[ES] El tiempo de floración es uno de los caracteres más importantes que influyen en la productividad y el rendimiento de los cultivos. La identificación de compuestos sintéticos que sean bioactivos en el control de la inducción floral es de gran interés. Su identificación podría permitirnos ajustar el tiempo de floración en los cultivos, adaptándolos a las condiciones ambientales más favorables. Para identificar estos compuestos, hemos tomado dos enfoques diferentes: un cribado genético químico y la caracterización del metaboloma de transición floral. En primer lugar, realizamos un rastreo de genética química para identificar moléculas pequeñas que tengan el potencial de controlar la expresión del florígeno, FLOWERING LOCUS T (FT) o la actividad o señalización de FT en Arabidopsis. Para ello, hemos utilizado plantas transgénicas que expresan el gen ß-GLUCURONIDASE (GUS) bajo el control del promotor FT para probar una librería de 360 moléculas preseleccionadas. Los resultados positivos obtenidos se volvieron a analizar mediante un cribado secundario basado en la expresión del gen reportero LUCIFERASE (LUC) bajo el control del promotor FT. Utilizando este enfoque, hemos identificado una molécula que induce con éxito la floración en condiciones de cultivo in vitro. En segundo lugar, hemos caracterizado la función del ácido pipecólico (Pip), una molécula previamente identificada como candidata a regular la floración. Hemos confirmado que las mutaciones en las enzimas responsables de la biosíntesis de Pip muestran una alteración en la respuesta del tiempo de floración. Además, hemos identificado un nuevo papel del Pip relacionado con el crecimiento y el tamaño de la roseta de Arabidopsis. Finalmente, utilizamos un sistema inducible basado en el promotor de CONSTANS (CO) que controla la expresión del gen endógeno de CO fusionado con el receptor de glucocorticoides de rata (CO::GR). De manera que con un solo tratamiento con dexametasona podemos inducir la floración. Con este sistema, realizamos un estudio del metaboloma de muestras de ápices y hojas mediante técnicas de metabolómica dirigida, lipidómica, cuantificación hormonal y transcriptómica. La integración de estos conjuntos de datos ómicos nos ha permitido identificar rutas metabólicas que se encuentran alteradas durante la transición floral. A su vez, la caracterización de mutantes de pérdida de función que codifican enzimas clave de esas vías metabólicas, reveló que algunos de estos mutantes mostraban un fenotipo afectado para el tiempo de floración. Entre ellos, nos enfocamos en la caracterización de los genes relacionados con el metabolismo de la rafinosa, un oligosacárido de reserva. Mutantes afectados en el gen RAFFINOSE SYNTHASE 5 (RS5) presentan un fenotipo de floración temprana y fertilidad reducida. En base a los resultados obtenidos, proponemos un modelo en el que, durante la transición floral, se produce una reestructuración de las ratios entre carbohidratos sencillos (monosacáridos y disacáridos) y de reserva, como la rafinosa. Estos cambios podrían ser modulados por el ácido abscísico (ABA) y por genes relacionados con la floración, desencadenando cambios en el metabolismo de la trehalosa y promoviendo una expresión temprana de FT. / [CA] El temps de floració és un dels caràcters amb més influència en la productivitat i el rendiment dels cultius. La identificació de compostos sintètics bioactius per al control de la inducció floral és de gran interés, ja que la seua identificació podria permetre ajustar el temps de floració dels cultius, aspecte que podria contribuir a l'adaptació a condicions ambientals més favorables. Per a identificar aquests compostos, hem portat a terme dues aproximacions diferents: un garbellat genètic químic i la caracterització del metaboloma de la transició floral. En primer lloc, hem realitzat un cribratge genètic-químicper a identificar xicotetes molècules amb potencial per a controlar l'expressió del florígen, FLOWERING LOCUS T (FT) o l'activitat o la senyalització de FT a Arabidopsis. Per a portar a terme aquest cribratge, hem utilitzat plantes transgèniques que expressen el gen ß-GLUCURONIDASE (GUS) sota el control del promotor de FT amb les quals hem assajat una llibreria de 360 molècules preseleccionades de manera prèvia. Els resultats positius obtinguts en aquest cribratge t s'han sotmés a un cribratge secundari basat en l'expressió del gen reporter LUCIFERASE (LUC) sota el control del promotor FT. La utilització d'aquesta primera aproximació ha permés la idenfiticació d'una molècula que indueix amb èxit la floració en condicions de cultiu in vitro. En En segon lloc, hem caracteritzat la funció de l'àcid pipecòlic (Pip), una molècula prèviament identificada com a candidata a regular la floració. Aquesta aproximació ens ha permet confirmar que mutacions als enzims responsables de la biosíntesi de Pip comporten una alteració al temps de floració. A més, en aquest treball hem identificat un nou paper del Pip relacionat amb el creixement i la grandària de la roseta d'Arabidopsis. Finalment, hem utilitzat un sistema induïble basat en el promotor de CONSTANS (CO) que controla l'expressió del gen endogen de CO fusionat al receptor de glucocorticoides de rata (CO::GR). Aquesta construcció ens proporciona una ferramenta amb la qual induir la floració amb un sol tractament amb dexametasona. A continuació, hem realitzat un estudi del metaboloma de mostres d'àpexs i fulles mitjançant tècniques de metabolòmica dirigida, lipidómica, quantificació hormonal i transcriptòmica. La integració d'aquest conjunt de dades ómiques ens ha permés identificar les rutes metabòliques que es troben alterades durant la transició floral. Al mateix temps, la caracterització de mutants de pèrdua de funció que codifiquen enzims clau per a aquestes rutes metabòliques, ha revelat que alguns d'aquests mutants mostren un fenotip afectat pel que fa al temps de floració. Dintre dels mutants analitzats, ens hem centrat en la caracterització dels gens relacionats amb el metabolisme de la rafinosa, un oligosacàrid de reserva. Els mutants del gen RAFFINOSE SYNTHASE 5 (RS5) presenten un fenotip de floració primerenca i fertilitat reduïda. Sobre la base dels resultats obtinguts, proposem un model en el qual, durant la transició floral, es produeix una reestructuració de les ràtios entre carbohidrats senzills (monosacàrids i disacàrids) i de reserva, com la rafinosa. Aquests canvis podrien ser modulats per l'àcid abscísic (ABA) i per gens relacionats amb la floració, i desencadenariencanvis al metabolisme de la trehalosa, així com la generació de l'expressió primerenca de FT. / [EN] Flowering time is one of the most important traits affecting crop productivity and yield. The identification of natural or synthetic bioactive compounds for the control of flowering induction is of great interest. The identification of compounds with the potential to regulate flowering could allow us to fine-tune flowering responses in crops and adapt them to the changing environmental conditions. To identify these compounds, we have taken two different approaches: a chemical genetic screening and the characterization of the metabolome of floral transition. First, we performed a chemical genetic screening to identify small molecules that have the potential to control the expression of the florigen FLOWERING LOCUS T (FT) or FT activity or signaling in Arabidopsis. We used transgenic plants expressing the ß-GLUCURONIDASE gene (GUS) under the control of the FT promoter to test a preselected library of 360 molecules. Positive hits were retested by a secondary screening based on the expression of the LUCIFERASE (LUC) reporter gene under the control of the FT promoter. Using this approach, we have identified one molecule that successfully induces flowering under in vitro culture conditions. Secondly, we have characterized the function of pipecolic acid (Pip), a molecule previously identified as a candidate to regulate flowering time. We have confirmed that mutations in enzymes responsible for Pip biosynthesis display an altered flowering response. A new role for Pip in rosette growth is also revealed in this work. Finally, we used an inducible system based on the promoter of CONSTANS (CO) driving the expression of CO fused to the rat glucocorticoid receptor (CO::GR). Such a construction provides a tool to induce flowering with a single dexamethasone treatment. We then performed a comprehensive metabolomic study of the shoot apex and leaf samples that included targeted metabolomics, lipidomics, hormone quantification, and transcriptomics. Integration of these omic datasets has allowed us to point out metabolic pathways that are altered during floral induction. Characterization of loss-of-function mutants coding key enzymes of those metabolic pathways revealed that some of these mutants showed a flowering time phenotype. Among them, we focused on the characterization of the contribution of the raffinose metabolism, a storage oligosaccharide, to the determination of flowering time. Mutants affecting RAFFINOSE SYNTHASE 5 (RS5) exhibit an early flowering phenotype and reduced fertility. We propose a model in which the balance between simple and storage carbohydrates in the apex changes during floral induction. This change could be modulated by ABA and flowering-related genes, and it triggers changes in trehalose metabolism, promoting flowering by an early FT upregulation. / Praena Tamayo, J. (2022). Identification of Bioactive Molecules in the Control of Flowering Time [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185177 / TESIS

The integrity of wetland ecosystems is largely determined by hydrological functionality, degree of connectivity to like ecosystems, and permeability to external influence. Land use changes in upland areas adjacent to wetland ecosystems may influence hydrology and connectivity while introducing novel biotic and abiotic materials. There is an increasing trend toward the use of remote assessment techniques to determine the degree of impact of external influences on adjacent wetlands. Remote assessment and predictive capabilities are provided by indices such as the Landscape Development Intensity Index (LDI) (Brown and Vivas 2005) which may be beneficial in determining site condition, and which have the added benefit of providing a quantitative gradient of human impact. This study assessed the predictive ability of the LDI in cypress ecosystems, by testing its correlations with plant community metrics including an index of floral quality calculated using coefficients of conservatism (CC)(Cohen et al. 2004), plant species diversity, and fluctuation in community composition assessed by changes in the wetland status and native status of component plant species. LDI was also compared against an independent measure of disturbance which was used to construct an a priori disturbance gradient. Overall, diversity measures showed little correlation with any of the disturbance indices, while CC scores were significantly correlated. Models were constructed in an attempt to explain each of the variables of plant community response to development in the surrounding landscape. The length of time since the development of the land adjacent to the cypress domes was a predictor of plant community response only when included in models with other variables. LDI was the strongest predictor in all models except where increases in land use associated with hydrological changes helped predict or better predicted proportions of exotic and upland species.

Current agroecosystem management practices have a negative effect on natural enemies and their ability to control insect pests. Conservation biological control through the addition of flowering resources can manage food resources for natural enemies. These floral resources can also provide multiple ecosystem services. Study goals were to determine if perennial Thymus vulgaris L. was attractive to natural enemies and if so, could it be a dual use resource encouraging pest management and providing harvestable product. In 2018 plots in three locations were used to examine the effect of habitat throughout the growing season on the attractiveness of T. vulgaris. Large numbers of Thysanoptera and Hemiptera were collected in all locations, represented by phytophagous Aphididae and Thripidae, and predatory Anthocoridae. Location influenced other families to varying degrees. Seasonal specimen counts were influenced by vegetation density, floral phenology, and predator/prey relationships. In 2019 replicated plots of three treatments were used to examine if harvesting plant material affected the attractiveness of T. vulgaris to natural enemies. Total specimens in 2019 were not significantly different among treatments, indicating removal of blooms did not significantly affect the attractiveness of T. vulgaris. Significant numbers of Thysanoptera and Hemiptera were again collected in all treatments, represented by phytophagous Aphididae and Thripidae. Greater numbers of Diptera and Hymenoptera were also collected. Significant numbers of Thripidae, Aphididae, Mymaridae, and Platygastridae were found in the Family level analyses. Results from both years indicate T. vulgaris was attractive to natural enemy and phytophagous Families. Data from 2018 suggest natural enemy families were attracted to alternative prey and hosts utilizing the foliage rather than flowers but the use of nectar and pollen cannot be ruled out. Data from 2019 suggest the presence of flowers played an important role in the attractiveness of T. vulgaris to micro-hymenopteran parasitoids, Syrphidae, and native Apidae. In conclusion, Thymus vulgaris has the potential to be a dual use floral resource that benefits growers through supporting native enemy populations and pollination services, as well as provide income from the harvest of foliage. It could also be used as a beneficial, harvestable floral resource in urban gardens to encourage pollinator conservation and natural pest control.

While pollinators and leaf herbivores have been a focus of research for decades, floral antagonists have been studied significantly less. Since floral antagonists can be as common as leaf herbivores and have strong impacts on plant reproduction, it is important to understand the role of floral antagonists in the ecology and evolution of flowers. I conducted four experiments to better understand the relationship between plants, floral traits, floral antagonists, and other plant-insect interactions. First, I manipulated resources (light and soil nutrients) that are known to have impacts on plants and floral traits to test how they affect floral antagonists and other plant-insect interactions. Plentiful resources increased the proportion of floral antagonists to visit flowers, but also increase tolerance of floral antagonists. Second, I manipulated flower bud gallers, a species-specific floral herbivore that destroys flowers, to test how it affected other plant-insect interactions, floral traits, and plant reproduction. Plants with flower bud gallers tended to have more pollinator visits, but this effect is due to a shared preference by gallers and pollinators for similar plants. Third, I manipulated florivory to examine how it affects subsequent plant-arthropod interactions, floral traits, and plant reproduction. Florivory had systemic effects on other plant-insect interactions, including leaf herbivores, and shifted the plant mating system towards more selfing. Additionally, I tested how several floral antagonists respond to floral attractive and defense traits to understand which floral traits are important in mediating antagonisms. Finally, I manipulated florivory, pollination, and nectar robbing to test for effects of multiple floral interactions on subsequent plant-insect interactions, floral traits, and plant reproduction. There were significant many-way interactions between the three treatments on subsequent plant-insect interactions and reproduction, indicating that the effect of one interaction depends on what other interactions are present. Understanding the role that floral antagonists play in plant ecology can help scientists determine which interactions are most important, and may help determine why some floral traits exist in their current state. Together, this work represents some of the most comprehensive research on the community consequences of floral antagonists, as well as the interplay between floral traits and floral interactions.

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<p> Many floral characters have can affect plant fitness by altering the frequency or efficiency of pollinator visits. More frequent pollinator visitation may lead to larger pollen loads and greater outcrossing rates, which in tern may lead to greater numbers of offspring, with higher genetic variability and less inbreeding depression. Mating systems may moderate the strength of natural selection on traits that attract pollinators through its effects on inbreeding depression, and the ability to self-pollinate autogamously. Natural selection is expected to be stronger in largely outcrossing systems where inbreeding depression is high, but reduced in largely selfing systems where deleterious alleles have been purged.</p> <p> Here I investigate the role of floral display height in attracting pollinators, and its consequences for plant fitness in two closely related monocarpic herbs (Hesperis matronalis and Alliaria petiolata) with different mating systems. Though others have looked at height as a potential trait attracting pollinators, no one has yet clearly separated the effects of floral display height from that of plant size. I experimentally altered plant height independently of plant size by placing potted plants into either elevated or non-elevated treatments and observed pollinator foraging behaviour and reproduction.</p> <p> Pollinators preferred taller floral displays in the predominant outcrosser Hesperis matronalis. Elevated plants produced more seeds than control plants although their seeds were smaller, reflecting a strong seed size-number trade-off. Path analysis showed seed number was affected directly by plant height, presumably though greater pollinator visitation, and indirectly by plant mass through increased fruit number.</p> <p> In Alliaria petiolata, a predominant self-pollinating species, pollinators visited taller displays in field observations and experimentally elevated plants had significantly greater seed per fruit. Experimentally outcrossed and self-pollinated plants showed no difference in seed production, but offspring quality was significantly higher in outcrossed plants for early traits. Measures of maternal fitness increased significantly with both maternal and seed mass. For individual seeds, seed mass was the most important factor in seed fitness, where larger seeds germinated sooner, more frequently, and had higher survivorship than smaller seeds, regardless of pollination treatment. Competition strongly affected all seedling traits, with increased density having a significant negative effect on seedling mass, total leaf area, and maximum leaf length; and, a positive effect on hypocotyl length and petiole length. Seedling mass at harvest was not significantly affected by elevation treatment, seed mass, or maternal mass, however, this may have been due to lower competition brought about by low germination and survival; thus, competition did not accentuate the effect of maternal size or elevation treatment on seedling. Taken together, the results of these two studies suggest the importance of height, in attracting pollinators, independent of size in both species. The consequences of pollinator attraction were highly significant in the predominant outcrosser Hesperis matronalis, while maternal size and seed size played more important roles in the fitness of the predominant selfer Alliaria petiolata.</p> / Thesis / Master of Science (MSc)