by Lui Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 143-166). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.iv / 摘要 --- p.viii / List of Tables --- p.xi / List of Figures --- p.xii / Contents --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Sulfur-Containing Amino Acids --- p.1 / Chapter 1.2 --- Metabolism of Sulfur-Containing Amino Acids --- p.3 / Chapter 1.2.1 --- The Metabolic Pathways --- p.3 / Chapter 1.2.2 --- Metabolic Regulation of Homocysteine --- p.7 / Chapter 1.3 --- Sulfur-Containing Amino acids and Health --- p.10 / Chapter 1.3.1 --- Sulfur-Containing Amino Acids and Renal Diseases --- p.11 / Chapter 1.3.2 --- Sulfur-Containing Amino Acids and the Nervous System --- p.13 / Chapter 1.3.3 --- Sulfur-Containing Amino Acids and Heart Diseases --- p.16 / Chapter 1.3.4 --- Sulfur-Containing Amino Acids and Liver Diseases --- p.20 / Chapter 1.4 --- Vitamin C (Ascorbic Acid) --- p.21 / Chapter 1.4.1 --- Biosynthesis 、 --- p.23 / Chapter 1.4.2 --- Vitamin C Transport System --- p.24 / Chapter 1.4.2.1 --- Uptake of Ascorbic Acid --- p.25 / Chapter 1.4.2.2 --- Uptake of Dehydroascorbic Acid --- p.26 / Chapter 1.5 --- Vitamin C and Health --- p.28 / Chapter 1.5.1 --- Vitamin C and Heart Diseases --- p.28 / Chapter 1.5.2 --- Vitamin C and Renal Diseases --- p.29 / Chapter 1.5.3 --- Vitamin C and Brain Diseases --- p.30 / Chapter 1.5.4 --- Vitamin C and Liver Diseases --- p.31 / Chapter 1.6 --- The Aims of Project --- p.33 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.35 / Chapter 2.2 --- Preparation of Materials --- p.37 / Chapter 2.2.1 --- Mobile Phase for Ascorbic Acid Analysis --- p.37 / Chapter 2.2.2 --- Mobile Phase for Amino Acid Analysis --- p.37 / Chapter 2.2.3 --- Reagents for RNA Extraction and Reverse Transcription- Polymerase Chain Reaction (RT-PCR) --- p.38 / Chapter 2.2.4 --- Sense and Antisense Primers --- p.38 / Chapter 2.2.5 --- Reagents for Electrophoresis --- p.40 / Chapter 2.3 --- Animals --- p.40 / Chapter 2.4 --- Experimental Studies --- p.41 / Chapter 2.5 --- Methods --- p.41 / Chapter 2.5.1 --- Ascorbic Acid Analysis --- p.41 / Chapter 2.5.1.1 --- Extraction of Ascorbic Acid --- p.41 / Chapter 2.5.1.2 --- Chromatography --- p.42 / Chapter 2.5.2 --- Amino Acid Analysis --- p.45 / Chapter 2.5.2.1 --- Sample Preparation --- p.45 / Chapter 2.5.2.2 --- Chromatography --- p.45 / Chapter 2.5.3 --- Determination of RNA Expression by RT-PCR Analysis --- p.50 / Chapter 2.5.3.1 --- RNA Isolation --- p.50 / Chapter 2.5.3.2 --- Measurement of RNA Yield --- p.51 / Chapter 2.5.3.3 --- Reverse Transcription --- p.51 / Chapter 2.5.3.4 --- Polymerase Chain Reaction (PCR) --- p.52 / Chapter 2.5.3.5 --- Separation of PCR Products by Agarose Gel Electrophoresis --- p.52 / Chapter 2.5.3.6 --- Quantification of Band Density --- p.53 / Chapter 2.6 --- Statistical Analysis --- p.53 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Effects of Sulfur-Containing Amino Acids and Leucine on Ascorbic Acid Distributions in Mice --- p.54 / Chapter 3.1.1 --- Effects of Sulfur-Containing Amino Acids and Leucine on Ascorbic Acid Concentrations in the Plasma --- p.55 / Chapter 3.1.2 --- Effects of Sulfur-Containing Amino Acids and Leucine on Ascorbic Acid Concentrations in the Kidney --- p.57 / Chapter 3.1.3 --- Effects of Sulfur-Containing Amino Acids and Leucine on Ascorbic Acid Concentrations in the Liver --- p.59 / Chapter 3.1.4 --- Effects of Sulfur-Containing Amino Acids and Leucine on Ascorbic Acid Concentrations in the Brain --- p.61 / Chapter 3.1.5 --- Effects of Sulfur-Containing Amino Acids and Leucine on Ascorbic Acid Concentrations in the Heart --- p.63 / Chapter 3.1.6 --- Summary --- p.65 / Chapter 3.2 --- Time-Response --- p.66 / Chapter 3.2.1 --- Effects of Sulfur-Containing Amino Acids and Leucine Administration for Various Periods on Ascorbic Acid Concentrations in the Plasma --- p.67 / Chapter 3.2.2 --- Effects of Sulfur-Containing Amino Acids and Leucine Administration for Various Periods on Ascorbic Acid Concentrations in the Kidney --- p.69 / Chapter 3.2.3 --- Effects of Sulfur-Containing Amino Acids and Leucine Administration for Various Periods on Ascorbic Acid Concentrations in the Liver --- p.71 / Chapter 3.2.4 --- Effects of Sulfur-Containing Amino Acids and Leucine Administration for Various Periods on Ascorbic Acid Concentrations in the Brain --- p.73 / Chapter 3.2.5 --- Effects of Sulfur-Containing Amino Acids and Leucine Administration for Various Periods on Ascorbic Acid / Chapter 3.2.6 --- Summary --- p.77 / Chapter 3.3 --- Effects of Sulfur-Containing Amino Acids and Leucine on SVCT and GLUT3 Gene Expressions --- p.78 / Chapter 3.3.1 --- Effects of Sulfur-Containing Amino Acids and Leucine Administration on SVCT mRNA Expression in the Kidney --- p.79 / Chapter 3.3.2 --- Effects of Sulfur-Containing Amino Acids and Leucine Administration on SVCT mRNA Expression in the Liver --- p.89 / Chapter 3.3.3 --- Effects of Sulfur-Containing Amino Acids and Leucine Administration on SVCT and GLUT3 mRNA Expression in the Brain --- p.98 / Chapter 3.3.4 --- Effects of Sulfur-Containing Amino Acids and Leucine Administration on SVCT mRNA Expression in the Heart --- p.109 / Chapter 3.3.5 --- Summary --- p.115 / Chapter 3.4 --- Sulfur-Containing Amino Acids Concentrations in the Plasma --- p.117 / Chapter 3.4.1 --- Effects of Administration of Sulfur-Containing Amino Acids and Leucine on Methionine Concentrations in the Plasma --- p.117 / Chapter 3.4.2 --- Effects of Administration of Sulfur-Containing Amino Acids and Leucine on Cystine Concentrations in the Plasma --- p.119 / Chapter 3.4.3 --- Effects of Administration of Sulfur-Containing Amino Acids and Leucine on Taurine Concentrations in the Plasma --- p.119 / Chapter 3.4.4 --- Effects of Administration of Sulfur-Containing Amino Acids and Leucine on Leucine Concentrations in the Plasma --- p.122 / Chapter 3.4.5 --- Summary --- p.124 / Chapter CHAPTER 4 --- DISCUSSION AND CONCLUSIONS / Chapter 4.1 --- Effects of Sulfur-Containing Amino Acids and Leucine on Ascorbic Acid Concentrations in Mice --- p.126 / Chapter 4.2 --- Effects of Sulfur-Containing Amino Acids and Leucine on SVCT and GLUT3 Gene Expressions --- p.131 / Chapter 4.3 --- Sulfur-Containing Amino Acids Concentrations in the Plasma --- p.136 / Chapter 4.4 --- Conclusions --- p.140 / REFERENCES --- p.143

Arid and semi-arid regions, mostly found in developing countries with exponentially increasing populations, are in chronic lack of water thereby severely limiting agricultural production. Irrigation with saline water, which is available in large quantities, could be an obvious solution, but current crops are all salt sensitive. Although major efforts are underway to breed salt tolerant crops, no breakthrough results have yet been obtained. One alternative could rely on plant-interacting microbiota communities. Indeed, rhizophere and endosphere microbial communities are distinct from those of the surrounding soils, and these specific communities contribute to plant growth and health by increasing nutrient availability or plant resistance towards abiotic and biotic stresses. Here we show that plant microbe interactions induce plant tolerance to multiple stresses. From a collection of strains isolated from the desert plant Indigofera argentea, we could identify at least four different strategies to induce salt stress tolerance in Arabidopsis thaliana. A deep analysis of Enterobacter sp. SA187 showed that it induces Arabidopsis tolerance to salinity through activation of the ethylene signaling pathway. Interestingly, although SA187 does not produce ethylene as such, the association of SA187 with plants induces the expression of the methionine salvage pathway in SA187 resulting in the conversion of bacterially produced 2-keto-4-methylthiobutyric acid (KMBA) to ethylene. In addition, a metabolic network characterization of both SA187 and Arabidopsis in their free-living and endophytic state revealed that the sulfur metabolic pathways are strongly upregulated in both organisms. Furthermore, plant genetic experiments verified the essential role of the sulfur metabolism and ethylene signaling in plant salt stress tolerance. Our findings demonstrate how successful plant microbes of a given community can help other plants to enhance tolerance to abiotic stress, and reveal a part of the complex molecular communication process during beneficial plant-microbe interaction.

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Tesis por compendio / [ES] El cambio climático ha aumentado la exposición de los cultivos a estreses como la sequía o la salinidad y, por tanto, tiene un impacto negativo en el rendimiento de las plantas y los cultivos en un mundo con una población en aumento en el que la seguridad alimentaria está amenazada. Existe un interés en desarrollar variedades y cultivares con mayor tolerancia a estreses abióticos. Con esta finalidad desarrollamos un abordaje, en cultivos con un alto valor económico y nutricional como brócoli y melón, para identificar rasgos fisiológicos y bioquímicos posiblemente limitantes de estrés abiótico y, por tanto, podrían ser dianas biotecnológicas para mejorar la tolerancia a estrés abiótico o que pueden ser útiles para predecir la tolerancia de variedades no caracterizadas previamente. En la presente tesis, empleamos fisiología y metabolómica para identificar como rasgos distintivos de tolerancia a estrés por sequía en brócoli, la fotosíntesis neta, la eficiencia en el uso del agua, la conductancia estomática, el ácido abscísico, metabolitos relacionados con el metabolismo del azufre y otras moléculas como la urea, el ácido quínico y el ácido glucónico lactona. Por otra parte, en melón encontramos el potencial hídrico y varios aminoácidos como serina, fenilalanina, glicina, isoleucina, asparagina y triptófano como rasgos distintivos entre cultivares tolerantes o sensibles bajo estrés por sequía. Al respecto del estrés salino, identificamos el ratio Na+/K+ como rasgo distintivo tanto para brócoli como para melón mientras que sólo en brócoli, la transpiración y la conductancia estomática como rasgos fisiológicos útiles y, en cuanto a rasgos bioquímicos, hormonas como el ácido abscísico, el ácido jasmónico y el ácido indol-3-acético así como metabolitos del ciclo de Krebs y el contenido total de los relacionados glutatión, cisteína y metionina y prolina, se mostraron como indicadores de respuestas a estrés salino en brócoli. Sin embargo, en melón, encontramos prolina, fenilalanina e histidina como rasgo distintivo de tolerancia a estrés salino. También utilizamos herramientas metabolómicas para identificar que el ácido γ-aminobutírico correlaciona con un mejor sabor del brócoli. Además, dada la importancia del metabolismo del azufre y la tolerancia al estrés, así como su relación con las propiedades nutricionales del brócoli, llevamos a cabo la caracterización de las enzimas serina O-acetiltransferasas de brócoli. La evidencia que encontramos sugiere que las dianas biotecnológicas más prometedoras para mejorar la tolerancia al estrés son los parálogos BoSAT1d y BoSAT3. / [CA] El canvi climàtic ha augmentat l'exposició dels cultius a estressos com la sequera o la salinitat i, per tant, té un impacte negatiu en el rendiment de les plantes i els cultius en un món amb una població en augment on la seguretat alimentària està amenaçada . Hi ha un interès a desenvolupar varietats i cultivars amb més tolerància a estressos abiòtics. A aquest efecte desenvolupem un abordatge, en cultius amb un alt valor econòmic i nutricional com ara bròquil i meló, per identificar trets fisiològics i bioquímics possiblement limitants d'estrès abiòtic i, per tant, podrien ser dianes biotecnològiques per millorar la tolerància a estrès abiòtic o que poden ser útils per predir la tolerància de varietats no prèviament caracteritzades. En aquesta tesi, utilitzem fisiologia i metabolòmica per identificar com a trets distintius de tolerància a estrès per sequera en bròquil, la fotosíntesi neta, l'eficiència en l'ús de l'aigua, la conductància estomàtica, l'àcid abscísic, metabòlits relacionats amb el metabolisme del sofre i altres molècules com la urea, l'àcid quínic i l'àcid glucònic lactona. D'altra banda, en meló trobem el potencial hídric i diversos aminoàcids com a serina, fenilalanina, glicina, isoleucina, asparagina i triptòfan com a trets distintius entre cultivars tolerants o sensibles sota estrès per sequera. Pel que fa a l'estrès salí, identifiquem la ràtio Na+/K+ com a tret distintiu tant per a bròquil com per a meló mentre que només en bròquil, la transpiració i la conductància estomàtica com a trets fisiològics útils i, quant a trets bioquímics, hormones com l'àcid abscísic , l'àcid jasmònic i l'àcid indol-3-acètic així com metabòlits del cicle de Krebs i el contingut total dels relacionats glutatió, cisteïna i metionina i prolina, es van mostrar com a indicadors de respostes a estrès salí en bròquil. Tot i això, en meló, trobem prolina, fenilalanina i histidina com a tret distintiu de tolerància a estrès salí. També utilitzem eines metabolòmiques per identificar que l'àcid γ-aminobutíric correlaciona amb un millor sabor del bròquil. A més, atesa la importància del metabolisme del sofre i la tolerància a l'estrès, així com la seva relació amb les propietats nutricionals del bròquil, duem a terme la caracterització dels enzims serina O-acetiltransferases de bròquil. La seva evidència suggereix que les dianes biotecnològiques més prometedores per millorar la tolerància a l'estrès són els paràlegs BoSAT1d i BoSAT3. / [EN] Climate change has increased the exposure of crops to stresses like drought and salinity and, thus, it has a negative impact on plant performance and crop yield in a world with an increasing population. This threatens food security. There is an interest in developing new varieties and cultivars of crops with enhanced tolerance to abiotic stresses. With this aim, we developed an approach in crops with high economic and nutritional values, such as broccoli and melon, to identify physiological and biochemical traits that could be limiting factors for abiotic stress and, thus are likely biotechnological targets for improving abiotic stress tolerance or may be useful for predicting tolerance of uncharacterized varieties. In the present thesis, we used physiology and metabolomics to identify as distinctive traits for drought stress tolerance in broccoli, net photosynthesis, water use efficiency, stomatal conductance, abscisic acid, metabolites related to sulfur metabolism and other molecules such as urea, quinic acid and gluconic acid lactone. On the other hand, in melon, we found water potential and several amino acids as serine, phenylalanine, glycine, isoleucine, asparagine and tryptophan to be differential traits among tolerant and sensitive cultivars under drought stress. Regarding salt stress tolerance, we identified Na+/K+ ratios as a distinctive trait for both broccoli and melon whereas in broccoli, but not melon, transpiration, and stomatal conductance were useful physiological traits. Concerning biochemical traits, hormones, such as abscisic acid, jasmonic acid and indole-3-acetic acid, metabolites of the Krebs cycle and total content of the related glutathione, cysteine and methionine and proline proved to be indicative of saline stress responses in broccoli. However, in melon, we found only proline, phenylalanine and histidine as a distinctive trait of salt stress tolerance. We also used metabolomic tools to identify that γ-Aminobutyric acid correlates with a better taste in broccoli. Given the importance of sulfur metabolism in stress tolerance, as well as its link with the nutritional properties of broccoli, we also carried out the characterization of the serine O-acetyltransferase enzymes in broccoli. Among the eight isoforms found in this species, our data suggest that the most promising biotechnological targets for enhancing stress tolerance are the BoSAT1d and the BoSAT3 paralogues. / I am a recipient of the FPU19/01977 grant from the Spanish Ministerio de Universidades. This work was funded by the RTC-2017-6468-2-AR project (APROXIMACIONES MOLECULARES PARA INCREMENTAR LA TOLERANCIA A SALINIDAD Y SEQUÍA DEL BRÓCOLI) awarded by the “Agencia estatal de Investigación” / Chevilly Tena, S. (2024). Molecular approaches to characterize and improve abiotic stress tolerance in broccoli and other crops [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/213681 / Compendio