Caractérisation d un récepteur kinase de type Leucine Rich Repeat dans le développement d Arabidopsis thaliana par une approche de génétique inverse

Le gène RLK7 appartient à la sous-famille LRR XI des récepteurs kinases de type LRR-RLK chez Arabidopsis thaliana. Afin de déterminer son rôle potentiel dans cette plante, son profil d expression a été étudié et une approche de génétique inverse a été entreprise.Le gène RLK7 a un profil d expression similaire à celui de certaines protéines LEA "Late Embryogenesis Abundant" lors de la phase tardive de maturation de la graine et lors de la germination. L analyse par génétique inverse montre que la vitesse de germination est retardée dans trois lignées d insertion ADN-T indépendantes et au contraire est plus précoce dans deux lignées possédant des formes tronquées de la protéine RLK7.Une approche de protéomique par 2-D DIGE révèle que certaines protéines impliquées dans la détoxification en réponse à un stress oxydant sont moins abondantes dans les trois lignées d insertion par rapport au sauvage. De plus, les plantules d une des lignées d insertion sont plus sensibles à un traitement à l H2O2, alors que les lignées possédant des formes tronquées de la protéine RLK7 sont plus résistantes.En conclusion, les lignées d insertion dans le gène RLK7 qui présentent un retard de germination sont aussi plus affectées par un stress oxydant et les lignées avec des formes tronquées de la protéine RLK7, qui germent plus précocement, sont plus tolérantes à un traitement à l H2O2. L accumulation des espèces actives de l oxygène lors de la post-maturation serait responsable de la levée de la dormance. Aussi, le gène RLK7 pourrait jouer un rôle dans ce processus de post-maturation, dans la perception et la régulation des espèces actives de l oxygène pour la levée de la dormance.The RLK7 gene belongs to class XI of the large gene family of the leucine-rich repeat receptor-like kinases in Arabidopsis. In order to define the potential role of this gene, its detailed expression pattern was determined and a reverse genetics strategy carried out.RT-PCR, reporter gene expression and in situ hybridization indicate that RLK7 is highly expressed during late embryogenesis of Arabidopsis seeds. Its expression pattern is similar to those of LEA Late Embryogenesis Abundant proteins during the desiccation and germination stages. Moreover, germination speed was delayed in three independent lines with T-DNA insertions in RLK7, whereas it was accelerated in lines containing two truncated forms of RLK7. So, the RLK7 gene presents a positive action on seed germination speed.A 2-D DIGE analysis indicates that some proteins involved in response to oxidative stress are less abundant in the three insertion lines than in the wild type. Moreover, seedlings of a T-DNA insertion line in RLK7 gene are more sensitive to hydrogen peroxide, whereas lines containing truncated forms of RLK7 are more resistant. So, the RLK7 gene is involved in the control of the redox balance.In conclusion, the insertion lines in RLK7 that present a delay of germination rate are also more sensitive to oxidative stress and the lines containing truncated forms of RLK7, that germinate more quickly, are more tolerant to an H2O2 treatment. Accumulation of reactive oxygen species during after-ripening may be involved in the dormancy breaking. Thus, the RLK7 gene may play a role in this process of after-ripening, in the perception and the regulation of reactive oxygen species to break the dormancy.PERPIGNAN-BU Sciences (661362101) / SudocSudocFranceF

AMP1 and CYP78A5/7 act through a common pathway to govern cell fate maintenance in Arabidopsis thaliana.

Higher plants can continuously form new organs by the sustained activity of pluripotent stem cells. These stem cells are embedded in meristems, where they produce descendants, which undergo cell proliferation and differentiation programs in a spatiotemporally-controlled manner. Under certain conditions, pluripotency can be reestablished in descending cells and this reversion in cell fate appears to be actively suppressed by the existing stem cell pool. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis causes defects in the suppression of pluripotency in cells normally programmed for differentiation, giving rise to unique hypertrophic phenotypes during embryogenesis as well as in the shoot apical meristem. A role of AMP1 in the miRNA-dependent control of translation has recently been established, however, how this activity is connected to its developmental functions is not resolved. Here we identify members of the cytochrome P450 clade CYP78A to act in parallel with AMP1 to control cell fate in Arabidopsis. Mutation of CYP78A5 and its close homolog CYP78A7 in a cyp78a5,7 double mutant caused suspensor-to-embryo conversion and ectopic stem cell pool formation in the shoot meristem, phenotypes characteristic for amp1. The tissues affected in the mutants showed pronounced expression levels of AMP1 and CYP78A5 in wild type. A comparison of mutant transcriptomic responses revealed an intriguing degree of overlap and highlighted alterations in protein lipidation processes. Moreover, we also found elevated protein levels of selected miRNA targets in cyp78a5,7. Based on comprehensive genetic interaction studies we propose a model in which both enzyme classes act on a common downstream process to sustain cell fate decisions in the early embryo and the shoot apical meristem

The small molecule hyperphyllin enhances leaf formation rate and mimics shoot meristem integrity defects associated with AMP1 deficiency

ALTERED MERISTEM PROGRAM1 (AMP1) is a member of the M28 family of carboxypeptidases with a pivotal role in plant development and stress adaptation. Its most prominent mutant defect is a unique hypertrophic shoot phenotype combining a strongly increased organ formation rate with enhanced meristem size and the formation of ectopic meristem poles. However, so far the role of AMP1 in shoot development could not be assigned to a specific molecular pathway nor is its biochemical function resolved. In this work we evaluated the level of functional conservation between AMP1 and its human homologue HsGCPII, a tumour marker of medical interest. We show that HsGCPII cannot substitute AMP1 in planta and that an HsGCPII-specific inhibitor does not evoke amp1-specific phenotypes. We used a chemical genetic approach to identify the drug hyperphyllin (HP), which specifically mimics the shoot defects of amp1, including plastochron reduction and enlargement and multiplication of the shoot meristem. We assessed the structural requirements of HP activity and excluded that it is a novel cytokinin analogue. HP-treated wild-type plants showed amp1-related tissue-specific changes of various marker genes and a significant transcriptomic overlap with the mutant. HP was ineffective in amp1 and elevated the protein levels of PHAVOLUTA, consistent with the postulated role of AMP1 in miRNA-controlled translation, further supporting an AMP1-dependent mode of action. Our work suggests that plant and animal members of the M28 family of proteases adopted unrelated functions. With HP we provide a novel tool to characterize the plant-specific functions of this important class of proteins

The small molecule hyperphyllin enhances leaf formation rate and mimics shoot meristem integrity defects associated with AMP1 deficiency

ALTERED MERISTEM PROGRAM1 (AMP1) is a member of the M28 family of carboxypeptidases with a pivotal role in plant development and stress adaptation. Its most prominent mutant defect is a unique hypertrophic shoot phenotype combining a strongly increased organ formation rate with enhanced meristem size and the formation of ectopic meristem poles. However, so far the role of AMP1 in shoot development could not be assigned to a specific molecular pathway nor is its biochemical function resolved. In this work we evaluated the level of functional conservation between AMP1 and its human homolog HsGCPII, a tumor marker of medical interest. We show that HsGCPII cannot substitute AMP1 in planta and that an HsGCPII-specific inhibitor does not evoke amp1-specific phenotypes. We used a chemical genetic approach to identify the drug hyperphyllin (HP), which specifically mimics the shoot defects of amp1, including plastochron reduction and enlargement and multiplication of the shoot meristem. We assessed the structural requirements of HP activity and excluded that it is a cytokinin analog. HP-treated wild-type plants showed amp1-related tissue-specific changes of various marker genes and a significant transcriptomic overlap with the mutant. HP was ineffective in amp1 and elevated the protein levels of PHAVOLUTA, consistent with the postulated role of AMP1 in miRNA-controlled translation, further supporting an AMP1-related mode of action. Our work suggests that plant and animal members of the M28 family of proteases adopted unrelated functions. With HP we provide a tool to characterize the plant-specific functions of this important class of proteins

Lipo-chitooligosaccharidic Symbiotic Signals Are Recognized by LysM Receptor-Like Kinase LYR3 in the Legume <i>Medicago truncatula</i>

While chitooligosaccharides (COs) derived from fungal chitin are potent elicitors of defense reactions, structurally related signals produced by certain bacteria and fungi, called lipo-chitooligosaccharides (LCOs), play important roles in the establishment of symbioses with plants. Understanding how plants distinguish between friend and foe through the perception of these signals is a major challenge. We report the synthesis of a range of COs and LCOs, including photoactivatable probes, to characterize a membrane protein from the legume <i>Medicago truncatula.</i> By coupling photoaffinity labeling experiments with proteomics and transcriptomics, we identified the likely LCO-binding protein as LYR3, a lysin motif receptor-like kinase (LysM-RLK). LYR3, expressed heterologously, exhibits high-affinity binding to LCOs but not COs. Homology modeling, based on the <i>Arabidopsis</i> CO-binding LysM-RLK AtCERK1, suggests that LYR3 could accommodate the LCO in a conserved binding site. The identification of LYR3 opens up ways for the molecular characterization of LCO/CO discrimination