Delphine Mieulet

Manipulation de la recombinaison chez une plante cultivée, le riz.L’accroissement prévisible de la population mondiale ainsi que les conséquences du changement climatique obligent les sélectionneurs à créer de nouvelles variétés plus productives et plus résilientes. Les nouvelles combinaisons d’allèles favorables sont issues de la recombinaison génétique entre chromosomes homologues dont le siège est la prophase de première division de méiose. De récentes avancées chez la plante modèle Arabidopsis ont montré que l'inactivation de certains gènes permet de manipuler la méiose pour abolir ou au contraire augmenter très significativement la recombinaison. Les mécanismes de la méiose étant relativement bien conservés chez les eucaryotes, l’objectif de cette thèse était de transposer ces avancées chez une plante cultivée importante, le riz. Abolir la recombinaison méiotique permettrait de propager de façon clonale par grain des formules variétales hybrides F1 dont le rendement est de 20% supérieur à celui des lignées pures chez le riz mais dont les semences restent peu utilisées par les riziculteurs de subsistance. Les travaux réalisés dans une première partie de la thèse ont montré que le cumul de trois mutations Ososd1, pair1 et Osrec8, permettait d’obtenir des gamètes clonaux diploïdes mâles et femelles. Le phénotype apoméiotique obtenu, appelé MiMe (Mitosis instead of meiosis) chez Arabidopsis, peut être utilisé pour tester différentes stratégies d’induction de la parthénogenèse afin de produire des grains formant des plantes diploïdes clonales apomictiques. Une optimisation du mécanisme permettrait d'envisager l'utilisation de l'apomixie pour fixer l'hétérosis dans les semences hybrides F1. Par ailleurs, une augmentation globale ou locale de la recombinaison méiotique est recherchée car elle permettrait de diminuer la taille des populations de sélection et de réduire la taille des segments chromosomiques introduits dans les variétés élite de riz. Nous avons montré dans une seconde partie, que la mutation du gène OsRECQl4 codant pour une hélicase permet d'augmenter le taux de recombinaison d'un facteur de 3,3 fois faisant passer la taille de la carte génétique de 1670 cM à 5538 cM sans affecter la fertilité de la plante ni le déroulement de la méiose. Chez les plantes affectées dans la fonction d’une autre hélicase, OsFANCM, le taux de recombinaison a été également augmenté mais dans une moindre mesure (x 2,2). L’augmentation de la recombinaison s’opère sur l'ensemble des bras chromosomiques sauf au niveau des centromères. Ces résultats confirment ceux obtenus chez A. thaliana qui ont montré le rôle de régulateur négatif des crossing-overs (CO) des protéines RECQ4 et FANCM. La combinaison en cours de ces mutations entre elles ou avec celle affectant l’AAA-ATPase FIGL1 permet d’espérer une augmentation de la recombinaison encore supérieure. Ces résultats ouvrent la voie à l'utilisation des gènes anti-COs pour augmenter de façon globale le nombre de recombinants dans les croisements chez le riz et sans doute chez les autres céréales. Pour offrir une possibilité concrète aux sélectionneurs d'utiliser les gènes anti-CO, nous avons montré que la technologie CRISPR/cas9 permet d'éteindre l'expression de OsFANCM OsRECQl4 et OsFIGL1.

BACKGROUND: Of the 130 known coffee (Coffea) species, very few have been properly evaluated for their beverage quality. The diversity of wild coffee species is considered critical to the long‐term sustainability of the coffee sector, particularly under climate change. The challenge is finding coffee crops that satisfy agronomic criteria, now and under the altered climatic conditions of the future, as well as consumer requirements for flavour. We evaluated the sensory characteristics of three wild coffee species with four independent sensory panels, and the key environmental/agronomic requirements of these wild species based on a literature review.RESULTS: Coffea congensis and C. stenophylla have a lower unroasted seed weight compared to C. arabica and C. canephora, while C. brevipes has the largest. Sensory analysis showed that the main differences between species was for the fruitiness attribute. Coffea stenophylla was the fruitiest wild species, and was considered an Arabica‐like coffee. The flavour profile range of C. stenophylla covers herb‐like, vegetal, floral and fruit; C. brevipes resembles C. stenophylla in some respects. Opinions concerning C. congensis were contradictory and several judges considered the industry‐standard coffee flavour wheel not suitable for the beverage produced from this species.CONCLUSION: The three wild species have the required sensory qualities for commercialization. According to published data, C. stenophylla has agronomic potential, especially in warmer climates than Arabica areas. Coffea brevipes and C. congensis have the potential to be easily crossed with C. canephora to form interspecific hybrids capable of adapting to different climatic and agronomic conditions. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Introducing asexual reproduction through seeds – apomixis – into crop species could revolutionize agriculture by allowing F1 hybrids with enhanced yield and stability to be clonally propagated. Engineering synthetic apomixis has proven feasible in inbred rice through the inactivation of three genes ( MiMe ), which results in the conversion of meiosis into mitosis in a line ectopically expressing the BABYBOOM1 (BBM1) parthenogenetic trigger in egg cells. However, only 10–30% of the seeds were clonal. We show here that synthetic apomixis can be induced in a F1 hybrid of rice by inducing MiMe mutations and egg cell expression of BBM1 in a single step. We generated hybrid plants that produced more than 95% of clonal seeds across multiple generations. Clonal apomictic plants maintained the phenotype of the F1 hybrid along successive generations. Our results demonstrate that there is no barrier to almost fully penetrant synthetic apomixis in an important crop species, rendering it compatible with use in agriculture.

Rice (Oryza sativa) stands among the world9s most important crop species. Rice is salt sensitive, and the undue accumulation of sodium ions (Na⁺) in shoots has the strongest negative correlation with rice productivity under long-term salinity. The plasma membrane Na⁺/H⁺ exchanger protein Salt Overly Sensitive 1 (SOS1) is the sole Na⁺ efflux transporter that has been genetically characterized to date. Here, the importance of SOS1-facilitated Na⁺ flux in the salt tolerance of rice was analyzed in a reverse-genetics approach. A sos1 loss-of-function mutant displayed exceptional salt sensitivity that was correlated with excessive Na⁺ intake and impaired Na⁺ loading into the xylem, thus indicating that SOS1 controls net root Na⁺ uptake and long-distance Na⁺ transport to shoots. The acute Na⁺ sensitivity of sos1 plants at low NaCl concentrations allowed analysis of the transcriptional response to sodicity stress without effects of the osmotic stress intrinsic to high-salinity treatments. In contrast with that in the wild type, sos1 mutant roots displayed preferential down-regulation of stress-related genes in response to salt treatment, despite the greater intensity of stress experienced by the mutant. These results suggest there is impaired stress detection or an inability to mount a comprehensive response to salinity in sos1. In summary, the plasma membrane Na⁺/H⁺ exchanger SOS1 plays a major role in the salt tolerance of rice by controlling Na⁺ homeostasis and possibly contributing to the sensing of sodicity stress.

Improved plant varieties are hugely significant in our attempts to face the challenges of a growing human population and limited planet resources. Plant breeding relies on meiotic crossovers to combine favorable alleles into elite varieties (1). However, meiotic crossovers are relatively rare, typically one to three per chromosome (2), limiting the efficiency of the breeding process and related activities such as genetic mapping. Several genes that limit meiotic recombination were identified in the model species Arabidopsis (2). Mutation of these genes in Arabidopsis induces a large increase in crossover frequency. However, it remained to be demonstrated whether crossovers could also be increased in crop species hybrids. Here, we explored the effects of mutating the orthologs of FANCM3, RECQ44 or FIGL15 on recombination in three distant crop species, rice (Oryza sativa), pea (Pisum sativum) and tomato (Solanum lycopersium). We found that the single recq4 mutation increases crossovers ~three-fold in these crops, suggesting that manipulating RECQ4 may be a universal tool for increasing recombination in plants. Enhanced recombination could be used in combination with other state-of-the-art technologies such as genomic selection, genome editing or speed breeding to enhance the pace and efficiency of plant improvement.

The DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1 (DOCS1) gene belongs to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) subfamily. It has been discovered few years ago in Oryza sativa (rice) in a screen to isolate mutants with defects in sensitivity to aluminum. The c68 (docs1–1) mutant possessed a nonsense mutation in the C-terminal part of the DOCS1 kinase domain. We have generated a new loss-of-function mutation in the DOCS1 gene (docs1–2) using the CRISPR-Cas9 technology. This new loss-of-function mutant and docs1–1 present similar phenotypes suggesting the original docs1–1 was a null allele. Besides the aluminum sensitivity phenotype, both docs1 mutants shared also several root phenotypes described previously: less root hairs and mixed identities of the outer cell layers. Moreover, our new results suggest that DOCS1 could also play a role in root cap development. We hypothesized these docs1 root phenotypes may affect gravity responses. As expected, in seedlings, the early gravitropic response was delayed. Furthermore, at adult stage, the root gravitropic set angle of docs1 mutants was also affected since docs1 mutant plants displayed larger root cone angles. All these observations add new insights into the DOCS1 gene function in gravitropic responses at several stages of plant development.

In the arbuscular mycorrhizal (AM) symbiosis, plants satisfy part of their nitrogen (N) requirement through the AM pathway. In sorghum, the ammonium transporters (AMT) AMT3;1, and to a lesser extent AMT4, are induced in cells containing developing arbuscules. Here, we have characterized orthologs of AMT3;1 and AMT4 in four other grasses in addition to sorghum. AMT3;1 and AMT4 orthologous genes are induced in AM roots, suggesting that in the common ancestor of these five plant species, both AMT3;1 and AMT4 were already present and upregulated upon AM colonization. An artificial microRNA approach was successfully used to downregulate either AMT3;1 or AMT4 in rice. Mycorrhizal root colonization and hyphal length density of knockdown plants were not affected at that time, indicating that the manipulation did not modify the establishment of the AM symbiosis and the interaction between both partners. However, expression of the fungal phosphate transporter FmPT was significantly reduced in knockdown plants, indicating a reduction of the nutrient fluxes from the AM fungus to the plant. The AMT3;1 knockdown plants (but not the AMT4 knockdown plants) were significantly less stimulated in growth by AM fungal colonization, and uptake of both ¹⁵N and ³³P from the AM fungal network was reduced. This confirms that N and phosphorus nutrition through the mycorrhizal pathway are closely linked. But most importantly, it indicates that AMT3;1 is the prime plant transporter involved in the mycorrhizal ammonium transfer and that its function during uptake of N cannot be performed by AMT4.

Cecropin A is a natural antimicrobial peptide that exhibits rapid, potent and long-lasting lytic activity against a broad spectrum of pathogens, thus having great biotechnological potential. Here, we report a system for producing bioactive cecropin A in rice seeds.