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Male and female unisexual flowers evolved from hermaphroditic ancestors, and control of flower sex is useful for plant breeding. We isolated a female-to-male sex transition mutant in melon and identified the causal gene as the carpel identity gene CRABS CLAW (CRC) . We show that the master regulator of sex determination in cucurbits, the transcription factor WIP1 whose expression orchestrates male flower development, recruits the corepressor TOPLESS to the CRC promoter to suppress its expression through histone deacetylation. Impairing TOPLESS-WIP1 physical interaction leads to CRC expression, carpel determination, and consequently the expression of the stamina inhibitor, the aminocyclopropane-1-carboxylic acid synthase 7 ( CmACS7 ), leading to female flower development. Our findings suggest that sex genes evolved to interfere with flower meristematic function, leading to unisexual flower development.

International audience; Shapes of vegetables and fruits are the result of adaptive evolution and human selection. Modules controlling organ shape have been identified. However, little is known about signals coordinating organ development and shape. Here, we describe the characterization of a melon mutation rf1, leading to round fruit. Histological analysis of rf1 flower and fruits revealed fruit shape is determined at flower stage 8, after sex determination and before flower fertilization. Using positional cloning, we identified the causal gene as the monoecy sex determination gene CmACS7, and survey of melon germplasms showed strong association between fruit shape and sexual types. We show that CmACS7-mediated ethylene production in carpel primordia enhances cell expansion and represses cell division, leading to elongated fruit. Cell size is known to rise as a result of endoreduplication. At stage 8 and anthesis, we found no variation in ploidy levels between female and hermaphrodite flowers, ruling out endoreduplication as a factor in fruit shape determination. To pinpoint the gene networks controlling elongated versus round fruit phenotype, we analyzed the transcriptomes of laser capture microdissected carpels of wild-type and rf1 mutant. These high-resolution spatiotemporal gene expression dynamics revealed the implication of two regulatory modules. The first module implicates E2FDP transcription factors, controlling cell elongation versus cell division. The second module implicates OVATE-and TRM5-related proteins, controlling cell division patterns. Our finding highlights the dual role of ethylene in the inhibition of the stamina development and the elongation of ovary and fruit in cucurbits.

International audience; In monoecious melon (Cucumis melo), sex is determined by the differential expression of sex determination genes (SDGs) and adoption of sex-specific transcriptional programs. Histone modifications such as H3K27me3 have been previously shown to be a hallmark associated to unisexual flower development in melon; yet, no genetic approaches have been conducted for elucidating the roles of H3K27me3 writers, readers, and erasers in this process. Here we show that melon homologs to Arabidopsis LHP1, CmLHP1A and B, redundantly control several aspects of plant development, including sex expression. Cmlhp1ab double mutants displayed an overall loss and redistribution of H3K27me3, leading to a deregulation of genes involved in hormone responses, plant architecture, and flower development. Consequently, double mutants display pleiotropic phenotypes and, interestingly, a general increase of the male:female ratio. We associated this phenomenon with a general deregulation of some hormonal response genes and a local activation of male-promoting SDGs and MADS-box transcription factors. Altogether, these results reveal a novel function for CmLHP1 proteins in maintenance of monoecy and provide novel insights into the polycomb-mediated epigenomic regulation of sex lability in plants.

Three amino acid loop extension homeodomain transcription factors (TALE HD TFs) act as life cycle regulators in green algae and land plants. In mosses these regulators are required for the deployment of the sporophyte developmental program. We demonstrate that mutations in either of two TALE HD TF genes, OUROBOROS or SAMSARA, in the brown alga Ectocarpus result in conversion of the sporophyte generation into a gametophyte. The OUROBOROS and SAMSARA proteins heterodimerise in a similar manner to TALE HD TF life cycle regulators in the green lineage. These observations demonstrate that TALE-HD-TF-based life cycle regulation systems have an extremely ancient origin, and that these systems have been independently recruited to regulate sporophyte developmental programs in at least two different complex multicellular eukaryotic supergroups, Archaeplastida and Chromalveolata.

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner., Author summary Plant shoot branching results from the precise regulation of bud growth versus dormancy. Positive and negative feedback mechanisms are likely involved in the dynamic control of this highly plastic trait. Strigolactones, the most recently discovered class of plant hormones, play a key role in controlling shoot branching. Negative feedback control of strigolactone biosynthesis has been observed in several species and was shown in pea to be mediated by a shoot-to-root signal that is RAMOSUS2 (RMS2)-dependent. The chemical nature of this feedback signal has been extensively discussed. Here, we demonstrate that the RMS2 protein belongs to the small family of auxin receptors and confirm that it behaves as an auxin receptor. Strigolactones decrease stem auxin levels by rapidly repressing transcript levels of auxin biosynthesis genes, thereby forming a long-distance feedback loop between auxin and strigolactones for the precise regulation of shoot branching in plants.

International audience; Fruit ripening and softening are key traits for many fleshy fruit. Since cell walls play a key role in the softening process, expansins have been investigated to control fruit over ripening and deterioration. In tomato, expression of Expansin I gene, SlExp1, during fruit ripening was associated with fruit softening. To engineer tomato plants with long shelf life, we screened for mutant plants impaired in Slexp1 function. Characterization of two induced mutations, Slexp1-6_14/211S, and Slexp1-7_Q213Stop, showed that Slexp1 loss of function leads to enhanced fruit firmness and delayed fruit ripening. Analysis of cell wall polysaccharide composition of Slexp1-7_Q213Stop mutant pointed out significant differences for uronic acid, neutral sugar and total sugar contents. Hemicelluloses chemistry analysis by endo-beta-1,4-D-glucanase hydrolysis and MALDI-TOF spectrometry revealed that xyloglucan structures were affected in the fruit pericarp of Slexp1 -7_Q213Stop mutant. Altogether, these results demonstrated that SlExp1 loss of function mutants yield firmer and late ripening fruits through modification of hemicellulose structure. These SlExp1 mutants represent good tools for breeding long shelf life tomato lines with contrasted fruit texture as well as for the understanding of the cell wall polysaccharide assembly dynamics in fleshy fruits.

International audience

How flowers separate males and females Most flowering plant families have bisexual flowers with both male and female function. However, most members of the Cucurbiticeae family, which includes melons, cucumbers, and gourds, have unisexual flowers. To understand this difference in sex expression, Boualem et al. identified a cucumber gene expressed in the female flowers. Mutations in this gene were associated with solely male flowers. By integrating this finding into a sex determination model, the authors explain how unisexual flowers can coexist in the same plant. Science, this issue p. 688

Background: [br/] Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics. [br/] Principal Findings: [br/] A population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147 Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G(33) may affect the protein function, whereas mutations at S-238 and S-249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation. [br/] Conclusions: [br/] We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.

International audience; BACKGROUND: Ordered collections of mutants serve as invaluable tools in biological research. TILLING (Targeting Induced Local Lesions IN Genomes) provides an efficient method to discover, in mutagenized populations, the possible phenotypes controlled by gene sequences whose function is unknown. This method can replace transgenic techniques for the functional validation of cloned genes, especially in the case of transformation-recalcitrant plants such as cucumber. RESULTS: We report the development of a TILLING cucumber population, generated by EMS mutagenesis in the Poinsett76 genetic background. The population was evaluated by screening for morphological mutations, and a range of developmental, pigmentation and spontaneous lesion mutants were observed. Suitability for detecting single nucleotide polymorphism in selected genes has been tested by screening a sample of amplicons, with detection rate of 1 SNP in ~1 Mbp. CONCLUSION: The population described in this Research Note represents a useful asset in cucumber research, to be exploited for forward genetic screens and functional genomics purposes.

The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called "BRACHYTIL", for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6, a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to assess the Genetically Effective Cell Number that was shown to be at least equal to 4 cells in Brachypodium distachyon. The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.

International audience; Late blight, caused by the pathogen Phytophthora spp. (Oomycete), is one of the most damaging diseases of Solanaceae. The deployment of resistant varieties is the most effective, economical and environmentally safe control of Phytophthora. Contrary to monogenic resistances that are easily overcome by the pathogen, polygenic resistances generally confer a durable control of disease severity. However, their molecular basis remains unknown since no resistance QTl to Phytophthora has ever been characterized. Phy-P5, the major effect Phytophthora capsici resistance QTl in pepper, has been mapped on the chromosome P5. It was detected in 3 pepper accessions and displayed a broad-spectrum resistance to several P. capsici isolates. We airn to determine the molecular basis of Phy-PS by isolating the responsible gene deploying a positional cloninq strategy.

Supplementary Informations : 15 p. ; Sex determination in plants leads to the development of unisexual flowers from an originally bisexual floral meristem. This mechanism results in the enhancement of outcrossing and promotes genetic variability, the consequences of which are advantageous to the evolution of a species. In melon, sexual forms are controlled by identity of the alleles at the andromonoecious (a) and gynoecious (g) loci. We previously showed that the a gene encodes an ethylene biosynthesis enzyme, mACS-7, that represses stamen development in female flowers. Here we show that the transition from male to female flowers in gynoecious lines results from epigenetic changes in the promoter of a transcription factor, CmWIP1. This natural and heritable epigenetic change resulted from the insertion of a transposon, which is required for initiation and maintenance of the spreading of DNA methylation to the CmWIP1 promoter. Expression of CmWIP1 leads to carpel abortion, resulting in the development of unisexual male flowers. Moreover, we show that CmWIP1 indirectly represses the expression of the andromonoecious gene, CmACS-7, to allow tamen development. Together our data indicate a model in which CmACS-7 and CmWIP1 interact to control the development of male, female and hermaphrodite flowers in melon.

La présente invention a pour objet une combinaison de deux éléments génétiques pour le contrôle du développement du type floral d'une plante dicotylédone, ladite combinaison comprenant, respectivement : un premier élément génétique de contrôle (A/a) présent dans une plante dicotylédone, sous la forme d'un allèle dominant (A), et d'un allèle récessif (a); et un second élément génétique de contrôle (G/g) présent dans une plante dicotylédone, sous la forme d'un allèle dominant (G), et d'un allèle récessif (g), étant entendu qu'au moins le second élément génétique de contrôle a été artificiellement introduit dans ladite plante dicotylédone. La combinaison ci-dessus permet de contrôler, et/ou de modifier le sexe des fleurs de plantes dicotylédones.

BackgroundAcidity is an essential component of the organoleptic quality of fleshy fruits. However, in these fruits, the physiological and molecular mechanisms that control fruit acidity remain unclear. In peach theDlocus controls fruit acidity; low-acidity is determined by the dominant allele. Using a peach progeny of 208 F2trees, theDlocus was mapped to the proximal end of linkage group 5 and co-localized with major QTLs involved in the control of fruit pH, titratable acidity and organic acid concentration and small QTLs for sugar concentration. To investigate the molecular basis of fruit acidity in peach we initiated the map-based cloning of theDlocus.ResultsIn order to generate a high-resolution linkage map in the vicinity of theDlocus, 1,024 AFLP primer combinations were screened using DNA of bulked acid and low-acid segregants. We also screened a segregating population of 1,718 individuals for chromosomal recombination events linked to theDlocus and identified 308 individuals with recombination events close toD. Using these recombinant individuals we delimited theDlocus to a genetic interval of 0.4 cM. We also constructed a peach BAC library of 52,000 clones with a mean insert size of 90 kb. The screening of the BAC library with markers tightly linked toDlocus indicated that 1 cM corresponds to 250 kb at the vicinity of theDlocus.ConclusionIn the present work we presented the first high-resolution genetic map ofDlocus in peach. We also constructed a peach BAC library of approximately 15× genome equivalent. This fine genetic and physical characterization of theDlocus is the first step towards the isolation of the gene(s) underlying fruit acidity in peach.

International audience

International audience