Search

Phenotypic plasticity, the response of a given genotype to its environment of development, is a ubiquitous feature of life, enabling organisms to cope with variation in their environment. Theoretical studies predict that, under stationary environmental variation, the level of plasticity should evolve to match the predictability of selection at the timing of development. However, we still lack critical empirical evidence on the extent to which selection on phenotypic plasticity cascades down from higher phenotypic levels to their underlying molecular basis. Here, we used experimental evolution under controlled environmental fluctuations, to test whether the evolution of phenotypic plasticity in responses to environmental predictability (ρ2) occurred across biological levels, going from DNA methylation to gene expression to cell morphology. Transcriptomic results indicate clear effects of salinity andρ2×salinity interaction on gene expression, thus identifying sets of genes involved in plasticity and its evolution. These transcriptomic effects were independent of DNA methylation changes incis. However we did findρ2-specific responses of DNA methylation to salinity change, albeit weaker than for gene expression. Overall, we found consistent evolution of reduced plasticity in less predictable environments for DNA methylation, gene expression, and cell morphology. Our results provide the first clear empirical signature of plasticity evolution at multiple levels in response to environmental predictability, and highlight the importance of experimental evolution to address predictions from evolutionary theory, as well as investigate the molecular basis of plasticity evolution.

Melon (Cucumis melo) has emerged as an alternative model to tomato for studying fruit ripening due to the coexistence of climacteric and non-climacteric varieties. Previous characterization of a major quantitative trait locus (QTL), ETHQV8.1, that is able to trigger climacteric ripening in a non-climacteric background resulted in the identification of a negative regulator of ripening CTR1-like (MELO3C024518) and a putative DNA demethylase ROS1 (MELO3C024516) that is the orthologue of DML2, a DNA demethylase that regulates fruit ripening in tomato. To understand the role of these genes in climacteric ripening, in this study we generated homozygous CRISPR knockout mutants of CTR1-like and ROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two growing seasons with an earlier ethylene production profile being observed compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single-cytosine methylome analyses of the ROS1-knockout mutant revealed changes in DNA methylation in the promoter regions of the key ripening genes such as ACS1, ETR1, and ACO1, and in transcription factors associated with ripening including NAC-NOR, RIN, and CNR, suggesting the importance of ROS1-mediated DNA demethylation for triggering fruit ripening in melon., This work was supported by Grant RTI2018-097665-B-C2 funded by MCIN/AEI/ 10.13039/501100011033, and by ‘ERDF A way of making Europe’, Severo Ochoa Programme for Centres of Excellence in R&D CEX2019-000902 also funded by MCIN/AEI/ 10.13039/501100011033, and the CERCA Programme/Generalitat de Catalunya to JG-M. AG was supported by the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie (grant agreement No. 793090). MSD was supported by a FPI grant from the Spanish Ministry of Economy and Competitiveness BES-2017-079956 funded by MCIN/AEI/ 10.13039/501100011033 and by ‘ESF Investing in your future’. Work in the Quadrana group is supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 948674)., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000902)

Transposable elements (TEs) are repetitive DNA sequences with the ability to replicate across genomes and generate mutations with major transcriptional effects. Epigenetic silencing mechanisms that target TEs to limit their activity, including DNA methylation, add to the range of gene expression variants generated by TEs. Here, using the iconic gene flowering locus C (FLC) as a case study I discuss the multiple ways by which TEs can affect the expression of genes and contribute to the adaptation of plants to changing environments

International audience; Transposable elements (TEs) constitute a major threat to genome stability and are therefore typically silenced by epigenetic mechanisms. In response, some TEs have evolved counteracting systems to suppress epigenetic silencing. In the model plant Arabidopsis thaliana, two such anti-silencing systems have been identified and found to be mediated by the VANC DNA-binding proteins encoded by VANDAL transposons. Here, we show that anti-silencing systems have rapidly diversified since their origin in eudicots by gaining and losing VANC-containing domains, such as DUF1985, DUF287, and Ulp1, as well as target sequence motifs. We further demonstrate that these motifs determine anti-silencing specificity by sequence, density, and helical periodicity. Moreover, such rapid diversification yielded at least 10 distinct VANC-induced anti-silencing systems in Arabidopsis. Strikingly, anti-silencing of non-autonomous VANDALs, which can act as reservoirs of 24-nt small RNAs, is critical to prevent the demise of cognate autonomous TEs and to ensure their propagation. Our findings illustrate how complex co-evolutionary dynamics between TEs and host suppression pathways have shaped the emergence of new epigenetic control mechanisms.

International audience; DNA methylation is a major epigenetic mark involved in the silencing of genes and transposable elements (TEs). DNA methylation varies significantly across the plant life cycle, but is efficiently reinforced during reproduction, ensuring stable silencing of TEs. Plants are remarkably flexible in their mode of reproduction and numerous species including crops can propagate asexually, skipping one or more of these critical reinforcement steps. In this review we summarize recent advances in the characterization of DNA methylation inheritance in sexual and asexual plants. We argue that because most epigenetic reinforcement appears to occur during seed formation, methylomes of asexual seeds should resemble that of their sexual counterparts. Conversely, clonally propagated plants are expected to be hypomethylated and undergo frequent stochastic epigenetic changes. Last, we provide insights on how the use of nonmodel organisms will advance our understanding of epigenetic inheritance in plants.

SUMMARYRNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNAAUXIN-REGULATED PROMOTER LOOP(APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. Here, we showed that in addition to the PRC1-component LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1),APOLOinteracts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). TheAPOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis geneYUCCA2by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrated that the lncRNAUHRF1 Protein Associated Transcript(UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology betweenUPATandAPOLO. In addition, we showed that increased levels ofAPOLOorUPAThamper VIM1 and LHP1 binding toYUCCA2promoter. Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation, and reveal that evolutionary unrelated lncRNAs may exert similar functions across kingdoms.

Background RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. Results Here, we show that in addition to the PRC1-component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrate that the lncRNA UHRF1 Protein Associated Transcript (UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO. In addition, we show that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter and globally alter the Arabidopsis transcriptome in a similar manner. Conclusions Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation through the interaction with VIM1, and indicates that evolutionary unrelated lncRNAs with potentially conserved structures may exert similar functions by interacting with homolog partners.

Because of their ability to replicate across genomes, transposable elements (TEs) represent major generators of large-effect mutations. As a result, chromatin-based mechanisms have evolved to control the mutational potential of TEs at multiple levels, from the epigenetic silencing of TE sequences, through the modulation of their integration space, up to the alleviation of the impact of new insertions. Although most TE insertions are highly deleterious, some can provide key adaptive variation. Together with their remarkable sensitivity to the environment and precise integration preferences, the unique characteristics of TEs place them as potent genomic engines of adaptive innovation. Herein, we review recent works exploring the regulation and impact of transposition in nature and discuss their implications for the evolutionary response of species to drastic environmental changes.

Transposable elements (TEs) are powerful generators of major-effect mutations, most of which are deleterious at the species level and maintained at very low frequencies within populations. As reference genomes can only capture a minor fraction of such variants, methods were developed to detect TE insertion polymorphisms (TIPs) in non-reference genomes from short-read sequencing data, which are becoming increasingly available. We present here a bioinformatic framework combining an improved version of the SPLITREADER and TEPID pipelines to detect non-reference TE presence and reference TE absence variants, respectively. We benchmark our method on ten non-reference Arabidopsis thaliana genomes and demonstrate its high specificity and sensitivity in the detection of TIPs between genomes.

Transposable elements (TEs) are repetitive DNA sequences that have the ability to mobilize in the genome and create major effect mutations. Despite the importance of transposition as a source of genetic novelty, we still know little about the rate, landscape, and consequences of TE mobilization. This situation stems in large part from the repetitive nature of TEs, which complicates their analysis. Moreover, TE mobilization is typically rare and therefore new TE (i.e., non-reference) insertions tend to be missed in small-scale population studies. This chapter describes a TE-sequence capture approach designed to identify transposition events for most of the TE families that are potentially active in Arabidopsis thaliana. We show that our TE-sequence capture design provides an efficient means to detect with high sensitivity and specificity insertions that are present at a frequency as low as 1/1000 within a DNA sample.

Tocochromanols constitute the different forms of vitamin E (VTE), essential components of the human diet, and display a high membrane protectant activity. By combining interval mapping and genome-wide association studies (GWAS), we unveiled the genetic determinants of tocochromanol accumulation in tomato (Solanum lycopersicum) fruits. To enhance the nutritional value of this highly consumed vegetable, we dissected the natural intraspecific variability of tocochromanols in tomato fruits and genetically engineered their biosynthetic pathway. These analyses allowed the identification of a total of 25 quantitative trait loci interspersed across the genome pinpointing the chorismate–tyrosine pathway as a regulatory hub controlling the supply of the aromatic head group for tocochromanol biosynthesis. To validate the link between the chorismate–tyrosine pathway and VTE, we engineered tomato plants to bypass the pathway at the arogenate branch point. Transgenic tomatoes showed moderate increments in tocopherols (up to approximately 20%) and a massive accumulation of tocotrienols (up to approximately 3400%). Gene expression analyses of these plants reveal a trade-off between VTE and natural variation in chorismate metabolism explained by transcriptional reprogramming of specific structural genes of the pathway. By restoring the accumulation of alpha-tocotrienols (α-t3) in fruits, the plants produced here are of high pharmacological and nutritional interest. Fil: Burgos, Estanislao. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: de Luca, Maria Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Diouf, Isidore. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: de Haro, Luis Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Albert, Elise. Michigan State University; Estados Unidos. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Sauvage, Christopher. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; . Syngenta France SAS; Francia Fil: Tao, Zhao J.. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Bermudez Salazar, Luisa Fernanda. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Asís, Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Nesi, Adriano N.. Universidade Federal de Viçosa.; Brasil Fil: Matringe, Michel. Universite Joseph Fourier; Francia. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Bréhélin, Claire. Universite Joseph Fourier; Francia. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Guiraud, Thomas. Universite de Bordeaux; Francia. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Ferrand, Carine. Universite de Bordeaux; Francia. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Atienza, Isabelle. Universite de Bordeaux; Francia. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Jorly, Joana. Universite de Bordeaux; Francia. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Mauxion, Jean P.. Universite de Bordeaux; Francia. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Baldet, Pierre. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; . Universite de Bordeaux; Francia Fil: Fernie, Alisdair R.. Institut Max Planck fur Molekulare Physiologie; Alemania Fil: Quadrana, Leandro. Inserm; Francia. Centre National de la Recherche Scientifique; Francia Fil: Rothan, Christophe. Universite de Bordeaux; Francia. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Causse, Mathilde. Centre Nouvelle - Aquitaine Bordeaux ; Instituto National de Recherches Agronomiques, Alimetation Et Environnement; Fil: Carrari, Fernando Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires; Argentina

BackgroundHow species can adapt to abrupt environmental changes, particularly in the absence of standing genetic variation, is poorly understood and a pressing question in the face of ongoing climate change. Here we leverage publicly available multi-omic and bio-climatic data for more than 1000 wildArabidopsis thalianaaccessions to determine the rate of transposable element (TE) mobilization and its potential to create adaptive variation in natural settings.ResultsWe demonstrate that TE insertions arise at almost the same rate as base substitutions. Mobilization activity of individual TE families varies greatly between accessions, in association with genetic and environmental factors as well as through complex gene-environment interactions. Although the distribution of TE insertions across the genome is ultimately shaped by purifying selection, reflecting their typically strong deleterious effects when located near or within genes, numerous recent TE-containing alleles show signatures of positive selection. Moreover, high rates of transposition appear positively selected at the edge of the species’ ecological niche. Based on these findings, we predict through mathematical modeling higher transposition activity in Mediterranean regions within the next decades in response to global warming, which in turn should accelerate the creation of large-effect alleles.ConclusionsOur study reveals that TE mobilization is a major generator of genetic variation inA. thalianathat is finely modulated by genetic and environmental factors. These findings and modeling indicate that TEs may be essential genomic players in the demise or rescue of native populations in times of climate crises.

International audience; Transposable elements (TEs) are repetitive DNA sequences that have the ability to mobilize in the genome and create major effect mutations. Despite the importance of transposition as a source of genetic novelty, we still know little about the rate, landscape, and consequences of TE mobilization. This situation stems in large part from the repetitive nature of TEs, which complicates their analysis. Moreover, TE mobilization is typically rare and therefore new TE (i.e., non-reference) insertions tend to be missed in small-scale population studies. This chapter describes a TE-sequence capture approach designed to identify transposition events for most of the TE families that are potentially active in Arabidopsis thaliana. We show that our TE-sequence capture design provides an efficient means to detect with high sensitivity and specificity insertions that are present at a frequency as low as 1/1000 within a DNA sample.

A mutation in Arabidopsis polycomb repressive complex 2 partially suppresses the transposon activity observed in a DNA methylation mutant, challenging expectations., In plants and mammals, DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3), which is deposited by the polycomb repressive complex 2, are considered as two specialized systems for the epigenetic silencing of transposable element (TE) and genes, respectively. Nevertheless, many TE sequences acquire H3K27me3 when DNA methylation is lost. Here, we show in Arabidopsis thaliana that the gain of H3K27me3 observed at hundreds of TEs in the ddm1 mutant defective in the maintenance of DNA methylation, essentially depends on CURLY LEAF (CLF), one of two partially redundant H3K27 methyltransferases active in vegetative tissues. Surprisingly, the complete loss of H3K27me3 in ddm1 clf double mutant plants was not associated with further reactivation of TE expression nor with a burst of transposition. Instead, ddm1 clf plants exhibited less activated TEs, and a chromatin recompaction as well as hypermethylation of linker DNA compared with ddm1. Thus, a mutation in polycomb repressive complex 2 does not aggravate the molecular phenotypes linked to ddm1 but instead partially suppresses them, challenging our assumptions of the relationship between two conserved epigenetic silencing pathways.

A comprehensive phenotypic analysis in five Arabidopsis accessions combined with methylome and transcriptome analyses in the Col-0 reference accession indicate plasticity to mild drought but no transgenerational effects or inherited stress memory., There is renewed interest in whether environmentally induced changes in phenotypes can be heritable. In plants, heritable trait variation can occur without DNA sequence mutations through epigenetic mechanisms involving DNA methylation. However, it remains unknown whether this alternative system of inheritance responds to environmental changes and if it can provide a rapid way for plants to generate adaptive heritable phenotypic variation. To assess potential transgenerational effects induced by the environment, we subjected four natural accessions of Arabidopsis thaliana together with the reference accession Col-0 to mild drought in a multi-generational experiment. As expected, plastic responses to drought were observed in each accession, as well as a number of intergenerational effects of the parental environments. However, after an intervening generation without stress, except for a very few trait-based parental effects, descendants of stressed and non-stressed plants were phenotypically indistinguishable irrespective of whether they were grown in control conditions or under water deficit. In addition, genome-wide analysis of DNA methylation and gene expression in Col-0 demonstrated that, while mild drought induced changes in the DNA methylome of exposed plants, these variants were not inherited. We conclude that mild drought stress does not induce transgenerational epigenetic effects.

In plants and mammals, DNA methylation is a hallmark of transposable element (TE) sequences that contributes to their epigenetic silencing. In contrast, histone H3 lysine 27 trimethylation (H3K27me3), which is deposited by the Polycomb Repressive Complex 2 (PRC2), is a hallmark of repressed genes. Nevertheless, there is a growing body of evidence for a functional interplay between these pathways. In particular, many TE sequences acquire H3K27me3 when they lose DNA methylation and it has been proposed that PRC2 can serve as a back-up silencing system for hypomethylated TEs. Here, we describe in the flowering plantArabidopsis thalianathe gain of H3K27m3 at hundreds of TEs in the mutantddm1, which is defective in the maintenance of DNA methylation specifically over TE and other repeat sequences. Importantly, we show that this gain essentially depends on CURLY LEAF (CLF), which is one of two otherwise partially redundant H3K27 methyltransferases active in vegetative tissues. Finally, our results challenge the notion that PRC2 can be a compensatory silencing system for hypomethylated TEs, as the complete loss of H3K27me3 inddm1 clfdouble mutant plants was not associated with further reactivation of TE expression nor with a burst of transposition. Instead, and surprisingly,ddm1 clfplants exhibited less activated TEs, and a chromatin recompaction as well as hypermethylation of linker DNA compared toddm1. Thus, we have described an unexpected genetic interaction between DNA methylation and Polycomb silencing pathways, where a mutation in PRC2 does not aggravate the molecular phenotypes linked to TE hypomethylation inddm1but instead partially suppresses them.Author summaryEpigenetic marks are covalent modifications of the DNA or its associated proteins (Histones) that impact gene expression in a heritable manner without changing DNA sequence. In plants and mammals, DNA methylation and trimethylation of Lysine 27 of Histone 3 (H3K27me3) are two conserved, major epigenetic systems that mediate the transcriptional silencing of transposons (invasive mobile genetic elements) and of developmental genes respectively. However, in the absence of DNA methylation, H3K27me3 marks can be recruited to transposons, suggesting that the two silencing systems can be compensatory. To test this hypothesis, we analyzed a compound DNA methylation and H3K27me3 mutant of the plant modelArabidopsis thaliana(importantly, mammals harboring equivalent mutations would not be viable). First, this approach allowed us to gain mechanistic insights into the recruitment of H3K27me3 at transposons. Furthermore, we also showed that transposon silencing release in the DNA methylation mutant was not enhanced, contrary to our initial hypothesis, but, surprisingly, partially suppressed by a mutation in H3K27me3 deposition. Thus, our genomic analysis revealed an unexpected and antagonistic genetic interaction between two major silencing pathways whose interplay is at the heart of many biological processes, including cancer.

Clubroot caused by the protistPlasmodiophora brassicaeis a major disease affecting cultivatedBrassicaceae. Here, we uncover the existence of a natural epigenetic variation that is associated with partial resistance to clubroot inArabidopsis,by using QTL fine mapping followed by extensive DNA sequence and methylation analyses. We show that at QTLPb-At5.2, DNA methylation variation is extensive across accessions and strictly correlates with expression variation of the two neighboring genes At5g47260 and At5g47280, which encode NLR-immune receptors. Moreover, these natural variants are stably inherited and are not consistently associated with any nucleotide variation. These findings suggest a direct role for epigenetic variation in quantitative resistance of plants to pathogen attacks.

Polyploidization is frequently associated with increased transposable element (TE) content. However, what drives TE dynamics following whole genome duplication (WGD) and the evolutionary implications remain unclear. Here, we leveraged whole-genome resequencing data of ∼300 individual Arabidopsis arenosa plants, a well characterized natural diploid-autotetraploid species, to address these questions. Based on 43,176 polymorphic TE insertions we detected in these genomes, we demonstrate that relaxed purifying selection rather than transposition bursts is the main driver of TE over-accumulation after WGD. Furthermore, the increased pool of TE insertions in tetraploids is especially enriched within or near abiotic and biotic stress response genes. Notably, we uncovered one such insertion in a major flowering-time repressor gene and found that the resulting allele is specific to the rapid-cycling tetraploid lineage that colonized mainland railways. Together, our findings indicate that tetrasomy by itself leads to an enhanced tolerance to accumulating genic TE variants, some of which can potentially contribute to local adaptation.

Polyploidization is frequently associated with increased transposable element (TE) content. However, what drives TE dynamics following whole genome duplication (WGD) and the evolutionary implications remain unclear. Here, we leverage whole-genome resequencing data available for ~300 individuals of Arabidopsis arenosa, a well characterized natural diploid-autotetraploid plant species, to address these questions. Based on 43,176 TE insertions we detect in these genomes, we demonstrate that relaxed purifying selection rather than transposition bursts is the main driver of TE over-accumulation after WGD. Furthermore, the increased pool of TE insertions in tetraploids is especially enriched within or near environmentally responsive genes. Notably, we show that the major flowering-time repressor gene FLC is disrupted by a TE insertion specifically in the rapid-cycling tetraploid lineage that colonized mainland railways. Together, our findings indicate that tetrasomy leads to an enhanced accumulation of genic TE insertions, some of which likely contribute to local adaptation., Nature Communications, 10 (1), ISSN:2041-1723

Inherited transposition events are important drivers of genome evolution but because transposable element (TE) mobilization is usually rare, its impact on the creation of genetic variation remains poorly characterized. Here, we used a population of A. thaliana epigenetic recombinant inbred lines (epiRILs) to characterize >8000 de novo insertions produced by several TEs families also active in nature. Integration was strongly biased towards genes, with evident deleterious effects. Biases were TE family-specific and associated with distinct chromatin features. Notably, we demonstrate that the histone variant H2A.Z guides the preferential integration of Ty1/Copia LTR-retrotransposons within environmentally responsive genes and that this guiding function is evolutionary conserved. Finally, we uncover an important role for epigenetic silencing in exacerbating or alleviating the effects of TE insertions on target genes. These findings establish chromatin as a major determinant of the spectrum and functional impact of TE-generated mutations, with important implications for adaptation and evolution.

Tocopherols are non-polar compounds synthesized in the plastids, which function as major antioxidants of the plant cells and are essential in the human diet. Both the intermediates and final products of the tocopherol biosynthetic pathway must cross plastid membranes to reach their sites of action. So far, no protein with tocopherol binding activity has been reported in plants. Here, we demonstrated that the tomato SlTBP protein is targeted to chloroplasts and able to bind α-tocopherol. SlTBP-knockdown tomato plants exhibited reduced levels of tocopherol in both leaves and fruits. Several tocopherol deficiency phenotypes were apparent in the transgenic lines, such as alterations in photosynthetic parameters, dramatic distortion of thylakoid membranes and significant variations in the lipid profile. These results, along with the altered expression of genes related to photosynthesis, and tetrapyrrole, lipid, isoprenoid, inositol/phosphoinositide and redox metabolism, suggest that SlTBP may act in conducting tocopherol (or its biosynthetic intermediates) between the plastid compartments and/or at the interface between chloroplast and endoplasmic reticulum membranes, affecting interorganellar lipid metabolism. Fil: Bermúdez, Luisa. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Del Pozo, Talía. Universidad Mayor; Chile Fil: Silvestre Lira, Bruno. Universidade de Sao Paulo; Brasil Fil: de Godoy, Fabiana. Universidade de Sao Paulo; Brasil Fil: Boos, Irene. Technical University of Denmark; Dinamarca Fil: Romanò, Cecilia. Technical University of Denmark; Dinamarca Fil: Previtali, Viola. Technical University of Denmark; Dinamarca Fil: Almeida, Juliana. Universidade de Sao Paulo; Brasil Fil: Bréhélin, Claire. Laboratoire de Biogénèse Membranaire; Francia Fil: Asis, Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Quadrana, Leandro Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Demarco, Diego Gabriel. Universidade de Sao Paulo; Brasil Fil: Alseekh, Saleh. Max Planck Institute Of Molecular Plant Physiology; Alemania Fil: Salinas Gamboa, Rigel. Universidad Autónoma Metropolitana - Iztapalapa; México Fil: Pérez-Flores, Laura. Universidad Autónoma Metropolitana - Iztapalapa; México Fil: Dominguez, Pia Guadalupe. Comisión Nacional de Investigación Científica y Tecnológica; Chile. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Rothan, Christophe. Biologie Du Fruit Et Pathologie; Francia Fil: Fernie, Alisdair Robert. Max Planck Institute Of Molecular Plant Physiology; Alemania Fil: González, Maurício. Universidad de Chile; Chile Fil: Stocker, Achim. University of Bern; Suiza Fil: Hemmerle, Andreas. University of Bern; Suiza Fil: Clausen, Mads Hartving. Technical University of Denmark; Dinamarca Fil: Carrari, Fernando Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Rossi, Magdalena. Universidade de Sao Paulo; Brasil

SummaryWhether environmentally induced changes in phenotypes can be heritable is a topic with revived interest, in part because of observations in plants that heritable trait variation can occur without DNA sequence mutations. This other system of inheritance, called transgenerational epigenetics, typically involves differences in DNA methylation that are stable across multiple generations. However, it remains unknown if such a system responds to environmental changes and if it could therefore provide a rapid way for plants to generate adaptive heritable phenotypic variation. Here, we used a well-controlled phenotyping platform and whole-genome bisulfite sequencing to investigate potential heritable effects of mild drought applied over two successive generations in Arabidopsis thaliana. Plastic phenotypic responses were observed in plants exposed to drought. After an intervening generation without stress, descendants of stressed and non-stressed plants were phenotypically indistinguishable, except for very few trait-based parental effects, and irrespective of whether they were grown in control conditions or under water deficit. Moreover, while mild drought induced changes to the DNA methylome of exposed plants, DNA methylation variants were not inherited. These findings add to the growing body of evidence indicating that transgenerational epigenetics is not a common response of plants to environmental changes.

Transgenerational epigenetics is defined in opposition to developmental epigenetics and implies an absence of resetting of epigenetic states between generations. Unlike mammals, plants appear to be particularly prone to this type of inheritance. In this review, we summarize our knowledge about transgenerational epigenetics in plants, which entails heritable changes in DNA methylation. We emphasize the role of transposable elements and other repeat sequences in the creation of epimutable alleles. We also argue that because reprogramming of DNA methylation across generations seems limited in plants, the inheritance of DNA methylation defects results from the failure to reinforce rather than reset this modification during sexual reproduction. We compare genome-wide assessments of heritable DNA methylation variation and its phenotypic impact in natural populations to those made using near-isogenic populations derived from crosses between parents with experimentally induced DNA methylation differences. Finally, we question the role of the environment in inducing transgenerational epigenetic variation and briefly present theoretical models under which epimutability is expected to be selected for.

Transposable elements (TEs) are powerful motors of genome evolution yet a comprehensive assessment of recent transposition activity at the species level is lacking for most organisms. Here, using genome sequencing data for 211 Arabidopsis thaliana accessions taken from across the globe, we identify thousands of recent transposition events involving half of the 326 TE families annotated in this plant species. We further show that the composition and activity of the 'mobilome' vary extensively between accessions in relation to climate and genetic factors. Moreover, TEs insert equally throughout the genome and are rapidly purged by natural selection from gene-rich regions because they frequently affect genes, in multiple ways. Remarkably, loci controlling adaptive responses to the environment are the most frequent transposition targets observed. These findings demonstrate the pervasive, species-wide impact that a rich mobilome can have and the importance of transposition as a recurrent generator of large-effect alleles. DOI: http://dx.doi.org/10.7554/eLife.15716.001

Tocopherols, compounds with vitamin E (VTE) activity, are potent lipid-soluble antioxidants synthesized only by photosynthetic organisms. Their biosynthesis requires the condensation of phytyl-diphosphate and homogentisate, derived from the methylerythritol phosphate (MEP) and shikimate pathways (SK), respectively. These metabolic pathways are central in plant chloroplast metabolism and are involved in the biosynthesis of important molecules such as chlorophyll, carotenoids, aromatic amino-acids and prenylquinones. In the last decade, few studies have provided insights into the regulation of VTE biosynthesis and its accumulation. However, the pathway regulatory mechanism/s at mRNA level remains unclear. We have recently identified a collection of tomato genes involved in tocopherol biosynthesis. In this work, by a dedicated qPCR array platform, the transcript levels of 47 genes, including paralogs, were determined in leaves and across fruit development. Expression data were analyzed for correlation with tocopherol profiles by coregulation network and neural clustering approaches. The results showed that tocopherol biosynthesis is controlled both temporally and spatially however total tocopherol content remains constant. These analyses exposed 18 key genes from MEP, SK, phytol recycling and VTE-core pathways highly associated with VTE content in leaves and fruits. Moreover, genomic analyses of promoter regions suggested that the expression of the tocopherol-core pathway genes is trancriptionally coregulated with specific genes of the upstream pathways. Whilst the transcriptional profiles of the precursor pathway genes would suggest an increase in VTE content across fruit development, the data indicate that in the M82 cultivar phytyl diphosphate supply limits tocopherol biosynthesis in later fruit stages. This is in part due to the decreasing transcript levels of geranylgeranyl reductase (GGDR) which restricts the isoprenoid precursor availability. As a proof of concept, by analyzing a collection of Andean landrace tomato genotypes, the role of the pinpointed genes in determining fruit tocopherol content was confirmed. The results uncovered a finely tuned regulation able to shift the precursor pathways controlling substrate influx for VTE biosynthesis and overcoming endogenous competition for intermediates. The whole set of data allowed to propose that 1-deoxy-D-xylulose-5-phosphate synthase and GGDR encoding genes, which determine phytyl-diphosphate availability, together with enzyme encoding genes involved in chlorophyll-derived phytol metabolism appear as the most plausible targets to be engineered aiming to improve tomato fruit nutritional value. Fil: Quadrana, Leandro Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Almeida de Souza, Juliana Beatriz. Universidade de Sao Paulo; Brasil Fil: Otaiza González, Santiago Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina Fil: Duffy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: da Silva, Junia V. Corrêa. Universidade de Sao Paulo; Brasil Fil: de Godoy, Fabiana. Universidade de Sao Paulo; Brasil Fil: Asis, Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Bermudez Salazar, Luisa Fernanda. Universidade de Sao Paulo; Brasil Fil: Fernie, Alisdair R.. Institut Max Planck fur Molekulare Physiologie; Alemania Fil: Carrari, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Rossi, Maria Magdalena. Universidade de Sao Paulo; Brasil

Post-splicing activities have been described for a subset of shuttling serine/arginine-rich splicing regulatory proteins, among them SF2/ASF. We showed that growth factors activate a Ras-PI 3-kinase-Akt/PKB signaling pathway that not only modifies alternative splicing of the fibronectin EDA exon, but also alters in vivo translation of reporter mRNAs containing the EDA binding motif for SF2/ASF, providing two co-regulated levels of isoform-specific amplification. Translation of most eukaryotic mRNAs is initiated via the scanning mechanism, which implicates recognition of the m7G cap at the mRNA 5′-terminus by the eIF4F protein complex. Several viral and cellular mRNAs are translated in a cap-independent manner by the action of cis-acting mRNA elements named internal ribosome entry sites that direct internal ribosome binding to the mRNA. Here we use bicistronic reporters that generate mRNAs carrying two open reading frames, one translated in a cap-dependent manner while the other by internal ribosome entry site-dependent initiation, to show that in vivo over-expression of SF2/ASF increases the ratio between cap-dependent and internal ribosome entry site-dependent translation. Consistently, knocking-down of SF2/ASF causes the opposite effect. Changes in expression levels of SF2/ASF also affect alternative translation of an endogenous mRNA, that one coding for fibroblast growth factor-2. These results strongly suggest a role for SF2/ASF as a regulator of alternative translation, meaning the generation of different proteins by the balance among these two translation initiation mechanisms, and expand the known potential of SF2/ASF to regulate proteomic diversity to the translation field. J. Cell. Biochem. 107: 826–833, 2009. © 2009 Wiley-Liss, Inc.

Serine/arginine-rich (SR) proteins are important regulators of mRNA splicing. Several postsplicing activities have been described for a subset of shuttling SR proteins, including regulation of mRNA export and translation. Using the fibronectin gene to study the links between signal-transduction pathways and SR protein activity, we show that growth factors not only modify the alternative splicing pattern of the fibronectin gene but also alter translation of reporter messenger RNAs in an SR protein-dependent fashion, providing two coregulated levels of isoform-specific amplification. These effects are inhibited by specific small interfering RNAs against SR proteins and are mediated by the AKT kinase, which elicits opposite effects to those evoked by overexpressing SR protein kinases Clk and SRPK. These results show how SR protein activity is modified in response to extracellular stimulation, leading to a concerted regulation of splicing and translation.

Background Transposable elements (TEs) are DNA sequences that are able to move from their location in the genome by cutting or copying themselves to another locus. As such, they are increasingly recognized as impacting all aspects of genome function. With the dramatic reduction in cost of DNA sequencing, it is now possible to resequence whole genomes in order to systematically characterize novel TE mobilization in a particular individual. However, this task is made difficult by the inherently repetitive nature of TE sequences, which in some eukaryotes compose over half of the genome sequence. Currently, only a few software tools dedicated to the detection of TE mobilization using next-generation-sequencing are described in the literature. They often target specific TEs for which annotation is available, and are only able to identify families of closely related TEs, rather than individual elements. Results We present TE-Tracker, a general and accurate computational method for the de-novo detection of germ line TE mobilization from re-sequenced genomes, as well as the identification of both their source and destination sequences. We compare our method with the two classes of existing software: specialized TE-detection tools and generic structural variant (SV) detection tools. We show that TE-Tracker, while working independently of any prior annotation, bridges the gap between these two approaches in terms of detection power. Indeed, its positive predictive value (PPV) is comparable to that of dedicated TE software while its sensitivity is typical of a generic SV detection tool. TE-Tracker demonstrates the benefit of adopting an annotation-independent, de novo approach for the detection of TE mobilization events. We use TE-Tracker to provide a comprehensive view of transposition events induced by loss of DNA methylation in Arabidopsis. TE-Tracker is freely available at http://www.genoscope.cns.fr/TE-Tracker. Conclusions We show that TE-Tracker accurately detects both the source and destination of novel transposition events in re-sequenced genomes. Moreover, TE-Tracker is able to detect all potential donor sequences for a given insertion, and can identify the correct one among them. Furthermore, TE-Tracker produces significantly fewer false positives than common SV detection programs, thus greatly facilitating the detection and analysis of TE mobilization events. Electronic supplementary material The online version of this article (doi:10.1186/s12859-014-0377-z) contains supplementary material, which is available to authorized users.

Vitamin E (VTE) content is a low heritability nutritional trait for which the genetic determinants are poorly understood. Here, we focus on a previously detected major tomato VTE quantitative trait loci (QTL; mQTL 9-2-6) and identify the causal gene as one encoding a 2-methyl-6-phytylquinol methyltransferase (namely VTE3(1)) that catalyses one of the final steps in the biosynthesis of γ 3- and α -tocopherols, which are the main forms of VTE. By reverse genetic approaches, expression analyses, siRNA profiling and DNA methylation assays, we demonstrate that mQTL9-2-6 is an expression QTL associated with differential methylation of a SINE retrotransposon located in the promoter region of VTE3(1). Promoter DNA methylation can be spontaneously reverted leading to different epialleles affecting VTE3(1) expression and VTE content in fruits. These findings indicate therefore that naturally occurring epialleles are responsible for regulation of a nutritionally important metabolic QTL and provide direct evidence of a role for epigenetics in the determination of agronomic traits. Fil: Quadrana, Leandro Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Centre National de la Recherche Scientifique. Ecole Normale Supérieure; Francia Fil: Almeida de Souza, Juliana Beatriz. Universidade de Sao Paulo; Brasil Fil: Asis, Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina Fil: Duffy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Dominguez, Pia Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Bermudez Salazar, Luisa Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Agronomía; Argentina Fil: Conti, Gabriela. Universidad de Buenos Aires. Facultad de Agronomía; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Silva, Junia V. Corrêa da. Universidade de Sao Paulo; Brasil Fil: Peralta, Iris Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Provincia de Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Universidad Nacional de Cuyo. Instituto Argentino de Investigaciones de las Zonas Áridas; Argentina Fil: Colot, Vincent. Centre National de la Recherche Scientifique. Ecole Normale Supérieure; Francia Fil: Asurmendi, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Fernie, Alisdair R.. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Rossi, Magdalena. Universidade de Sao Paulo; Brasil Fil: Carrari, Fernando Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomía; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina

Limitations in our understanding about the mechanisms underlying source-sink assimilate partitioning are increasingly becoming a major hurdle for crop yield enhancement via metabolic engineering. By means of a comprehensive approach, this work reports the functional characterization of a DnaJ chaperone related-protein (named as SPA; sugar partition-affecting) involved in assimilate partitioning in tomato plants. SPA protein was found to be targeted to the chloroplast thylakoid membranes. SPA-RNAi tomato plants produced more and heavier fruits, thus resulting in a considerable increment in harvest index. The transgenic plants also displayed increased pigment levels and reduced sucrose, glucose and fructose contents in leaves. Detailed metabolic and enzymatic activities analyses showed that sugar phosphate intermediates are increased while the activity of phosphoglucomutase, sugar kinases and invertases are reduced in the photosynthetic organs of the silenced plants. These changes would be anticipated to promote carbon export from foliar tissues. The combined results suggest that the tomato SPA protein plays an important role in plastid metabolism and mediates the source-sink relationships by affecting the rate of carbon translocation to fruits. Fil: Bermudez Salazar, Luisa Fernanda. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Universidade de Sao Paulo; Brasil Fil: de Godoy, Fabiana. Universidade de Sao Paulo; Brasil Fil: Baldet, Pierre. Universite de Bordeaux; Francia. Institut National de la Recherche Agronomique; Francia Fil: Demarco, Diego. Universidade de Sao Paulo; Brasil Fil: Osorio, Sonia. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Quadrana, Leandro Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Almeida de Souza, Juliana Beatriz. Universidade de Sao Paulo; Brasil Fil: Asis, Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Gibon, Yves. Universite de Bordeaux; Francia. Institut National de la Recherche Agronomique; Francia Fil: Fernie, Alisdair. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Rossi, Maria Magdalena. Universidade de Sao Paulo; Brasil Fil: Carrari, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Rac1b is an alternatively spliced isoform of the small GTPase Rac1 that includes the 57-nucleotide exon 3b. Rac1b was originally identified through its over-expression in breast and colorectal cancer cells, and has subsequently been implicated as a key player in a number of different oncogenic signaling pathways, including tumorigenic transformation of mammary epithelial cells exposed to matrix metalloproteinase-3 (MMP-3). Although many of the cellular consequences of Rac1b activity have been recently described, the molecular mechanism by which MMP-3 treatment leads to Rac1b induction has not been defined. Here we use proteomic methods to identify heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a factor involved in Rac1 splicing regulation. We find that hnRNP A1 binds to Rac1 exon 3b in mouse mammary epithelial cells, repressing its inclusion into mature mRNA. We also find that exposure of cells to MMP-3 leads to release of hnRNP A1 from exon 3b and the consequent generation of Rac1b. Finally, we analyze normal breast tissue and breast cancer biopsies, and identify an inverse correlation between expression of hnRNP A1 and Rac1b, suggesting the existence of this regulatory axis in vivo. These results provide new insights on how extracellular signals regulate alternative splicing, contributing to cellular transformation and development of breast cancer. Fil: Pelisch, Federico Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Khauv, Davitte. Mayo Clinic Cancer Center; Estados Unidos Fil: Risso, Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Stallings Mann, Melody. Mayo Clinic Cancer Center; Estados Unidos Fil: Blaustein Kappelmacher, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Quadrana, Leandro Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Radisky, Derek C.. Mayo Clinic Cancer Center; Estados Unidos Fil: Srebrow, Anabella. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina

Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for α-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (α, β, γ, and δ) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical. Instituto de Biotecnología Fil: Almeida de Souza, Juliana Beatriz. Universidade de Sao Paulo. Instituto de Biociências. Departamento de Botânica; Brasil Fil: Quadrana, Leandro Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Asis, Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina Fil: Setta, Nathalia. Universidade de Sao Paulo. Instituto de Biociências. Departamento de Botânica; Brasil Fil: Godoy, Fabiana de. Universidade de Sao Paulo. Instituto de Biociências. Departamento de Botânica; Brasil Fil: Bermúdez, Luisa. Universidade de Sao Paulo. Instituto de Biociências. Departamento de Botânica; Brasil Fil: Otaiza, Santiago N. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina Fil: Silva, Junia V. Corrêa da. Universidade de Sao Paulo. Instituto de Biociências. Departamento de Botânica; Brasil Fil: Fernie, Alisdair R. Max Planck Institute for Molecular Plant Physiology; Alemania Fil: Carrari, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fil: Rossi, Magdalena. Universidade de São Pablo. Instituto de Biociências. Departamento de Botânica; Brasil

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of “sectoring” the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation. Fil: Quadrana, Leandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Rodriguez, Maria Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bermudez Salazar, Luisa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Nunes Nesi, Adriano. Max-Planck-Institut fur Molekulare Pflanzenphysiologie; Alemania Fil: Fernie, Alisdair R. Max-Planck-Institut fur Molekulare Pflanzenphysiologie; Alemania Fil: Descalzo, Adriana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina Fil: Asis, Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, Universidad Nacional de Córdoba; Argentina Fil: Rossi, Magdalena. Universidade de São Paulo. Instituto de Biociências. Departamento de Botânica; Brasil Fil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Carrari, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Cátedra de Genética; Argentina

International audience