the antioxidant capacity of the hydrolysate, (ii) the use of the fractionation operating parameters to modify the membrane selectivity and to obtain peptide fractions with defined molecular weight, and (iii) the development of analytical tools adapted to peptide quantification and to the follow-up of peptide retention on the UF membrane.LORIENT-BU (561212106) / SudocSudocFranceF

CARACTERISATION FONCTIONNELLE D'UN GENE RADIO-INDUIT CHEZ ARABIDOPSIS THALIANA (LE GENE CODANT LA POLY(ADP-RIBOSE)POLYMERASE-1 (ATHPARP-1))

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Identification d'un opéron phase variable chez "Pseudomonas brassicacearum" NFM421, effet de "Pseudomonas brassicacearum" NFM421 sur le transcriptome partiel d'"Arabidopsis thaliana" écotype WS par la technique des filtres à haute densité

PSEUDOMONAS BRASSICACEARUM NFM421 EST UNE RHIZOBACTERIE ISOLEE DE RACINES D'ARABIDOPSIS THALIANA CULTIVEE SUR DIFFERENTS SOLS. ELLE REPRESENTE L'UNE DES POPULATIONS DOMINANTES FERMEMENT ATTACHEES AUX RACINES DE CETTE PLANTE. CETTE ESPECE, COMME BEAUCOUP D'AUTRES ESPECES BACTERIENNES ASSOCIEES AUX PLANTES ET AUX ANIMAUX, SE CARACTERISE PAR L'APPARITION D'UNE VARIATION DE PHASE. LES VARIANTS DE P. BRASSICACEARUM NFM421 (CELLULES EN PHASE II) APPARAISSENT EN BORDURE DES COLONIES FORMEES PAR LES CELLULES EN PHASE I, SUR MILIEU DE CULTURE. LES COLONIES FORMEES DE CELLULES EN PHASE II SONT LISSES, NON MUQUEUSES SANS ACTIVITE PROTEASIQUE ET LIPASIQUE, CONTRAIREMENT AUX COLONIES DE LA PHASE I, ET BEAUCOUP PLUS GRANDES QUE CELLES EN PHASE I. D'UNE PART, POUR ESSAYER D'ELUCIDER LE MECANISME DE VARIATION DE PHASE, NOUS AVONS REALISE UNE BANQUE GENOMIQUE DES CELLULES EN PHASE I. LE CRIBLAGE DE LA BANQUE PAR DIFFERENTES SONDES, NOUS A PERMIS D'ISOLER UN OPERON CONTENANT LES GENES CODANT LA PROTEASE, LA LIPASE, UN HOMOLOGUE AUX SERINES PROTEASES ET LE SYSTEME DE SECRETION DE TYPE I. L'ETUDE PAR FUSION TRANSCRIPTIONNELLE ENTRE LE PROMOTEUR DU GENE APRA ET LE GENE LACZ A MIS EN EVIDENCE LA NON TRANSCRIPTION DE CET OPERON CHEZ LES CELLULES EN PHASE II ; CELA SUGGERANT L'INTERVENTION D'UN ELEMENT REGULATEUR. D'AUTRE PART, NOUS AVONS EGALEMENT ETUDIE L'EFFET DE PSEUDOMONAS BRASSICACEARUM NFM421 PHASE I ET II SUR LE TRANSCRIPTOME PARTIEL D'ARABIDOPSIS THALIANA PAR LA TECHNIQUE DES FILTRES A HAUTE DENSITE. LES PREMIERS RESULTATS ONT MONTRE QUE CERTAINS GENES ETAIENT ACTIVES PAR LES CELLULES EN PHASE I ALORS QUE D'AUTRES ETAIENT REPRIMES PAR LES CELLULES EN PHASE II.AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF