Metabolite profiling and quantitative genetics of natural variation for flavonoids in Arabidopsis

Little is known about the range and the genetic bases of naturally occurring variation for flavonoids. Using Arabidopsis thaliana seed as a model, the flavonoid content of 41 accessions and two recombinant inbred line (RIL) sets derived from divergent accessions (Cvi-0×Col-0 and Bay-0×Shahdara) were analysed. These accessions and RILs showed mainly quantitative rather than qualitative changes. To dissect the genetic architecture underlying these differences, a quantitative trait locus (QTL) analysis was performed on the two segregating populations. Twenty-two flavonoid QTLs were detected that accounted for 11–64% of the observed trait variations, only one QTL being common to both RIL sets. Sixteen of these QTLs were confirmed and coarsely mapped using heterogeneous inbred families (HIFs). Three genes, namely TRANSPARENT TESTA (TT)7, TT15, and MYB12, were proposed to underlie their variations since the corresponding mutants and QTLs displayed similar specific flavonoid changes. Interestingly, most loci did not co-localize with any gene known to be involved in flavonoid metabolism. This latter result shows that novel functions have yet to be characterized and paves the way for their isolation

Oxydation des flavonoïdes dans les graines d'Arabidopsis thaliana. Analyse fonctionnelle du gène TRANSPARENT TESTA 10 codant une polyphénoloxydase de type laccase

Arabidopsis seeds accumulate flavonoids (proanthocyanidins and flavonols) during their development. A previous study has shown that a laccase (AtLAC15) encoded by the TRANSPARENT TESTA 10 (TT10 ) gene could trigger flavonoid oxidation in the seed coat. If both proanthocyanidins (PAs) and flavonols appear to be TT10 protein substrates, only PA oxidation leads to brown pigments responsible for the mature seed coat color. An important consequence of TT10 activity on seed flavonoid metabolism is an increased ratio of insoluble to soluble PAs. The physiological functions of TT10 are still unknown, however defense against biotic and abiotic stresses, either constitutive or induced, may be predicted on the basis of present knowledge on polyphenoloxidases.The purpose of this thesis was to perform a functional characterization of the TT10 gene. A part of the work was devoted to the analysis of the regulatory mechanisms controlling the developmental pattern of TT10 gene expression in seeds and vegetative plant parts. The functional 5’-dissection of a 2.0-kb promoter realized with the uidA reporter gene encoding β-glucuronidase (GUS) was performed to identify regions responsible for activation in seed and other plant organs. TT10 promoter happens to be activated exclusively in seed coat and siliques. Directed mutagenesis was undertaken to precise the regulatory role of in silico-detected cis-acting regulatory elements (CAREs) located in a 194-bp region necessary for expression in seed coat. TT10 gene expression assessed in different tissues at various stages of development using qRT-PCR matched promoter activity pattern. Natural variation for TT10 expression among Arabidopsis accessions was also detected, with the levels of TT10 mRNA in Cvi, Ler and Sha being strongly reduced compared to the ones in Ws, Col and Bay. The impact of this molecular polymorphism on seed flavonoid composition, as analyzed on mature seeds with LC-MS, is discussed. In silico analysis of the TT10 promoter revealed the presence of putative CAREs potentially involved in signaling and response to biotic and abiotic stresses. Howeverhistochemical analysis of GUS activity in transgenic Arabidopsis plantlets expressing pT T102.0−kb : GUS failed to detect any ectopic activity when submitted to a variety of stresses. This result suggests that transcriptional response to environmental stimuli is highly constrained by developmental parameters. TT10 appeared to be the only member of the laccase gene family to be strongly expressed in seeds. TT10 function may have evolved towards flavonoid oxidation by co-localization with these substrates, which is ensured by tissue-specific gene expression.Les graines d’Arabidopsis accumulent des flavonoïdes (proanthocyanidines et flavonols) durant leur développement. Une étude précédente a montré qu’une laccase (AtLAC15) codée par le gène TRANSPARENT TESTA 10 (TT10 ) induisait l’oxydation des flavonoïdes au niveau des téguments. Si les proanthocyanidines (PAs) et les flavonols sont des substrats de la protéine TT10, seule l’oxydation des PAs conduit à la formation de pigments bruns responsables de la couleur de la graine mature. Une importante conséquence de l’activité de TT10 sur les flavonoïdes de la graine est l’augmentation du rapport PAs insolubles / PAs solubles. Les fonctions physiologiques de TT10 sont toujours inconnues, mais un rôle dans la défense contre des stress biotiques et abiotiques constitutifs ou induits peut être prédit sur la base des connaissances actuelles sur les polyphénoloxydases.L’objectif de cette thèse était de réaliser une analyse fonctionnelle du gène TT10. Une partie de l’étude était consacrée à l’analyse des mécanismes de régulation contrôlant le pattern développemental d’expression du gène TT10 dans les graines et les parties végétatives. Une dissection fonctionnelle en 5’ d’un promoteur de 2.0-kb réalisée à l’aide du gène rapporteur uidA codant la β-glucuronidase (GUS) a été réalisée pour identifier les régions responsables de l’activation du promoteur dans les graines et les autres parties de la plante. Le promoteur de TT10 est activé exclusivement dans les téguments de la graine et les siliques. Une mutagénèse dirigée a été réalisée dans une région de 194 pb nécessaire à l’expression dans les téguments, pour préciser la fonctionnalité des éléments régulateurs agissant en cis (ERACs) identifiés in silico. L’expression du gène TT10 mesurée par qRT-PCR dans différents tissus et à différents stades de développement est en accord avec le pattern d’activité du promoteur. Une variation naturelle pour l’expression de TT10 a aussi été détectée parmi plusieurs accessions d’Arabidopsis, avec des niveaux d’ARNm mesurés chez Cvi, Ler et Sha fortement réduits par rapport à ceux relevés chez Ws, Col et Bay. L’impact de ce polymorphisme moléculaire sur la composition en flavonoïdes de la graine, analysée sur graines matures par LC-MS, est discuté. L’analyse in silico du promoteur de TT10 a révélé la présence d’ERACs potentiellement impliqués dans la signalisation et la réponse aux stress biotiques et abiotiques. Cependant l’analyse histochimique de l’activité GUS de plantes transgéniques exprimant pT T102.0−kb : GUS n’a pas permis de détecter d’activité ectopique en présence de stress variés. Ce résultat suggère que la réponse transcriptionnelle aux stimuli environnementaux est fortement conditionnée par les paramètres développementaux. TT10 est le seul membre de la famille des laccases à être exprimé fortement dans les graines. La fonction de TT10 a probablement évolué versl’oxydation des flavonoïdes par la co-localisation avec ces substrats, qui est assurée par la spécificité tissulaire d’expression du gène

Flavonoid oxidation in Arabidopsis thaliana seeds (Functional analysis of the TRANSPARENT TESTA 10 gene encoding a polyphenoloxidase of the laccase type)

L objectif de cette thèse était de réaliser une analyse fonctionnelle du gène TRANSPARENT TESTA 10 (TT10) codant une laccase (AtLAC15) impliquée dans l oxydation des flavonoïdes de la graine d Arabidopsis. Une partie de l étude était consacrée à l analyse des mécanismes de régulation contrôlant le pattern développemental d expression du gène TT10 dans les graines et les parties végétatives. Une dissection fonctionnelle en 5 d un promoteur de 2,0 kb réalisée à l aide du gène rapporteur uidA codant la -glucuronidase (GUS) a été réalisée pour identifier les régions responsables de l activation du promoteur dans les graines et les autres parties de la plante. Le promoteur de TT10 est activé exclusivement dans les téguments de la graine et les siliques. Une mutagénèse dirigée a été réalisée dans une région de 194 pb nécessaire à l expression dans les téguments, pour préciser la fonctionnalité des éléments régulateurs agissant en cis identifiés in silico. L expression du gène TT10 mesurée par qRT-PCR dans différents tissus et à différents stades de développement est en accord avec le pattern d activité du promoteur. Une variation naturelle pour l expression de TT10 a aussi été détectée parmi plusieurs accessions d Arabidopsis, avec des niveaux d ARNm mesurés chez Cvi, Ler et Sha fortement réduits par rapport à ceux relevés chez Ws, Col et Bay. L impact de ce polymorphisme moléculaire sur la composition en flavonoïdes de la graine, analysée sur graines matures par LC-MS, est discuté. TT10 est le seul membre de la famille des laccases à être exprimé fortement dans les graines.The purpose of this thesis was to perform a functional characterization of the TRANSPARENT TESTA 10 (TT10) gene encoding a laccase (AtLAC15) involved in flavonoid oxidation in Arabidopsis seed. A part of the work was devoted to the analysis of the regulatory mechanisms controlling the developmental pattern of TT10 gene expression in seeds and vegetative plant parts. The functional 5 -dissection of a 2.0-kb promoter realized with the uidA reporter gene encoding b-glucuronidase (GUS) was performed to identify regions responsibles for activation in seed and other plant organs. TT10 promoter happens to be activated exclusively in seed coat and siliques. Directed mutagenesis was undertaken to precise the regulatory role of in silico-detected cis-acting regulatory elements located in a 194-bp region necessary for expression in seed coat. TT10 gene expression assessed in different tissues at various stages of development using qRT-PCR matched promoter activity pattern. Natural variation for TT10 expression among Arabidopsis accessions was also detected, with the levels of TT10 mRNA in Cvi, Ler and Sha being strongly reduced compared to the ones in Ws, Col and Bay. The impact of this molecular polymorphism on seed flavonoid composition, as analyzed on mature seeds with LC-MS, is discussed. TT10 appeared to be the only member of the laccase gene family to be strongly expressed in seeds.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Rice (Oriza sativa) defense against rice blast disease (Magnaporthe Oryzae) under drought stress

National audienc

Rising serum CA-125 levels within the normal range is strongly associated recurrence risk and survival of ovarian cancer

Abstract Background In clinical practice alterations in CA-125 concentration within normal range in patients with ovarian cancer after first-line treatment are common. Even minor increase in CA-125 concentration is associated with patients’ anxiety and difficult interpretation and counselling for clinicians. The aim of this study was to evaluate the significance of CA-125 fluctuations within reference level in patients who suffered from ovarian cancer with complete response after first-line treatment. Results 168 patients with epithelial ovarian cancer, who achieved complete remission after first line treatment were enrolled in the study. CA-125 concentration assessment was carried out during follow up visits. The recurrence of the disease was diagnosed on the first appearance of symptoms: clinical, radiological or histopathological/cytological. PFS and 5-year survival rate was calculated with Kaplan-Meier plots. Statistical analysis was performed with SAS / STAT® 9.4 / 14.4, SAS Institute Inc., Cary, NC, USA, 2017. Median concentration of CA-125 after first-line therapy was 10 U/ml. Increasing CA-125 concentration by > 5 U/ml, 3 and 6 months after the treatment was associated with higher risk of relapse (HR = 7.6, p  5 U/ml within normal range, 3 and 6 months after treatment was unfavorable prognostic factor in ovarian cancer patients with complete response to primary therapy

Transcriptional basis of drought-induced susceptibility to the rice blast fungus Magnaporthe oryzae

BGPI : équipe 4Plants are often facing several stresses simultaneously. Understanding how they reactand the way pathogens adapt to such combinational stresses is poorly documented.Here, we developed an experimental system mimicking field intermittent drought onrice followed by inoculation by the pathogenic fungus Magnaporthe oryzae. Thisexperimental system triggers an enhancement of susceptibility that could be correlatedwith the dampening of several aspects of plant immunity, namely the oxidative burst andthe transcription of several pathogenesis-related genes. Quite strikingly, the analysisof fungal transcription by RNASeq analysis under drought reveals that the fungus isgreatly modifying its virulence program: genes coding for small secreted proteins weremassively repressed in droughted plants compared to unstressed ones whereas genescoding for enzymes involved in degradation of cell-wall were induced. We also showthat drought can lead to the partial breakdown of several major resistance genes byaffecting R plant gene and/or pathogen effector expression.We propose a model wherea yet unknown plant signal can trigger a change in the virulence program of the pathogento adapt to a plant host that was affected by drought prior to infection

Disruption of LACCASE4 and 17 Results in Tissue-Specific Alterations to Lignification of Arabidopsis thaliana Stems[W]

This study provides evidence that two laccases, LAC4 and LAC17, participate in the polymerization of lignins in Arabidopsis stems. These findings suggest that the genetic engineering of lignin-specific laccases is a potentially innovative and promising tool for the fine-tuning of lignin content and structure

The Rice DNA-Binding Protein ZBED Controls Stress Regulators and Maintains Disease Resistance After a Mild Drought

Background: Identifying new sources of disease resistance and the corresponding underlying resistance mechanisms remains very challenging, particularly in Monocots. Moreover, the modification of most disease resistance pathways made so far is detrimental to tolerance to abiotic stresses such as drought. This is largely due to negative cross-talks between disease resistance and abiotic stress tolerance signaling pathways. We have previously described the role of the rice ZBED protein containing three Zn-finger BED domains in disease resistance against the fungal pathogen Magnaporthe oryzae. The molecular and biological functions of such BED domains in plant proteins remain elusive.Results: Using Nicotiana benthamiana as a heterologous system, we show that ZBED localizes in the nucleus, binds DNA, and triggers basal immunity. These activities require conserved cysteine residues of the Zn-finger BED domains that are involved in DNA binding. Interestingly, ZBED overexpressor rice lines show increased drought tolerance. More importantly, the disease resistance response conferred by ZBED is not compromised by drought-induced stress.Conclusions: Together our data indicate that ZBED might represent a new type of transcriptional regulator playing simultaneously a positive role in both disease resistance and drought tolerance. We demonstrate that it is possible to provide disease resistance and drought resistance simultaneously