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Ptilotus nobilis is a short-lived perennial wildflower, native to semi-arid and arid areas of Australia. Propagation by vegetative means is constrained by the early onset and a continuous flowering habit. Despite being defined as the main barrier for vegetative propagation no published research on floral induction and initiation was found. The aim of this study is to provide the first insights into floral evocation in P. nobilis, with the general objectives being to investigate floral induction and initiation, find the means to maintain plants in the vegetative phase, enhance leaf initiation and to gain knowledge of growth and development with an emphasis on light intensity, temperature and daylength. Early experiments aimed to increase the understanding of growth and development. The main cardinal events were identified including the onset of branching (axillary stem growth), visible bud stage, first floret opening and maturity of the inflorescence. To aid future cultivation schedules, four growing degree days (GDD) and one chronological model, using days after sowing (DAS), were established and validated. All GDD models were accurate in predicting first floret opening and maturity, but not in predicting visible bud stage. Best prediction was achieved by using an upper temperature threshold of 18.2 ºC and an own base temperature of 5.0 ºC. Days after sowing were an accurate means of prediction, indicating that temperature and other variables such as light intensity regulate development. A series of defoliation treatments investigated the maintenance of P. nobilis plants in the vegetative phase. Within all treatments, reproductive structures were observed and all plants with more than two true leaves entered the reproductive phase. Plants with less than two true leaves showed a delayed floral bud appearance by up to 20 days. Floral development was affected by most defoliation treatments resulting in vegetative growth within the inflorescences. It is hypothesized that plants have a very short juvenile phase and that a constant floral stimulus may be needed for floral evocation. Using scanning electron and light microscopy a template for the transition from vegetative to reproductive phase was developed. Bract initiation was accompanied by a significant increase in meristem area and diameter, and was defined as the onset of flowering. The established template was used in a subsequent glasshouse trial, which revealed that floral initiation occurred very early and at 25 DAS all plants had entered the reproductive phase. Branching and leaf area expansion were identified as post- initiation processes. Plants were exposed to different light intensities (229.3, 398.6 and 909.3 µmol m-2 s-1) in a glasshouse. Under low light, final leaf number increased by up to three leaves, indicating that the vegetative phase was prolonged. Cardinal events were delayed but all plants reached maturity. It was concluded that a light intensity of 229.3 µmol m-2 s-1 was not low enough to prevent floral initiation and that a further decrease of light intensity in combination with temperature could be more effective. Interactions of light intensity, temperature and daylength were investigated. Plants were grown under high light and low light (< 1.2 MJ m-2) intensities, 25/10 ºC and 35/20 ºC and daylengths of 11 h and 16 h. During the trial period (42 days), low light intensity suppressed floral initiation and high temperatures maintained more plants (70%) in the vegetative phase. However daylength treatments had no effect on the time of bract initiation or the percentage of vegetative plants. After 83 days floral buds and axillary stems were observed on some plants under low light intensity, indicating the onset of the reproductive phase and showing that P. nobilis could not be maintained in the vegetative phase indefinitely. Plants under high light were harvested at maturity and effects of temperature and daylength were analysed. Final leaf number increased under 35/20 ºC supporting the previously established results that floral initiation was delayed and leaf production enhanced by high temperature. Morphological data was collected to classify the photoperiodic response of P. nobilis. Plants under 25/10 ºC and 11 h had the longest inflorescences and greatest number of buds and flowers, which was also reflected in the buds and flowers dry weight, with an increase of up to 3.4 fold under these conditions. The difficulty of classifying some plants accordingly to their photoperiodic response and the proposal that P. nobilis may be a facultative short day plant under 25/10 ºC is discussed. In summary, this study presents the first evidence that P. nobilis has a very short juvenile phase and that growth and development are mainly driven by light intensity and temperature. It was possible to enhance vegetative growth by defoliation, low light intensity and high temperatures, however this did not fully prevent flowering, which indicates that P. nobilis has a very strong flowering response or signal.

Ptilotus nobilis is a short-lived perennial wildflower, native to semi-arid and arid areas of Australia. Propagation by vegetative means is constrained by the early onset and a continuous flowering habit. Despite being defined as the main barrier for vegetative propagation no published research on floral induction and initiation was found. The aim of this study is to provide the first insights into floral evocation in P. nobilis, with the general objectives being to investigate floral induction and initiation, find the means to maintain plants in the vegetative phase, enhance leaf initiation and to gain knowledge of growth and development with an emphasis on light intensity, temperature and daylength. Early experiments aimed to increase the understanding of growth and development. The main cardinal events were identified including the onset of branching (axillary stem growth), visible bud stage, first floret opening and maturity of the inflorescence. To aid future cultivation schedules, four growing degree days (GDD) and one chronological model, using days after sowing (DAS), were established and validated. All GDD models were accurate in predicting first floret opening and maturity, but not in predicting visible bud stage. Best prediction was achieved by using an upper temperature threshold of 18.2 ºC and an own base temperature of 5.0 ºC. Days after sowing were an accurate means of prediction, indicating that temperature and other variables such as light intensity regulate development. A series of defoliation treatments investigated the maintenance of P. nobilis plants in the vegetative phase. Within all treatments, reproductive structures were observed and all plants with more than two true leaves entered the reproductive phase. Plants with less than two true leaves showed a delayed floral bud appearance by up to 20 days. Floral development was affected by most defoliation treatments resulting in vegetative growth within the inflorescences. It is hypothesized that plants have a very short juvenile phase and that a constant floral stimulus may be needed for floral evocation. Using scanning electron and light microscopy a template for the transition from vegetative to reproductive phase was developed. Bract initiation was accompanied by a significant increase in meristem area and diameter, and was defined as the onset of flowering. The established template was used in a subsequent glasshouse trial, which revealed that floral initiation occurred very early and at 25 DAS all plants had entered the reproductive phase. Branching and leaf area expansion were identified as post- initiation processes. Plants were exposed to different light intensities (229.3, 398.6 and 909.3 µmol m-2 s-1) in a glasshouse. Under low light, final leaf number increased by up to three leaves, indicating that the vegetative phase was prolonged. Cardinal events were delayed but all plants reached maturity. It was concluded that a light intensity of 229.3 µmol m-2 s-1 was not low enough to prevent floral initiation and that a further decrease of light intensity in combination with temperature could be more effective. Interactions of light intensity, temperature and daylength were investigated. Plants were grown under high light and low light (< 1.2 MJ m-2) intensities, 25/10 ºC and 35/20 ºC and daylengths of 11 h and 16 h. During the trial period (42 days), low light intensity suppressed floral initiation and high temperatures maintained more plants (70%) in the vegetative phase. However daylength treatments had no effect on the time of bract initiation or the percentage of vegetative plants. After 83 days floral buds and axillary stems were observed on some plants under low light intensity, indicating the onset of the reproductive phase and showing that P. nobilis could not be maintained in the vegetative phase indefinitely. Plants under high light were harvested at maturity and effects of temperature and daylength were analysed. Final leaf number increased under 35/20 ºC supporting the previously established results that floral initiation was delayed and leaf production enhanced by high temperature. Morphological data was collected to classify the photoperiodic response of P. nobilis. Plants under 25/10 ºC and 11 h had the longest inflorescences and greatest number of buds and flowers, which was also reflected in the buds and flowers dry weight, with an increase of up to 3.4 fold under these conditions. The difficulty of classifying some plants accordingly to their photoperiodic response and the proposal that P. nobilis may be a facultative short day plant under 25/10 ºC is discussed. In summary, this study presents the first evidence that P. nobilis has a very short juvenile phase and that growth and development are mainly driven by light intensity and temperature. It was possible to enhance vegetative growth by defoliation, low light intensity and high temperatures, however this did not fully prevent flowering, which indicates that P. nobilis has a very strong flowering response or signal.

[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