Integration of multiple environmental stresses for compound gene regulation in Arabidopsis thaliana

Plants are known to respond to various types of environmental stresses arising from physicochemical changes and other organisms. As plants often simultaneously experience multiple stress factors due to their immobility, capacity to appropriately regulate gene expression by integrating multiple stress signals is crucial for successful adaptation to hostile environments. Although significant progress has been made in elucidating the molecular mechanisms for regulation of stress response genes under single stress, little is known about the effects of combined stress signals on gene regulation and their associated mechanisms. This thesis aimed to contribute to the understanding of plant stress response by studying the signal integration mechanisms under various perspectives: first, the thesis explored how multiple stress signals affect the choices over discretised regulatory outcomes, such as up-regulation or down-regulation. We propose that processing of multiple signals can be described as logical operations, and subsequently investigate the mechanisms for each signal integration outcome by constructing logical model of intracellular signalling network. The resulting insight was applied to analyse a transcriptomic dataset from the model plant Arabidopsis thaliana, leading to novel hypotheses about potential crosstalk interactions that are missing between multiple stress signalling pathways. In parallel, the thesis also explored the cases where integration of multiple stress signals modulates dynamics of gene expression. An experimental study of the expression profile of Response-to-Dehydration 29A (RD29A), a model stress response gene, was conducted to show that combination of multiple stress inputs introduces a unique qualitative effect on dynamics of gene expression. The origin of this behaviour was investigated via a dynamical model of the RD29A regulatory network, which subsequently revealed potential interactions in the regulatory network that are currently unknown. Taken together, this thesis argues that systematic comparison between gene regulatory outcomes under single and combined stress inputs provides a crucial source of information for discovering functionally significant regulatory interactions in the stress signalling network.Open Acces

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest

Predicting Phenotypes From Novel Genomic Markers Using Deep Learning

Genomic selection (GS) is a powerful method concerned with predicting the phenotypes of individuals from genome-wide markers to select candidates for the next breeding cycle. Previous studies in GS have used single nucleotide polymorphism (SNP) markers to predict phenotypes using conventional statistical or deep learning models. However, these predictive models face challenges due to the high dimensionality of genome-wide SNP marker data and interactions between alleles. Thanks to recent breakthroughs in DNA sequencing and decreased sequencing cost, the study of novel genomic variants such as structural variations (SVs) and transposable elements (TEs) became increasingly prevalent. Here, we present a one-dimensional deep convolutional neural network, NovGMDeep, to predict phenotypes using novel genomic markers, such as SVs and TEs. The model is designed to use novel genomic markers to reduce the curse of dimensionality of the SNP genotypic data for GS. The proposed model is trained and tested on the samples of Arabidopsis thaliana and Oryza sativa using 3-fold cross-validation. The prediction accuracy is evaluated using Pearson’s Correlation Coefficient (PCC), Mean Absolute Error (MAE), and Standard Deviation (SD) of MAE on the testing sets. The predicted results showed a higher correlation when the model is trained with SVs and TEs than SNPs. NovGMDeep also has higher prediction accuracy when compared with conventional statistical models. We also included an extended study which describes sample size effects when the proposed model is trained on different number of samples for SVs. The results show better PCC values when the model was trained on more than 700 samples. This work sheds light on the unrecognized function of SVs and TEs in genotype-to-phenotype associations, as well as their extensive significance and value in crop development. Moreover, the predictions identified here using SVs and TEs will be useful to investigate the evolution and trait architecture of A. thaliana and O. sativa

Reuniting philosophy and science to advance cancer research

Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: (i) the role of mutations in cancer; (ii) the clonal evolution of cancer cells; (iii) the relationship between cancer and multicellularity; (iv) the tumour microenvironment; (v) the immune system; and (vi) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer

Holocentric plants of the genus Rhynchospora as a new model to study meiotic adaptations to chromosomal structural rearrangements

Climate change, world hunger and overpopulation are some of the biggest challenges the world is currently facing. Moreover, they are part of a multidimensional single scenario: as climate change continues to modify our planet, we might see a decrease of arable land and increase in extreme weather patterns, posing a threat to food security. This has a direct impact on regions with high population growth, where food security is already scarce. Considering additionally the unsustainability of intensive global food production and its contribution to greenhouse emissions and biodiversity loss, it´s clear that all these factors are interconnected (Cardinale et al., 2012; Prosekov & Ivanova, 2018; Wiebe et al., 2019). Plants are the main source of staple food in the world and are also the main actors in carbon fixation, they are therefore key protagonists in controlling climate change. Plants are also an essential habitat-defining element balancing our ecosystem. Thus, how we grow plants and crops will, aside from the obvious implications for food security, also have a profound impact on the climate and biodiversity. The natural variability of species is considered an immense pool of genes and traits, and their understanding is key to generate new useful knowledge. For instance, natural populations can be more tolerant to abiotic and biotic stresses, or carry traits that combined together in hybrids, might achieve a higher seed number, or a faster growth. Classical breeding has exploited unrelated varieties to achieve traits of interest like dwarfism and higher grain production. However, only a limited number of crop species have been the focus of recent scientific and technological approaches, and they do not represent the extremely vast natural diversity of species that could generate useful knowledge for future applications (Castle et al., 2006; Pingali, 2012). The key to this natural variability is a process called meiotic recombination, the exchange of genomic material between homologous parental chromosomes. Meiotic recombination takes place during meiosis, a specialized cell division in which sexually reproducing organisms reduce the genomic complement of their gametes by half in preparation for fertilization. Meiotic recombination takes place at the beginning of meiosis, in a stage called prophase I. To exchange DNA sequences, the strands of two homologous chromosomes must be fragmented. This specific process of physiologically induced DNA fragmentation is conserved in the vast majority of eukaryotes (Keeney et al., 1997). After the formation of double-strand breaks, the 3’ ends that are left are targeted by recombinases that help the strands search and invade templates for repair. After invasion, the 3’ end is extended by DNA synthesis, exposing sequences on the opposite strand that can anneal to the other 3’ end of the original double strand break. DNA synthesis at both ends generates a new structure called a double Holliday Junction (dHJ), forming a physical link between homologous chromosomes, named chiasma (Wyatt & West, 2014). The resolutions of these structures are called crossovers (COs), which is the molecular event representing the outcome of meiotic recombination. Other outcomes are possible, like noncrossovers (NCOs). In this case, the invading strand is ejected and anneals to the single-strand 3´end of the original double-strand break (Allers & Lichten, 2001). Crossovers can be divided into two main groups, called class I and class II. COs of the first group are considered to be sensitive to interference, which means that there are mechanisms that prevent two class I COs from happening in proximity of each other. Class II is insensitive to interference. Class I COs are the result of a pathway called ZMM, which involves a group of specialised proteins that are highly conserved among eukaryotes (Lambing et al., 2017; Mercier et al., 2015). Class I COs are the most common, studied and important type of COs. Centromeres are structures, located on regions of the chromosomes, that allow proper chromosome segregation during mitosis and meiosis. Centromeres have a profound effect on plant breeding and crop improvement, as it is known that meiotic recombination is suppressed at centromeres in most eukaryotes. This represents a great limitation for crop improvement, as many possibly useful traits might be in regions not subject to recombination and thus might not be available for breeding purposes. Additionally, the mechanisms behind how recombination is regulated and prevented from happening at centromeres are still unclear. In most model organisms centromeres are single entities localized on specific regions on the chromosomes. This configuration is called monocentric. However, another type of configuration can be found in nature, but is less studied. In fact, some organisms harbour multiple centromeric determinants distributed over their whole chromosomal length. This configuration is called holocentric. The Cyperaceae comprise a vast, diverse family of plants, with a cosmopolitan distribution in all habitats (Spalink et al., 2016). Despite the presence of this family worldwide, knowledge about it is limited. Few genomes are available and molecular insights are scarce. This family is also known to be mainly formed by holocentric species (Melters et al., 2012). Understanding if and how meiotic recombination is achieved in holocentric plants will generate new knowledge that in the future might unlock new traits in elite crops, previously unavailable to breeding, that could help humanity face global climatic, economic and social challenges. Recent studies have reported new knowledge about important meiotic, chromosome and genome adaptions found in species of the Cyperaceae family and in particular the genus Rhynchospora (Marques et al., 2015, 2016a). With the recent publication of the first reference genomes for several Rhynchospora species, we could already perform a comprehensive analysis of their unique genome features and trace the evolutionary history of their karyotypes and how these have been determined by chromosome fusions (Hofstatter et al., 2021, 2022). This new resource paves the way for future research utilising Rhynchospora as a model genus to study adaptations to holocentricity in plants. With this work, my intention is to shed light on the underexplored topic of holocentricity in plants. Using cutting edge techniques, I examine the conservation of meiotic recombination together with other species-specific adaptations like achiasmy and polyploidy in holocentrics. My results reveal new insights into how plant meiotic recombination is regulated when small centromere units are found distributed chromosome-wide, challenging the classic dogma of suppression of recombination at centromeres

Applied Photosynthesis

Using the energy from sunlight, photosynthesis usually converts carbon dioxide into organic compounds, which are important for all living creatures. Photosynthesis is one of the most important reactions on Earth, and it is a scientific field that is intrinsically interdisciplinary, and many research groups have considered photosynthesis. The aim of this book is to provide new progresses on applied aspects of photosynthesis, and different research groups collected their voluble results from study of this interesting process. All sections have been written by experts in their fields, and book chapters present different and new subjects on photosynthesis

Behavioural variation of acellular slime moulds

Protists are represented in every biome and have a diverse range of ecosystem roles.However, protists are severely under-represented in the scientific literature with fewstudies on how their diversity or behaviour affects ecosystem functioning. One group ofprotists, the acellular slime moulds, have been extensively studied for the behaviouralabilities of the model species Physarum polycephalum. Although the decision-making andproblem-solving abilities of P. polycephalum are well known, it is unclear whether thebehaviour of P. polycephalum is representative of acellular slime mould species andwhether their behaviour varies within individuals or strains. I investigated variations inacellular slime moulds at the species, strain and individual level and found a wide range ofvariability. Age affected two strains of P. polycephalum in a non-linear pattern and Iobserved age-related fluctuations in behaviour, physiology and cellular measures (Chapter1). I found a non-linear relationship between age and decision-making, as well as distinctdifferences in decision-making between strains (Chapter 2). I found variation in foragingbehaviour between three species of acellular slime moulds and each species also showed behavioural variation depending on the foraging environment as well as variations ininteractions between species (Chapter 3). I was able to observe facilitation betweenspecies where foraging success improved in the presence of other acellular slime mouldspecies (Chapter 3). The diversity of behaviour and physiology found within individualsand strains of P. polycephalum demonstrates the importance of including information onstrains and age in future behavioural investigations. In addition, variation in behaviourbetween species demonstrates the diversity of behaviour within this group of protists andhighlights the need for further research to understand how the behaviour of these protistsaffect species diversity and ecosystem functioning

Comparative genomics of recent adaptation in Candida pathogens

[eng] Fungal infections pose a serious health threat, affecting >1,000 million people and causing ~1.5 million deaths each year. The problem is growing due to insufficient diagnostic and therapeutic options, increased number of susceptible patients, expansion of pathogens partly linked to climate change and the rise of antifungal drug resistance. Among other fungal pathogens, Candida species are a major cause of severe hospital-acquired infections, with high mortality in immunocompromised patients. Various Candida pathogens constitute a public health issue, which require further efforts to develop new drugs, optimize currently available treatments and improve diagnostics. Given the high dynamism of Candida genomes, a promising strategy to improve current therapies and diagnostics is to understand the evolutionary mechanisms of adaptation to antifungal drugs and to the human host. Previous work using in vitro evolution, population genomics, selection inferences and Genome Wide Association Studies (GWAS) have partially clarified such recent adaptation, but various open questions remain. In the three research articles that conform this PhD thesis we addressed some of these gaps from the perspective of comparative genomics. First, we addressed methodological issues regarding the analysis of Candida genomes. Studying recent adaptation in these pathogens requires adequate bioinformatic tools for variant calling, filtering and functional annotation. Among other reasons, current methods are suboptimal due to limited accuracy to identify structural variants from short read sequencing data. In addition, there is a need for easy-to-use, reproducible variant calling pipelines. To address these gaps we developed the “personalized Structural Variation detection” pipeline (perSVade), a framework to call, filter and annotate several variant types, including structural variants, directly from reads. PerSVade enables accurate identification of structural variants in any species of interest, such as Candida pathogens. In addition, our tool automatically predicts the structural variant calling accuracy on simulated genomes, which informs about the reliability of the calling process. Furthermore, perSVade can be used to analyze single nucleotide polymorphisms and copy number-variants, so that it facilitates multi-variant, reproducible genomic studies. This tool will likely boost variant analyses in Candida pathogens and beyond. Second, we addressed open questions about recent adaptation in Candida, using perSVade for variant identification. On the one hand, we investigated the evolutionary mechanisms of drug resistance in Candida glabrata. For this, we used a large-scale in vitro evolution experiment to study adaptation to two commonly-used antifungals: fluconazole and anidulafungin. Our results show rapid adaptation to one or both drugs, with moderate fitness costs and through few mutations in a narrow set of genes. In addition, we characterize a novel role of ERG3 mutations in cross-resistance towards fluconazole in anidulafungin-adapted strains. These findings illuminate the mutational paths leading to drug resistance and cross-resistance in Candida pathogens. On the other hand, we reanalyzed ~2,000 public genomes and phenotypes to understand the signs of recent selection and drug resistance in six major Candida species: C. auris, C. glabrata, C. albicans, C. tropicalis, C. parapsilosis and C. orthopsilosis. We found hundreds of genes under recent selection, suggesting that clinical adaptation is diverse and complex. These involve species-specific but also convergently affected processes, such as cell adhesion, which could underlie conserved adaptive mechanisms. In addition, using GWAS we predicted known drivers of antifungal resistance alongside potentially novel players. Furthermore, our analyses reveal an important role of generally-overlooked structural variants, and suggest an unexpected involvement of (para)sexual recombination in the spread of resistance. Taken together, our findings provide novel insights on how Candida pathogens adapt to human-related environments and suggest candidate genes that deserve future attention. In summary, the results of this thesis improve our knowledge about the mechanisms of recent adaptation in Candida pathogens, which may enable improved therapeutic and diagnostic applications.[cat] Les infeccions fúngiques representen una greu amenaça per a la salut, afectant a més de 1.000 milions de persones i causant aproximadament 1,5 milions de morts cada any. El problema està augmentant a causa d’unes opcions terapèutiques i diagnòstiques insuficients, l'increment del nombre de pacients susceptibles, l'expansió dels patògens parcialment vinculada al canvi climàtic i l'augment de la resistència als fàrmacs antifúngics. D’entre diversos fongs patògens, els llevats del gènere Candida són una causa important d'infeccions nosocomials, amb una alta mortalitat en pacients immunodeprimits. Diverses espècies de Candida constitueixen un problema de salut pública, cosa que requereix més esforços per a desenvolupar nous medicaments, optimitzar els tractaments disponibles i millorar els diagnòstics. Tenint en compte el dinamisme genòmic d’aquests patògens, una estratègia prometedora per millorar les teràpies i diagnòstics actuals és comprendre els mecanismes evolutius d'adaptació als fàrmacs antifúngics i a l’hoste humà. Treballs anteriors utilitzant l'evolució in vitro, la genòmica de poblacions, les inferències de selecció i els estudis d'associació de genoma complet (GWAS, per les sigles en anglès) han aclarit parcialment aquesta adaptació recent, però encara hi ha diverses preguntes obertes. En els tres articles que conformen aquesta tesi doctoral, hem abordat algunes d'aquestes preguntes des de la perspectiva de la genòmica comparativa. En primer lloc, hem abordat qüestions metodològiques relatives a l'anàlisi dels genomes de les espècies Candida. L'estudi de l'adaptació recent en aquests patògens requereix eines bioinformàtiques adequades per a la detecció, filtratge i anotació funcional de variants genètiques. Entre altres raons, els mètodes actuals són subòptims a causa de la limitada precisió per identificar variants estructurals a partir de dades de seqüenciació amb lectures curtes. A més, hi ha una necessitat d’eines computacionals per a la detecció de variants que siguin senzilles d'utilitzar i reproduibles. Per abordar aquestes mancances, hem desenvolupat el mètode bioinformàtic "personalized Structural Variation detection" (perSVade), una eina que permet la detecció, filtratge i anotació de diversos tipus de variants, incloent-hi les variants estructurals, directament des de les lectures. PerSVade permet la identificació precisa de les variants estructurals en qualsevol espècie d'interès, com ara els patògens Candida. A més, la nostra eina prediu automàticament la precisió de la detecció d’aquestes variants en genomes simulats, la qual cosa informa sobre la fiabilitat del procés. Finalment, perSVade es pot utilitzar per analitzar altres tipus de variants, com els polimorfismes de nucleòtid únic o els canvis en el nombre de còpies, facilitant així estudis genòmics integrals i reproduibles. Aquesta eina probablement impulsarà les anàlisis genòmiques en els patògens Candida i també en altres espècies. En segon lloc, hem abordat algunes de les preguntes obertes sobre l'adaptació recent en els llevats Candida, utilitzant perSVade per a la identificació de variants. D'una banda, hem investigat els mecanismes evolutius de resistència als fàrmacs antifúngics en Candida glabrata. Per a això, hem utilitzat un experiment d'evolució in vitro a gran escala per estudiar l'adaptació a dos antifúngics comuns: el fluconazol i l’anidulafungina. Els nostres resultats mostren una adaptació ràpida a un o ambdós fàrmacs, amb un cost per al creixement moderat i a través de poques mutacions en un nombre reduït de gens. A més, hem caracteritzat un paper nou de les mutacions en ERG3 en la resistència creuada al fluconazol en soques adaptades a anidulafungina. Aquests descobriments aclareixen els processos mutacionals que condueixen a la resistència als fàrmacs i a la resistència creuada en els patògens Candida. D'altra banda, hem re-analitzat aproximadament 2.000 genomes i fenotips disponibles en repositoris públics per a comprendre els senyals genòmics de selecció recent i de resistència a fàrmacs antifúngics, en sis espècies rellevants de Candida: C. auris, C. glabrata, C. albicans, C. tropicalis, C. parapsilosis i C. orthopsilosis. Hem trobat centenars de gens sota selecció recent, suggerint que l'adaptació clínica és diversa i complexa. Aquests gens estan relacionats amb funcions específiques de cada espècie, però també trobem processos alterats de manera similar en diferents patògens, com per exemple l’adhesió cel·lular, cosa que indica fenòmens d’adaptació conservats. A part, utilitzant GWAS hem predit mecanismes esperats de resistència a antifúngics i també possibles nous factors. A més, les nostres anàlisis revelen un paper important de les variants estructurals, generalment poc estudiades, i suggereixen una implicació inesperada de la recombinació (para)sexual en la propagació de la resistència. En conjunt, els nostres descobriments proporcionen noves perspectives sobre com els patògens Candida s'adapten als entorns humans, i suggereixen gens candidats que mereixen investigacions futures. En resum, els resultats d’aquesta tesi milloren el nostre coneixement sobre els mecanismes d'adaptació recent en els patògens Candida, cosa que pot permetre el disseny de noves teràpies i diagnòstics