Crosstalks between Myo-Inositol Metabolism, Programmed Cell Death and Basal Immunity in Arabidopsis

BACKGROUND: Although it is a crucial cellular process required for both normal development and to face stress conditions, the control of programmed cell death in plants is not fully understood. We previously reported the isolation of ATXR5 and ATXR6, two PCNA-binding proteins that could be involved in the regulation of cell cycle or cell death. A yeast two-hybrid screen using ATXR5 as bait captured AtIPS1, an enzyme which catalyses the committed step of myo-inositol (MI) biosynthesis. atips1 mutants form spontaneous lesions on leaves, raising the possibility that MI metabolism may play a role in the control of PCD in plants. In this work, we have characterised atips1 mutants to gain insight regarding the role of MI in PCD regulation. METHODOLOGY/PRINCIPAL FINDINGS: - lesion formation in atips1 mutants depends of light intensity, is due to PCD as evidenced by TUNEL labelling of nuclei, and is regulated by phytohormones such as salicylic acid - MI and galactinol are the only metabolites whose accumulation is significantly reduced in the mutant, and supplementation of the mutant with these compounds is sufficient to prevent PCD - the transcriptome profile of the mutant is extremely similar to that of lesion mimic mutants such as cpr5, or wild-type plants infected with pathogens. CONCLUSION/SIGNIFICANCE: Taken together, our results provide strong evidence for the role of MI or MI derivatives in the regulation of PCD. Interestingly, there are three isoforms of IPS in Arabidopsis, but AtIPS1 is the only one harbouring a nuclear localisation sequence, suggesting that nuclear pools of MI may play a specific role in PCD regulation and opening new research prospects regarding the role of MI in the prevention of tumorigenesis. Nevertheless, the significance of the interaction between AtIPS1 and ATXR5 remains to be established

Caractérisation d'un inhibiteur de kinases cycline-dépendantes de N. tomentosiformis. Analyses de son rôle au cous du développement de la plante.

Plant development requires stringent controls between cell proliferation and cell differentiation. Proliferation is positively regulated by cyclin-dependent kinases (CDKs), whose activity is regulated at several levels including inhibition by CDK inhibitors (CKIs). The screen of a two-hybrid BY-2 cell suspension library with a CDKA as bait, allows the isolation of two cDNAs, named NtKIS1a and NtKIS1b. NtKIS1a and NtKIS1b mRNAs arise from the same N. tomentosiformis gene by alternative splicing. The deduced polypeptide from NtKIS1a shares strong sequence similarity with mammalian CIP/KIP inhibitors, which is not the case for NtKIS1b. Consistent with this, NtKIS1a but not NtKIS1b inhibits in vitro the kinase activity of CDK/cyclin complexes. To gain insight into the role of NtKIS1a and NtKIS1b during plant development, their overexpression in different species was achieved. Arabidopsis thaliana plants overexpressing NtKIS1b display a wild-type phenotype, whereas plants overexpressing NtKIS1a display strong morphological modifications. Our results suggest that the inhibition of cell division is responsible for the phenotypic modifications and thus that NtKIS1a is a cell division inhibitor in planta. Plants overexpressing simultaneously NtKIS1a and AtCycD3;1 were achieved. Their analyse demonstrate that overexpression of the CKI NtKIS1a restores essentially normal development in AtCycD3;l overexpressing plants, providing for the first time, evidence of cyclinD-CKI co-operationwithin the context of a living plant. At the aim of highlighting the links between cell cycle and development, the expression of two genes was modified simultaneously in planta: KNAT1 (knotted1-like from Arabidopsis thaliana), involved in shoot apical meristem development and function, and NtKIS1a involved in cell cycle regulation. The analysis of the F1 plants shows that co-expression of NtKIS1a and KNAT1 enhances the KNAT1 phenotype, suggesting that the two gene products co-operate with each other during plant development.Le développement d'une plante nécessite un contrôle précis entre prolifération cellulaire et différenciation. Le cycle cellulaire est contrôlé par des kinases dépendantes des cyclines (CDKs) dont l'activité est régulée à plusieurs niveaux, en particulier par des inhibiteurs (CKIs, cyclin dépendent kinase inhibitors). Le criblage d'une banque double hybride de la suspension cellulaire de tabac BY-2 avec une CDKA comme appât à permis l'isolement de deux ADNc, nommés NtKIS1a et NtKIS1b. Les deux ARNm proviennent d'un même gène de N. Tomentosiformis par épissage alternatif. La séquence protéique déduite de NtKIS1a présente de fortes similarités de séquence avec les CKIs de mammifères de la famille CIP/KIP, alors que ce n'est pas le cas de NtKIS1b. En accord avec cette observation, NtKIS1a mais pas NtKIS1b inhibe in vitro l'activité kinase de complexes CDK/cycline. Pour élucider le rôle de NtKIS1a et NtKIS1b au cours du développement, leur surexpression dans différentes espèces végétales a été réalisée. Les plantes d'Arabidopsis thaliana surexprimant NtKIS1b ont un phénotype sauvage, tandis que celles surexprimant NtKIS1a présentent d'importantes modifications morphologiques. L'ensemble de nos résultats suggèrent que les modifications phénotypiques proviennent d'une inhibition de la division et montrent donc que NtKIS1a est un inhibiteur de la division in planta. Des plantes surexprimant simultanément NtKIS1a et AtCycD3;1 ont été obtenues. Leur analyse montre que la surexpression du CKI NtKIS1a restaure un développement normal des plantes surexprimant AtCycD3;l, fournissant la première évidence d'une coopération CKI-cycline in planta. Dans le but d'appréhender les liens qui existent entre le cycle cellulaire et le développement, l'expression de deux gènes a été modifiée simultanément in planta : KNAT1 (knottedl-like from Arabidopsis thaliana), impliqué dans le développement et la fonction du méristème apical caulinaire, et NtKIS1a, impliqué dans l'inhibition du cycle cellulaire. L'analyse des plantes F1 montre que la co-expression de NtKIS1a et KNAT1 renforce le phénotype des plantes 35S::KNAT1, suggérant que les produits des deux gènes coopèrent au cours du développement

Caractérisation d'un inhibiteur de kinases cycline-dépendantes de N. tomentosiformis (analyse de son rôle au cours du développement de la plante)

Le développement d'une plante nécessite un contrôle précis entre prolifération cellulaire et différenciation. Le cycle cellulaire est contrôlé par des kinases dépendantes des cyclines (CDKs) dont l'activité est régulée à plusieurs niveaux, en particulier par des inhibiteurs (CKIs, cyclin dépendent kinase inhibitors). Le criblage d'une banque double hybride de la suspension cellulaire de tabac BY-2 avec une CDKA comme appât à permis l'isolement de deux ADNc, nommés NtKIS1a et NtKIS1b. Les deux ARNm proviennent d'un même gène de N. tomentosiformis par épissage alternatif. La séquence protéique déduite de NtKIS1a présente de fortes similarités de séquence avec les CKIs de mammifères de la famille CIP/KIP, alors que ce n'est pas le cas de NtKIS1b. En accord avec cette observation, NtKIS1a mais pas NtKIS1b inhibe in vitro l'activité kinase de complexes CDK/cycline. Pour élucider le rôle de NtKIS1a et NtKIS1b au cours du développement, leur surexpression dans différentes espèces végétales a été réalisée. Les plantes d'Arabidopsis thaliana surexprimant NtKIS1b ont un phénotype sauvage, tandis que celles surexprimant NtKIS1a présentent d'importantes modifications morphologiques. L'ensemble de nos résultats suggèrent que les modifications phénotypiques proviennent d'une inhibition de la division et montrent donc que NtKIS1a est un inhibiteur de la division in planta. Des plantes surexprimant simultanément NtKIS1a et AtCycD3;1 ont été obtenues. Leur analyse montre que la surexpression du CKI NtKIS1a restaure un développement normal des plantes surexprimant AtCycD3;l, fournissant la première évidence d'une coopération CKI-cycline in planta. Dans le but d'appréhender les liens qui existent entre le cycle cellulaire et le développement, l'expression de deux gènes a été modifiée simultanément in planta : KNAT1 (knottedl-like from Arabidopsis thaliana), impliqué dans le développement et la fonction du méristème apical caulinaire, et NtKIS1a, impliqué dans l'inhibition du cycle cellulaire. L'analyse des plantes F1 montre que la co-expression de NtKIS1a et KNAT1 renforce le phénotype des plantes 35S::KNAT1, suggérant que les produits des deux gènes coopèrent au cours du développement.Plant development requires stringent controls between cell proliferation and cell differentiation. Proliferation is positively regulated by cyclin dependent kinases (CDKs), whose activity is regulated at several levels including inhibition by CDK inhibitors (CKIs). The screen of a two-hybrid BY-2 cell suspension library with a CDKA as a bait, allows the isolation of two cDNA, named NtKIS1a and NtKIS1b. NtKIS1a and NtKIS1b mRNAs arise from the same N. tomentosiformis gene by alternative splicing. The deduced polypeptide from NtKIS1a shares strong sequence similarity with mammalian CIP/KIP inhibitors, which is not the case for NtKIS1b. Consistent with this, NtKIS1a but not NtKIS1b inhibits in vitro the kinase activity of CDK/cyclin complexes. To gain insight into the role of NtKIS1a and NtKIS1b during plant development, their overexpression in different species was achieved. Arabidopsis thaliana plants overexpressing NtKIS1b display a wild type phenotype, whereas plants overexpressing NtKIS1a display strong morphological modifications. Our results suggest that the inhibition of cell division is responsible for the phenotypic modifications and thus that NtKIS1a is a cell division inhibitor in planta. Plants overexpressing simultaneously NtKIS1a and AtCycD3;1 were achieved. Their analyze demonstrates that overexpression of the CKI NtKIS1a restores essentially normal development in AtCycD3;1 overexpressing plants, providing for the first time, evidence of Cyclin D-CKI co-operation within the context of a living plant. At the aim of highlighting the links between cell cycle and development, the expression of two genes was modify simultaneously in planta: KNAT1 (knotted1-like from Arabidopsis thaliana) involved in shoot apical meristem development and function, and NtKIS1a involved in cell cycle regulation. The analysis of the F1 plants shows that co-expression of NtKIS1a and KNAT1 enhance the KNAT1 phenotype, suggesting that the two gene products co-operate with each other during plant development.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Improving seed oil and protein content in

Western Europe oleoproteaginous species like rapeseed mainly accumulate oil and protein in their seeds. To become competitive with soybean, seed protein quantity and quality should be improved in rapeseed. The negative correlation existing between seed protein and oil content apparently prevents the possibility to increase protein content without affecting oil content. Exploration of natural and induced genetic variability in the model plant Arabidopsis thaliana allows the identification of several genotypes impaired in this negative correlation. Different genetic approaches have been undertaken in order to isolate genetic factors responsible for the tight control of seed oil and protein homeostasis and this negative correlation. Once isolated in this model plant, such genetic determinants will be identified in important crops such as rapeseed or other oilseed crops in order to manipulate both components independently and thus produce on purposed seeds. In the long term, this research will help breed new varieties that could contribute to reduce Europe’s dependence on US soybean import

The evolutionary-developmental analysis of plant microRNAs

MicroRNAs (miRNAs) control many important aspects of plant development, suggesting these molecules may also have played key roles in the evolution of developmental processes in plants. However, evolutionary-developmental (evo-devo) studies of miRNAs have been held back by technical difficulties in gene identification. To help solve this problem, we have developed a two-step procedure for the efficient identification of miRNA genes in any plant species. As a test case, we have studied the evolution of the MIR164 family in the angiosperms. We have identified novel MIR164 genes in three species occupying key phylogenetic positions and used these, together with published sequence data, to partially reconstruct the evolution of the MIR164 family since the last common ancestor of the extant flowering plants. We use our evolutionary reconstruction to discuss potential roles for MIR164 genes in the evolution of leaf shape and carpel closure in the angiosperms. The techniques we describe may be applied to any miRNA family and should thus enable plant evo-devo to begin to investigate the contributions miRNAs have made to the evolution of plant development

Improving seed oil and protein content in Brassicaceae: some new genetic insights from Arabidopsis thaliana

Western Europe oleoproteaginous species like rapeseed mainly accumulate oil and protein in their seeds. To become competitive with soybean, seed protein quantity and quality should be improved in rapeseed. The negative correlation existing between seed protein and oil content apparently prevents the possibility to increase protein content without affecting oil content. Exploration of natural and induced genetic variability in the model plant Arabidopsis thaliana allows the identification of several genotypes impaired in this negative correlation. Different genetic approaches have been undertaken in order to isolate genetic factors responsible for the tight control of seed oil and protein homeostasis and this negative correlation. Once isolated in this model plant, such genetic determinants will be identified in important crops such as rapeseed or other oilseed crops in order to manipulate both components independently and thus produce on purposed seeds. In the long term, this research will help breed new varieties that could contribute to reduce Europe’s dependence on US soybean import

Architecture génétique de la qualité de la graine chez Arabidopsis et le colza

International audienc

The Mitogen-Activated Protein Kinase 12 is involved in Arabidopsis seed filling

International audienc

Lien entre composition en protéines de réserve et teneurs en glucosinolates dans la graine de colza : vers l’identification de déterminants génétiques et moléculaires

National audienceIn rapeseed, the seed storage proteins are cruciferins (12S globulins) and napins (2S albumins) which represent more than 70% of the total proteins. As napins are richer in sulfur and aromatic residues than cruciferins, they are of greater nutritional interest. However, several studies show that in modern varieties '00' (no erucic acid and low glucosinolates), the napins content is lower than in old varieties '++' (high erucic acid and glucosinolates). The aim of our study is to identify the genetic and molecular bases of the relationship between the glucosinolate and 2S contents in rapeseed.Association genetic analysis identified eight genomic regions controlling both napin and glucosinolate contents. These regions cover nearly 650 genes, seven are described involved in sulfur metabolism pathways and were targeted in this study. In particular, the MYB28 gene, located on chromosome C09, shows a 4-bp insertion polymorphism between the '00' and '++' varieties. Furthermore, MYB28 insertion mutants in Arabidopsis produce seeds with significantly reduced sulfur, glucosinolates and napins contents. These results could provide clues for improving the reserve protein composition of rapeseed.Chez le colza, les protéines de réserve de la graine sont les cruciférines (globulines 12S) et les napines (albumines 2S) qui représentent plus de 70% des protéines totales. Les napines étant plus riches en résidus soufrés et aromatiques que les cruciférines, elles présentent un intérêt supérieur sur le plan nutritionnel. Cependant plusieurs études montrent que chez les variétés modernes de colza, ‘00’, dont les graines sont dépourvues en acide érucique et pauvres en glucosinolates, la teneur en napines est plus faible que chez les variétés anciennes ‘++’. L’objectif de notre étude est d’identifier les bases génétiques et moléculaires de la relation entre la teneur en glucosinolates et en napines dans la graine de colza.Une analyse par génétique d’association a permis d’identifier huit régions génomiques contrôlant à la fois les teneurs en napines et en glucosinolates. Ces régions couvrent près de 650 gènes dont sept sont décrits comme étant impliqués dans les voies du métabolisme soufré et ont été ciblés pour la suite de cette étude. En particulier, le gène MYB28, localisé sur le chromosome C09, montre un polymorphisme d’insertion de 4 pb entre les variétés ‘00’ et ‘++’. Par ailleurs, des mutants d’insertion MYB28 chez Arabidopsis produisent des graines dont les teneurs en soufre, glucosinolates et napines sont significativement réduites. Ces résultats pourraient fournir des pistes pour améliorer la composition en protéines de réserve de la graine de colza

Arabidopsis seed content QTL mapping using high-throughput phenotyping: the assets of Near Infrared Spectroscopy

Seed storage compounds are of crucial importance for human diet, feed and industrial uses. In oleo-proteaginous species like rapeseed, seed oil and protein are the qualitative determinants that conferred economic value to the harvested seed. To date, although the biosynthesis pathways of oil and storage protein are rather well known, the factors that determine how these types of reserves are partitioned in seeds have to be identified. With the aim of implementing a quantitative genetics approach, requiring phenotyping of hundreds of plants, our first objective was to establish near-infrared reflectance spectroscopic (NIRS) predictive equations in order to estimate oil, protein, carbon and nitrogen content in Arabidopsis seed with high-throughput level. Our results demonstrated that NIRS is a powerful non-destructive, high-throughput method to assess the content of these four major components studied in Arabidopsis seed. With this tool in hand, we analysed Arabidopsis natural variation for these four components and illustrated that they all displayed a wide range of variation. Finally, NIRS was used in order to map QTL for these four traits using seeds from the Arabidopsis thaliana Ct-1 x Col-0 recombinant inbred line population. Some QTL co-localised with QTL previously identified, but others mapped to chromosomal regions never identified so far for such traits. This paper illustrates the usefulness of NIRS predictive equations to perform accurate high-throughput phenotyping of Arabidopsis seed content, opening new perspectives in gene identification following QTL mapping and Genome Wide Association Studies