In this diploma thesis the impacts of both nitrogen fertilization and drought on the content of proteins of the gliadin fraction in case of a winter wheat variety Tiguan were observed. Selected samples were cultivated at two locations in a total duration of two years. Two different conditions were selected: one without the nitrogen fertilization (0 kg N/ ha) and the other with the nitrogen fertilization in 140 kg N/ ha concentration. Second observed factor was the drought which was ensured by roofs above the crops and their cultivation in common climatic environment. Gliadin fraction was separated by the A-PAGE method and the protein quantification carried out by a computer densitometry. Such nitrogen fertilization caused a significant increase of gliadins, especially -1,2 and -5 fractions. Stress induced by the drought caused an increase of gliadin content compared to the control sample, particularly in case of - and - fractions. The most important factor influencing the gliadin content in grains was therefore the nitrogen fertilization in a dry environment. The observed increase of gliadin content was lower in case of the sample which was fertilized in a humid environment than in case of the sample which was fertilized in the dry one.

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Water availability and arable lands are increasingly limiting resources in many parts of the U.S., particularly in semi-arid and arid regions. As a means of addressing food and fuel demands associated with burgeoning population growth, highly productive and water-use efficient crops need to be identified. One potential crop, Agave, merits consideration and evaluation due to its putative capability to provide sustenance and energy despite growing in water-limited regions and on marginal soils. However, little is known regarding the productivity these succulent plants will have under growing conditions of the Southwest, where high concentrated saline soils are abundant, and water is often limited. The objectives of these studies were to determine the effects of high levels of salinity and different volumetric water content levels (VWC) on plant growth, biomass accumulation, and nutrient uptake. I used a hydroponic study to compare the effects of four salinity treatments (0.5, 3, 6, and 9 dS m-1) on productivity of four Agave species (Agave parryi, Agave utahensis ssp. kaibabensis, Agave utahensis ssp. utahensis, and Agave weberi). In a second study, an automated irrigation system was established to examine four pre-determined VWC threshold set-points and simulated a gradient of well-watered to drought conditions, to evaluate how A. weberi would respond to varying levels of water availability. Salinity concentrations did not significantly affect root and plant dry weight accumulation in A. weberi, but all other agave plants experienced less biomass accumulation under high saline conditions (>6 dS m-1). Seedlings of A. utahensis were two times more likely to die in the two highest saline treatments (6 and 9 dS m-1) than the two lower treatments (0.5 dS m-1 and 3 dS m-1). Calcium, Mg, S, Mn levels decreased in both A. parryi and A. weberi at higher salinity levels. Agave weberi was able to tolerate salinity, but it also experienced lower biomass production ≤3 dS m-1. In the water-stress study, Agave weberi plants experienced a decrease of 2.11 g as compared to plants in the highest treatment. Plants in the intermediate VWC treatments had similar dry mass values as those in the highest treatment, which suggests that this species could have moderately high yields under limited water conditions, and consequently should be evaluated as a potential bioenergy crop for semi-arid regions, such as the U.S. Southwest. Agave shows considerable potential to be grown in arid and semi-arid regions that are moderately high in salinity and have limited water availability. Indeed, the cultivation of Agave as a crop appears to be a viable option for many areas of the Southwest. While some of the Agave species evaluated were quite productive under moderate salt and water stress, it is uncertain if growth will be significantly reduced if under these stress conditions for periods longer than 3 months.

This master’s thesis project investigates the utilization of a low-cost hyperspectral (HS) imaging rig to identify and classify drought stress in pine plants. Drought stress is a widespread environmental challenge affecting global forestry, requiring more resources as the industry grows and global warming rises. This provokes a need for affordable, and efficient monitoring methods. HS imaging, with its ability to capture a wide range of spectral information, offers promising methods for quick and precise measurements of plant stress. The project methodology is comprised of redesigning an existing HS imaging rig, with the camera employing push-broom technology, to yield precise and consistent HS images. This involved exploring the camera’s spectral range, designing components to ensure consistent artificial lighting using blackbody radiation sources, and calibrating the HS camera for focal depth and aberrations like smile and keystone. Two experiments were conducted to obtain the data for pine stress detection, first for two binary categories: Control, and 100% Drought, and later introducing a third semi-drought category in the second experiment. The data analysis encompassed preprocessing the HS images to correct the lighting intensity distributions and normalization of pixel values. Accompanied by filtering, resampling spectral data, and feature extraction facilitating consistent drought identification, and data management. To identify stress patterns in pine plants and temporal decay rates, methods such as spectral reflectance analysis, various vegetation indices (VI), and statistical learning techniques like discriminant analysis and logistic regression were evaluated for distinguishing between stressed and healthy plants. The results demonstrate the accuracy of the HS imaging rig in measuring spectral reflectances from plants, capturing changes between 550 − 670 nm in the visible spectrum and 750 − 890 nm in the near-infrared (NIR) spectrum due to increasing stress affecting chlorophyll levels. Both well-established VIs and empirically designed indices indicate reliable early detection. Comparing multiple VIs to statistical learning models shows similar performances in binary classification tasks. Feature selection methods using correlation matrices, and L1 penalty for logistic regression support stress effects visible in the data, paving the way for cost-effective strategies in sustainable forestry management.

Gene expression regulation is dynamic and specific to various factors such as developmental stages, environmental conditions, and stimulation of pathogens. Nowadays, a tremendous amount of transcriptome data sets are available from diverse species. This trend enables us to perform comparative transcriptome analysis that identifies conserved or diverged gene expression responses across species using transcriptome data. The goal of this dissertation is to develop and apply approaches of comparative transcriptomics to transfer knowledge from model species to non-model species with the hope that such an approach can contribute to the improvement of crop yield and human health. First, we presented a comprehensive method to identify cross-species modules between two plant species. We adapted the unsupervised network-based module finding method to identify conserved patterns of co-expression and functional conservation between Arabidopsis, a model species, and soybean, a crop species. Second, we compared drought-responsive genes across Arabidopsis, soybean, rice, corn, and Populus in order to explore the genomic characteristics that are conserved under drought stress across species. We identified hundreds of common gene families and conserved regulatory motifs between monocots and dicots. We also presented a BLS-based clustering method which takes into account evolutionary relationships among species to identify conserved co-expression genes. Last, we analyzed single-cell RNA-seq data from monocytes to attempt to understand regulatory mechanism of innate immune system under low-grade inflammation. We identified novel subpopulations of cells treated with lipopolysaccharide (LPS), that show distinct expression patterns from pro-inflammatory genes. The data revealed that a promising therapeutic reagent, sodium 4-phenylbutyrate, masked the effect of LPS. We inferred the existence of specific cellular transitions under different treatments and prioritized important motifs that modulate the transitions using feature selection by a random forest method. There has been a transition in genomics research from bulk RNA-seq to single-cell RNA-seq, and scRNA-seq has become a widely used approach for transcriptome analysis. With the experience we gained by analyzing scRNA-seq data, we plan to conduct comparative single-cell transcriptome analysis across multiple species. / Doctor of Philosophy / All cells in an organism have the same set of genes, but there are different cell types, tissues, organs with different functions as the organism ages or under different conditions. Gene expression regulation is one mechanism that modulates complex, dynamic, and specific changes in tissues or cell types for any living organisms. Understanding gene regulation is of fundamental importance in biology. With the rapid advancement of sequencing technologies, there is a tremendous amount of gene expression data (transcriptome) from individual species in public repositories. However, major studies have been reported from several model species and research on non-model species have relied on comparison results with a few model species. Comparative transcriptome analysis across species will help us to transform knowledge from model species to non-model species and such knowledge transfer can contribute to the improvement of crop yields and human health. The focus of my dissertation is to develop and apply approaches for comparative transcriptome analysis that can help us better understand what makes each species unique or special, and what kinds of common functions across species have been passed down from ancestors (evolutionarily conserved functions). Three research chapters are presented in this dissertation. First, we developed a method to identify groups of genes that are commonly co-expressed in two species. We chose seed development data from soybean with the hope to contribute to crop improvement. Second, we compared gene expression data across five plant species including soybean, rice, and corn to provide new perspectives about crop plants. We chose drought stress to identify conserved functions and regulatory factors across species since drought stress is one of the major stresses that negatively impact agricultural production. We also proposed a method that groups genes with evolutionary relationships from an unlimited number of species. Third, we analyzed single-cell RNA-seq data from mouse monocytes to understand the regulatory mechanism of the innate immune system under low-grade inflammation. We observed how innate immune cells respond to inflammation that could cause no symptoms but persist for a long period of time. Also, we reported an effect of a promising therapeutic reagent (sodium 4-phenylbutyrate) on chronic inflammatory diseases. The third project will be extended to comparative single-cell transcriptome analysis with multiple species.

Soybean (Glycine max L.) is an important legume crop often exposed to heat and drought stresses during reproductive and early-seed setting stages, resulting in lower yields and seed quality. Ten soybean cultivars were phenotyped for individual (drought or heat) and combined stress tolerance. Under drought, reduced stomatal conductance and increased canopy temperature significantly reduced seed number (46%) and weight (35%). Heat stress alone reduced seed number (19%) and weight (23%) compared to control. Moreover, a degree increase in daytime temperature above 32 °C during the reproductive stage reduced seed weight by 4% and 7% under well-watered and drought conditions, respectively. Seed protein was increased under drought, while it declined under heat and combined stress compared to control. In contrast, oil content showed the opposite trend. Weak correlations between phenotypic traits under individual and combined stress suggest that selecting cultivars for individual stress tolerance may not work under combined stress conditions.

Tesis por compendio / [ES] Anteriormente, investigadores del Instituto Valenciano de Investigaciones Agrarias (IVIA) y la Universidad Politécnica de Valencia (UPV) evaluaron la respuesta a estrés hídrico en pimiento injertado para obtener patrones de pimiento que confieran tolerancia a la variedad injertada en estrés hídrico. Los mejores patrones se estudian en campo en condiciones de estrés hídrico a largo plazo. Se patentó el patrón híbrido de pimiento NIBER® tolerante a estrés hídrico, las plantas injertadas sobre NIBER® mostraron menor impacto en la biomasa y el rendimiento en déficit hídrico a largo plazo. La tolerancia de NIBER® se atribuyó al mantenimiento de la actividad fotosintética y una mejor distribución de la biomasa radicular en estrés hídrico. Además, la respuesta sostenida de tolerancia observada en las plantas injertadas sobre NIBER® podría relacionarse con una respuesta rápida en la fase inicial en estrés hídrico, que no se conoce a corto plazo. Así, estudiar la contribución de la respuesta a corto plazo de NIBER® sobre la tolerancia de la variedad injertada en estrés hídrico arrojaría luz en las estrategias de tolerancia en plantas injertadas de pimiento. Además, estudiar la modulación génica, el balance hormonal y el perfil metabólico ampliará el conocimiento sobre los mecanismos moleculares en la respuesta a estrés hídrico. En esta tesis doctoral, observamos que los mecanismos constitutivos en ausencia de estrés hídrico influyen en la respuesta a estrés hídrico en plantas injertadas de pimiento, y que las estrategias constitutivas de NIBER® incluyen la estimulación del sistema antioxidante y la inducción sostenida de ABA. En estrés hídrico, las raíces de NIBER® muestran un menor impacto que las raíces de A10, dado su menor contenido de GSSG por menor daño oxidativo. NIBER® promueve la síntesis de osmolitos en la raíz y vitamina B6 en las hojas de la variedad injertada protegiendo al aparato fotosintético del daño oxidativo producido por el estrés hídrico. Además, la prolina, implicada en la protección del aparato fotosintético, se acumula en las plantas injertadas sobre el híbrido de pimiento H92, capaces de mantener la actividad fotosintética a largo plazo en estrés hídrico. Este rol de la prolina en la tolerancia a estrés hídrico a largo plazo no se observó a corto plazo y podría ser una estrategia tardía. La respuesta al estrés hídrico a corto plazo incluye la regulación del movimiento estomático en NIBER® en la fase inicial de estrés (5 h), evitando el cierre estomático hasta las 48 h mediante cambios en la expresión génica de reguladores negativos de ABA y acuaporinas, seguido del cierre estomático a las 48 h asociado a una síntesis previa de ABA en las raíces y transporte a las hojas. El JA también aumentó en las hojas de la variedad injertada sobre NIBER® (48 h) en estrés hídrico, y se regula por señales a larga distancia desde la raíz, que promueven la síntesis en hoja y el transporte a la raíz, y su resíntesis. El JA está implicado en el cierre estomático y la señalización en estrés, causando la activación de factores de transcripción de respuesta a la deshidratación. En las raíces de NIBER® el ratio auxinas/citoquininas se regula en la respuesta inicial al estrés hídrico, favoreciendo el crecimiento de la raíz respecto al tallo a las 5 h, después aumentando las citoquininas y disminuyendo las auxinas a 24 h y finalmente aumentando las auxinas y reduciendo las citoquininas para una mayor biomasa radicular y capacidad exploratoria. En las hojas de la variedad injertada, NIBER® aumenta los metabolitos protectores clorofila a, ácido esteárico, antocianinas y metabolitos implicados en la síntesis de suberina y cutina. Estos últimos también aumentan en la raíz y tienen un papel antioxidante o bien como constituyentes de barreras celulares controlando los flujos de agua, gases y solutos. Finalmente, el contenido en sirohemo aumenta en la raíz y posiblemente está relacionado con una asimilación del nitrógeno más eficiente. / [CA] Prèviament, investigadors de l'Institut Valencià d'Investigacions Agràries (IVIA) i de la Universitat Politècnica de València (UPV) avaluaren la resposta a estrés hídric en pebrera empeltada per obtenir patrons de pebrera que conferisquen tolerància a la varietat empeltada en estrés hídric. Els millors patrons s'estudien al camp en condicions d'estrés hídric a llarg termini. Es va patentar el patró híbrid de pebrera NIBER® com a patró tolerant, les plants empeltades sobre NIBER® mostraren menor impacte en la biomassa i el rendiment en condicions de dèficit hídric. La tolerància de NIBER® es va atribuir al manteniment de l'activitat fotosintètica i a una millor distribució de la biomassa radicular en estrés hídric. A més, la resposta sostinguda de tolerància observada a les plantes empeltades sobre NIBER® podria relacionar-se amb una resposta ràpida a la fase inicial en estrés hídric, que no s'ha estudiat a curt termini. Així, estudiar la contribució de la resposta a curt termini de NIBER® sobre la tolerància de la varietat empeltada en estrés hídric aclariria els mecanismes de tolerància en plantes empeltades de pebrera. A més, estudiar la modulació gènica, el balanç hormonal i el perfil metabòlic ampliaria el coneixement sobre els mecanismes moleculars en la resposta a estrés hídric. En aquesta tesi doctoral comprovarem que els mecanismes constitutius en absència d'estrés hídric influeixen en la resposta a estrés hídric en plantes empeltades de pebrera i que les estratègies constitutives de NIBER® inclouen l'estimulació del sistema antioxidant i la inducció sostinguda d'ABA. En estrés hídric, les arrels de NIBER® mostraren menor impacte en comparació amb A10, amb un menor contingut de GSSG per menor dany oxidatiu. NIBER® promou la síntesis d'osmòlits a les arrels i de vitamina B6 a les fulles de la varietat empeltada per a protegir a l'aparell fotosintètic del dany oxidatiu per l'estrés hídric. A més, la prolina, implicada en la protecció de l'aparell fotosintètic, s'acumula a les plantes empeltades sobre l'híbrid de pebrera H92, que mantenen l'activitat fotosintètica en condicions d'estrés hídric a llarg termini. Aquest paper de la prolina no es va observar en condicions d'estrés hídric a curt termini i podria ser una estratègia tardana. Les respostes a l'estrés hídric a curt termini inclouen la regulació del moviment estomàtic en NIBER® a la fase inicial d'estrés hídric (5 h), evitant el tancament estomàtic fins a les 48 h mitjançant canvis a l'expressió gènica de reguladors negatius d'ABA i acuaporines, seguit d'un tancament estomàtic a les 48 h associat a una síntesis prèvia d'ABA a les arrels i transport a les fulles. L'JA també va augmentar a les fulles de la varietat empeltada sobre NIBER® a les 48 h en estrés hídric i està regulat per senyals a llarga distància des de les arrels que promouen la síntesis a les fulles i el transport a les arrels i resíntesi. L'JA està implicat al tancament estomàtic i la senyalització en condicions d'estrés, promovent l'activació de factors de transcripció de resposta a la deshidratació. El rati auxines/citoquinines es regula a les arrels de NIBER® a la resposta inicial a l'estrés hídric, primer afavorint el creixement de l'arrel sobre el creixement de la part aèria a les 5 h, després augmentant les citoquinines i disminuint les auxines a les 24 h i finalment augmentant les auxines i reduint les citoquinines per obtenir major biomassa radicular i capacitat exploratòria. A les fulles de la varietat empeltada, NIBER® augmenta el contingut dels metabòlits protectors clorofil·la a, àcid esteàric, antocianines i metabòlits relacionats amb la síntesi de suberina i cutina. Aquests últims també augmenten a les arrels i tenen un paper antioxidant o actuen com a constituents de barreres cel·lulars controlant els fluxos d'aigua, gasos i soluts. Finalment, el contingut en siroheme augmenta a les arrels i possiblement està relacionat amb una assimilació de nitrogen més eficient. / [EN] Previously, research groups at the Valencian Institute of Agricultural Research (IVIA) and the Polytechnic University of Valencia (UPV) evaluated the water stress responses in grafted pepper plants to obtain tolerant pepper rootstocks that make the grafted variety able to overcome water stress. The best rootstocks are studied in the field under long-term water stress conditions. In this way, the NIBER® pepper hybrid rootstock was obtained and patented as water stress-tolerant rootstock, because plants grafted onto NIBER® had a lower impact on biomass and yield under long-term deficit irrigation conditions. NIBER® tolerance response was attributed to sustained photosynthetic activity and improved root biomass distribution under long-term water stress. However, the sustained tolerance response observed in plants grafted onto NIBER® may be linked to prompt responses in the early phase of water stress conditions, but the short-term modulation and behavior of NIBER® water stress response has not been studied. Hence, studying the contribution of the NIBER® short-term water stress responses to tolerance in the grafted variety would shed light into tolerance mechanisms in grafted pepper plants. Moreover, understanding the modulation of the gene expression, phytohormones balance and metabolic profile will also broad the knowledge on the molecular mechanisms implicated in water stress response. In the present doctoral thesis, we stated that the constitutive mechanisms taking place under non-water stress conditions dispose the response to water stress in grafted pepper plants, and NIBER® constitutive strategies include an enhanced ROS detoxification system and maintained ABA induction. When the water stress comes into play, its impact was minor in NIBER® roots in relation to A10 roots, which is reflected in lesser GSSG content from lower oxidative damage. NIBER® promotes the synthesis of osmolytes in roots and vitamin B6 in the grafted variety leaves to protect the plants from the oxidative damage resulting from water stress. Moreover, proline has a role in photosynthetic apparatus protection, because it is accumulated in plants grafted onto pepper hybrid H92, which showed sustained photosynthetic activity under long-term water stress conditions. The proline role in water stress tolerance is not observed under short-term water stress and may constitute a late strategy in grafted pepper plants. Short-term responses to water stress include stomatal movements in NIBER® during early phases (5h) of water stress, starting with the avoidance of stomatal closure up to 48 h by gene expression changes in ABA negative regulators and aquaporins, and followed by stomatal closure at 48 h associated with previous ABA synthesis in roots and transport to leaves. JA is also increased in the leaves of the variety grafted onto NIBER® at 48 h under water stress, and is regulated by long-distance signals from roots that promote its synthesis on leaves and transport to roots and resynthesis. JA is involved in stomatal closure and stress signaling, which leads to dehydration-responsive transcription factors activation. The auxins/cytokinins ratio is also fine-tuned by NIBER® roots during the early water stress response, beginning with promotion of root over shoot growth at 5 h, then increases the cytokinins and reduces the auxins content at 24 h, and finally increases the auxins and reduces the cytokinins content to obtain higher root biomass and greater water exploring ability. In the leaves of the grafted variety, NIBER® increases protective metabolites as chlorophyll a, stearic acid, anthocyanins and suberin and cutin biosynthesis-related metabolites, being the latter also increased in the roots. The mentioned metabolites have an antioxidant role or act as cellular barrier constituents that can control fluxes of water, gases and solutes. Lastly, siroheme increases in roots and it is possibly linked to a more effective nitrogen assimilation. / Padilla Herrero, YG. (2023). Study of the Physiological, Metabolomic and Transcriptional Changes Mediated by Rootstocks to Explain the Water Stress Tolerance of Grafted Pepper Plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/199992 / Compendio

The aims of this work were 1) to assess whether sorghum (Sorghum bicolor (L.) Moench) genotypes originating from the India can be grown and analyzed in the climatic conditions of central Europe and 2) to find out the utilization potential of selected non- destructive and destructive methods based mostly on the chlorophyll a fluorescence measurements and the determination of photosynthetic pigments' content for the differentiation of sorghum genotypes based on their presumed drought tolerance. Field experiments made during 2 years compared 15 genotypes of this species (2 stay-green parental lines, 2 senescent parental lines and 11 introgression lines with stay-green loci), 2 of these genotypes were further analyzed in greenhouse conditions where the water deficit was induced by a cessation of watering for 12 days. The field-grown plants showed some differences between individual genotypes in all measured parameters; however, for the majority of the genotypes these differences were not statistically significant. The stay-green parental genotype B35 differred the most from the other ones in both field seasons, but the other stay-green genotypes usually did not differ from the senescent genotypes. No significant differences between both greenhouse-tested genotypes (presumably contrasting in their...

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