The influence of genetic architecture on responses to selection under drought in rice

Accurately predicting responses to selection is a major goal in biology and important for successful crop breeding in changing environments. However, evolutionary responses to selection can be constrained by such factors as genetic and cross-environment correlations, linkage, and pleiotropy, and our understanding of the extent and impact of such constraints is still developing. Here, we conducted a field experiment to investigate potential constraints to selection for drought resistance in rice (Oryza sativa) using phenotypic selection analysis and quantitative genetics. We found that traits related to drought response were heritable, and some were under selection, including selection for earlier flowering, which could allow drought escape. However, patterns of selection generally were not opposite under wet and dry conditions, and we did not find individual or closely linked genes that influenced multiple traits, indicating a lack of evidence that antagonistic pleiotropy, linkage, or cross-environment correlations would constrain selection for drought resistance. In most cases, genetic correlations had little influence on responses to selection, with direct and indirect selection largely congruent. The exception to this was seed mass under drought, which was predicted to evolve in the opposite direction of direct selection due to correlations. Because of this indirect effect on selection on seed mass, selection for drought resistance was not accompanied by a decrease in seed mass, and yield increased with fecundity. Furthermore, breeding lines with high fitness and yield under drought also had high fitness and yield under wet conditions, indicating that there was no evidence for a yield penalty on drought resistance. We found multiple genes in which expression influenced both water use efficiency (WUE) and days to first flowering, supporting a genetic basis for the trade-off between drought escape and avoidance strategies. Together, these results can provide helpful guidance for understanding and managing evolutionary constraints and breeding stress-resistant crops

An atlas of over 90.000 conserved noncoding sequences provides insight into crucifer regulatory regions

Despite the central importance of noncoding DNA to gene regulation and evolution, understanding of the extent of selection on plant noncoding DNA remains limited compared to that of other organisms. Here we report sequencing of genomes from three Brassicaceae species (Leavenworthia alabamica, Sisymbrium irio and Aethionema arabicum) and their joint analysis with six previously sequenced crucifer genomes. Conservation across orthologous bases suggests that at least 17% of the Arabidopsis thaliana genome is under selection, with nearly one-quarter of the sequence under selection lying outside of coding regions. Much of this sequence can be localized to approximately 90,000 conserved noncoding sequences (CNSs) that show evidence of transcriptional and post-transcriptional regulation. Population genomics analyses of two crucifer species, A. thaliana and Capsella grandiflora, confirm that most of the identified CNSs are evolving under medium to strong purifying selection. Overall, these CNSs highlight both similarities and several key differences between the regulatory DNA of plants and other species

The origin, evolution and function of Mustang-A, a family of angiosperm-specific host genes derived from transposable elements

Transposable elements (TEs) are mobile genetic elements that have successfully populated eukaryotic genomes. Although first viewed solely as selfish, TEs are now known as important vectors to drive the adaptation and evolution of their host genome, such as sources for novel genes. The latter is called exaptation, or TE- co-option, or molecular domestication, and refers to the exaptation of TE genes that evolve to contribute directly to the phenotypic function of the host organism. The frequency of TE exaptation remains unresolved and consequently the full evolutionary and developmental impact of this adaptive process cannot be assessed. Among the few cases of TE exaptation reported in plants, we find the MUSTANG-A (MUG-A) family, which was identified in Arabidopsis thaliana and is derived from the transposon superfamily Mutator-like elements (MULEs). The main goal of my thesis is to investigate the process of TE exaptation in plants by exploring the evolution of the MUG-A, family and its function(s) in A. thaliana, as well as novel putative co-opted TE families recently uncovered using bioinformatic approaches. In Chapter 2, I present the results for the phenotypic characterization of the MUG-A family, with a particular emphasis on two genes, MUG1 and MUG2. Under standard growth conditions, T-DNA knockout mutants for both mug1 and mug2 reveal a pleiotropic phenotype that strongly reduces fitness. These results experimentally validate that genes of the MUG-A family are functional exapted TEs (ETEs). In Chapter 3, I further explore the potential function(s) for MUG1 and MUG2 with the working hypothesis that MUG genes may encode for transcription factors (TFs), a function recurrently selected for in known ETEs. Using an approach combining biochemistry, RNA-seq, and ChIP-seq, I show that MUG genes encode proteins that likely function as TFs, as they localize to the nucleus, have a conserved DNA-binding domain and bind specific DNA regions that correlates in part with transcriptome changes in MUG mutants. In Chapter 4, I present the results for a high throughput path involving reverse genetics approach and abiotic stress assays to reveal potential functions for known and novel co-opted TE families previously uncovered using direct systematic searches. These co-opted TEs are conserved among the Brassicaceae and were tested for inherent response to phosphate limitation and tolerance to high salt concentration, freezing temperature, and arsenic exposure/toxicity. The results reveal that most of the co-opted TEs tested show a response under stress conditions. In Chapter 5, I explore the potential scenarios for TE exaptation by looking at the evolution of the MUG-A family, using a progressive phylogenetic TE search for MUG homologs and related MULE sequences in angiosperm, gymnosperm, and moss genomes. The results from the phylogeny reveal that MUG-A was likely exapted in early angiosperm evolution preceding the most extant angiosperm (at least 160 million years ago). Taken together, the findings of my thesis contribute to increase our understanding of the MUG-A family and the process of TE exaptation in plant genomes. Furthermore, the work presented in this thesis represents one of the first instances of combining bioinformatics and experimental validation to address the process of TE exaptation.Les éléments transposables, appelés parfois transposons (ETs) sont des séquences d’ADN qui ont réussi à peupler la plupart des génomes nucléaires eucaryotes. Malgré le fait qu’ils aient été longtemps considérés comme des éléments égoïstes, parasites, ou encore de l’ADN «poubelle», les Ets sont maintenant reconnus comme des vecteurs d’évolution importants pouvant contribuer à l’adaptation et à l’évolution de leur génome hôte, par exemple comme source de nouveaux gènes. Ce dernier est un processus appelé exaptation, domestication moléculaire, ou encore la co-option de ETs et s’applique à l’exaptation des gènes ETs qui évoluent pour contribuer directement à la fonction phénotypique de l’organisme hôte. La fréquence d’exaptation des ETs demeure inconnue et conséquemment, le véritable impact de ce processus adaptif au niveau évolutif et développemental demeure difficile à évaluer. Parmi les quelques exemples d’exaptation des ETs dans les génomes de plantes, nous retrouvons la famille de gènes MUSTANG-A (MUG-A), découverte dans la plante Arabidopsis thaliana et provenant d’un ET de la superfamille Mutator-like elements (MULEs). L’objectif principal de cette thèse est d’examiner le processus d’exaptation des ETs chez les plantes en explorant l’évolution et les fonctions possibles de la famille de gènes MUG-A dans la plante modèle A. thaliana, ainsi que des familles putatives de ETs exaptés (ETEs) récemment découvertes à l’aide d’analyses bioinformatiques. En premier lieu (Chapitre 2), je présente les résultats de la caractérisation des phénotypes de la famille MUG-A, en mettant l’emphase sur deux gènes, MUG1 et MUG2. Dans des conditions de croissance normales, l’invalidation de ces deux gènes par l’insertion aléatoire d’une séquence d’ADN (T-DNA) induit des phénotypes qui réduisent fortement la valeur sélective des plantes mutantes. Ces résultats démontrent de manière expérimentale que les ETEs de la famille MUG-A sont fonctionnels. En second lieu (Chapitre 3), j’explore les fonctions possibles de MUG1 et MUG2, en partant de l’hypothèse que ces gènes codent des protéines facteurs de transcription, étant donné que plusieurs ETEs connus ont été sélectionné pour cette fonction. En utilisant une approche combinant des techniques en biochimie, ainsi que des techniques appelées ChIP-seq et RNA-seq, je démontre pour la première fois que MUG1 et MUG2 code des protéines et que celles-ci fonctionnent probablement comme facteurs de transcription. En effet, la localisation sous-cellulaire de ces protéines se trouve dans le noyau, elles possèdent un domaine de liaison à l’ADN, et se lient à un morceau d’ADN présent dans des régions du génome qui corrèlent avec des gènes présentant des changements d’expression lorsque les gènes MUG1 et MUG2 sont mutants. En troisième lieu (Chapitre 4), je décris les résultats d’un pipeline de criblage à haut débit combinant une approche de génétique inverse et une série de criblage à l’aide d’expériences impliquant des stress abiotiques (salinité, restriction de phosphate, arsenic et gel) dans le but de valider la fonctionnalité de nouvelles familles de ETEs précédemment découvertes à l’aide de techniques en bioinformatique. En quatrième lieu, (Chapitre 5), j’explore les scénarios évolutifs ayant potentiellement amené l’exaptation de la famille MUG-A à l’aide d’une nouvelle approche qui consiste en une recherche progressive phylogénétique d’éléments transposables et de séquences homologues à MUG dans plusieurs génomes d’espèces d’angiospermes, de gymnospermes et de mousses. En terminant, les résultats présentés dans cette thèse contribuent à améliorer notre compréhension de la famille MUG-A et plus généralement du processus d’exaptation des ETs dans des génomes de plantes. De plus, cette thèse constitue un des premiers exemples d’une approche alliant des techniques de bioinformatique et de validations expérimentales afin d’étudier le processus d’exaptation de ETs

Abiotic Stress Phenotypes Are Associated with Conserved Genes Derived from Transposable Elements

Plant phenomics offers unique opportunities to accelerate our understanding of gene function and plant response to different environments, and may be particularly useful for studying previously uncharacterized genes. One important type of poorly characterized genes is those derived from transposable elements (TEs), which have departed from a mobility-driven lifestyle to attain new adaptive roles for the host (exapted TEs). We used phenomics approaches, coupled with reverse genetics, to analyze T-DNA insertion mutants of both previously reported and novel protein-coding exapted TEs in the model plant Arabidopsis thaliana. We show that mutations in most of these exapted TEs result in phenotypes, particularly when challenged by abiotic stress. We built statistical multi-dimensional phenotypic profiles and compared them to wild-type and known stress responsive mutant lines for each particular stress condition. We found that these exapted TEs may play roles in responses to phosphate limitation, tolerance to high salt concentration, freezing temperatures, and arsenic toxicity. These results not only experimentally validate a large set of putative functional exapted TEs recently discovered through computational analysis, but also uncover additional novel phenotypes for previously well-characterized exapted TEs in A. thaliana

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in plants self-organizes in vitro. The recombinant apoprotein, denatured in dodecyl sulfate, spontaneously folds when it is mixed with its pigments, chlorophylls, and carotenoids in detergent solution, and assembles into structurally authentic LHCII in the course of several minutes. Pulse EPR techniques, specifically double-electron-electron resonance (DEER), have been used to analyze protein folding during this process. Pairs of nitroxide labels were introduced site-specifically into recombinant LHCII and shown not to affect the stability and function of the pigment-protein complex. Interspin distance distributions between two spin pairs were measured at various time points, one pair located on either end of the second transmembrane helix (helix 3), the other one located near the luminal ends of the intertwined transmembrane helices 1 and 4. In the dodecyl sulfate-solubilized apoprotein, both distance distributions were consistent with a random-coil protein structure. A rapid freeze-quench experiment on the latter spin pair indicated that 1 s after initiating reconstitution the protein structure is virtually unchanged. Subsequently, both distance distributions monitored protein folding in the same time range in which the assembly of chlorophylls into the complex had been observed. The positioning of the spin pair spanning the hydrophobic core of LHCII clearly preceded the juxtaposition of the spin pair on the luminal side of the complex. This indicates that superhelix formation of helices 1 and 4 is a late step in LHCII assembly

A Gene Family Derived from Transposable Elements during Early Angiosperm Evolution Has Reproductive Fitness Benefits in <em>Arabidopsis thaliana</em>

<div><p>The benefits of ever-growing numbers of sequenced eukaryotic genomes will not be fully realized until we learn to decipher vast stretches of noncoding DNA, largely composed of transposable elements. Transposable elements persist through self-replication, but some genes once encoded by transposable elements have, through a process called molecular domestication, evolved new functions that increase fitness. Although they have conferred numerous adaptations, the number of such domesticated transposable element genes remains unknown, so their evolutionary and functional impact cannot be fully assessed. Systematic searches that exploit genomic signatures of natural selection have been employed to identify potential domesticated genes, but their predictions have yet to be experimentally verified. To this end, we investigated a family of domesticated genes called <em>MUSTANG</em> (<em>MUG</em>), identified in a previous bioinformatic search of plant genomes. We show that <em>MUG</em> genes are functional. Mutants of <em>Arabidopsis thaliana MUG</em> genes yield phenotypes with severely reduced plant fitness through decreased plant size, delayed flowering, abnormal development of floral organs, and markedly reduced fertility. <em>MUG</em> genes are present in all flowering plants, but not in any non-flowering plant lineages, such as gymnosperms, suggesting that the molecular domestication of <em>MUG</em> may have been an integral part of early angiosperm evolution. This study shows that systematic searches can be successful at identifying functional genetic elements in noncoding regions and demonstrates how to combine systematic searches with reverse genetics in a fruitful way to decipher eukaryotic genomes.</p> </div

<i>MUSTANG</i> phylogeny and gene structure in <i>A. thaliana</i>.

<p>(A) <i>MUG</i> phylogeny in nine angiosperm species. At, <i>Arabidopsis thaliana</i>; Bd, <i>Brachypodium distachyon</i>; Cp, <i>Carica papaya</i>; Mg, <i>Mimulus guttatus</i>; Mt, <i>Medicago truncatula</i>; Os, <i>Oryza sativa</i>; Sb, <i>Sorghum bicolor</i>; Vv, <i>Vitis vinifera</i>; Zm, <i>Zea mays</i>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002931#pgen.1002931.s001" target="_blank">Figure S1</a> for sequences and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002931#pgen.1002931.s008" target="_blank">Table S5</a> for locus IDs. All bootstrap values are >70% (not shown). Cp7 is truncated; its position is approximate. (B) Graphical representation of <i>At-MUG1</i>, <i>At-MUG2</i>, <i>At-MUG7</i>, and <i>At-MUG8</i> gene transcripts. Bold horizontal lines represent transcripts, dips introns, and rectangles coding sequences.</p

MT1-MMP Cooperates with TGF-&beta; Receptor-Mediated Signaling to Trigger SNAIL and Induce Epithelial-to-Mesenchymal-like Transition in U87 Glioblastoma Cells

Epithelial-to-mesenchymal transition (EMT) recapitulates metastasis and can be induced in vitro through transforming growth factor (TGF)-&beta; signaling. A role for MMP activity in glioblastoma multiforme has been ascribed to EMT, but the molecular crosstalk between TGF-&beta; signaling and membrane type 1 MMP (MT1-MMP) remains poorly understood. Here, the expression of common EMT biomarkers, induced through TGF-&beta; and the MT1-MMP inducer concanavalin A (ConA), was explored using RNA-seq analysis and differential gene arrays in human U87 glioblastoma cells. TGF-&beta; triggered SNAIL and fibronectin expressions in 2D-adherent and 3D-spheroid U87 glioblastoma cell models. Those inductions were antagonized by the TGF-&beta; receptor kinase inhibitor galunisertib, the JAK/STAT inhibitors AG490 and tofacitinib, and by the diet-derived epigallocatechin gallate (EGCG). Transient gene silencing of MT1-MMP prevented the induction of SNAIL by ConA and abrogated TGF-&beta;-induced cell chemotaxis. Moreover, ConA induced STAT3 and Src phosphorylation, suggesting these pathways to be involved in the MT1-MMP-mediated signaling axis that led to SNAIL induction. Our findings highlight a new signaling axis linking MT1-MMP to TGF-&beta;-mediated EMT-like induction in glioblastoma cells, the process of which can be prevented by the diet-derived EGCG

Reproducing on Time When Temperature Varies: Shifts in the Timing of Courtship by Fiddler Crabs

<div><p>Many species reproduce when conditions are most favorable for the survival of young. Numerous intertidal fish and invertebrates release eggs or larvae during semilunar, large amplitude, nocturnal tides when these early life stages are best able to escape predation by fish that feed near the shore during the day. Remarkably, some species, including the fiddler crabs <i>Uca terpsichores</i> and <i>Uca deichmanni</i>, maintain this timing throughout the year as temperature, and thus the rate of embryonic development, vary. The mechanisms that allow such precision in the timing of the production of young are poorly known. A preliminary study suggested that when temperature decreases, <i>U. terpsichores</i> mate earlier in the tidal amplitude cycle such that larvae are released at the appropriate time. We tested this idea by studying the timing of courtship in <i>U. terpsichores</i> and <i>U. deichmanni</i> as temperature varied annually during two years, at 5 locations that differed in the temperature of the sediment where females incubate their eggs. <i>Uca terpsichores</i> courted earlier at locations where sediment temperature declined seasonally but not where sediment temperature remained elevated throughout the year. In contrast, clear shifts in courtship timing were not observed for <i>U. deichmanni</i> despite variation in sediment temperature. We discuss other mechanisms by which this species may maintain reproductive timing. These two species are likely to be affected differently by changes in the frequency and intensity of cold periods that are expected to accompany climate change.</p></div