One of the major challenges for agricultural research in the 21st century is to increase crop productivity to meet the growing demand for food and feed. Biotic (e.g. plant pathogens) and abiotic stresses (e.g. soil salinity) have detrimental effects on agricultural productivity, with yield losses being as high as 60% for major crops such as barley, corn, potatoes, sorghum, soybean and wheat, especially in semi-arid regions such as Saudi Arabia. Plant growth promoting bacteria isolated from pioneer desert plants could serve as an eco-friendly, sustainable solution for improving plant growth, stress tolerance and health. In this dissertation, culture-independent amplicon sequencing of bacterial communities revealed how native desert plants influence their surrounding bacterial communities in a phylogeny-dependent manner. By culture-dependent isolation of the plant endosphere compartments and a number of bioassays, more than a hundred bacterial isolates with various biochemical properties, such as nutrient acquisition, hormone production and growth under stress conditions were obtained. From this collection, five phylogenetically diverse bacterial strains were able to promote the growth of the model plant Arabidopsis thaliana under salinity stress conditions in a common mechanism of inducing transcriptional changes of tissue-specific ion transporters and lowering Na+/K+ ratios in the shoots. By combining a number of in vitro bioassays, plant phenotyping and volatile-mediated inhibition assays with next-generation sequencing technology, gas chromatography–mass spectrometry and bioinformatics tools, a candidate strain was presented as a multi-stress tolerance promoting bacterium with potential use in agriculture. Since recent research showed the importance of microbial partners for enhancing the growth and health of plants, a review of the different factors influencing plant-associated microbial communities is presented and a framework for the successful application of microbial inoculants in agriculture is proposed. The presented work demonstrates a holistic approach for tackling agricultural challenges using microbial inoculants from desert plants by combining culturomics, phenomics, genomics and transcriptomics. Microbial inoculants are promising tools for studying abiotic stress tolerance mechanisms in plants, and they provide an eco-friendly solution for increasing crop yield in arid and semi-arid regions, especially in light of a dramatically growing human population and detrimental effects of global warming and climate change.

Lipids maintain fluidity of the cell membrane during the lifetime of all organisms. The moss Physcomitrium patens, an early land plant, enters reproductive phase under cold (15°C) conditions relative to its gametophytes (22°C). Thus, we hypothesized that their lipid content and composition would be distinct. Using ESI-MS/MS, we showed that the content and acyl composition of 11 lipid classes varied during development. Galactolipids were abundant in gametophytes but insignificant in sporophytes; among phospholipids, phosphatidylcholine was predominant in both phases. Although, sporophytes contained around five-fold less lipids than the gametophyte, their phosphatidic acid content, which accumulates during stress, was 18-fold higher. Furthermore, comparative analyses of lipidomes revealed that the 38C and 40C acyl species are abundant in the moss and mouse, which are mostly absent in vascular plants. The occurrence of long-chain, highly unsaturated lipids perhaps contribute to membrane stability that was necessary for the evolution of early land plants.

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.

[EN] The general aim of this thesis is to study the dormancy process from a molecular point of view identifying mechanisms and targeting genes that control it. In order to do that we have focused on the study of three genes that are differentially expressed during reproductive bud development within the conceptual framework of the three major processes that converge spatially and temporally in a reproductive bud: dormancy, stress tolerance and flower development. The first gene is down-regulated in dormancy release flower buds and encodes a STRESS ASSOCIATED PROTEIN (PpSAP1) that contains Zn-finger domains A20 and AN1. SAP proteins have been related to stress tolerance response in both plants and animals and in fact, we have shown that drought stress induces its expression in buds, resembling other SAP genes in plants. Moreover, the constitutive expression of PpSAP1 in plum increases its tolerance to water stress by increasing water retention. Likewise, transgenic plum plants show leaf alterations related to reduced cell size concomitant with the down-regulation of genes involved in cell growth. All these studies suggest a dual role of PpSAP1 in stress tolerance response and cell growth during peach dormancy. The second gene is PpeS6PDH, coding for an enzyme with sorbitol-6-phosphate dehydrogenase activity. PpeS6PDH is differentially regulated during bud development, highly expressed in dormant buds consistently with sorbitol accumulation. Concomitantly with PpeS6PDH down-regulation in dormancy-released flower buds, chromatin around the translation start site of the gene shows changes in the methylation state of specific residues of histone H3 (H3K4 and H3K27). These data suggest the transcriptional regulation of PpeS6PDH expression by chromatin modification mechanisms. Moreover, abiotic stresses affect PpeS6PDH expression. Low temperature treatments induce gene expression in buds and leaves, whereas desiccation up-regulates PpeS6PDH in buds and represses the gene in leaves. These data suggest the participation of PpeS6PDH in tolerance against cold and water deficit stresses in buds. Finally, the third gene is PpeDAM6, one of the major regulators of bud dormancy in peach. PpeDAM6 is sharply down-regulated during bud development concomitantly with dormancy release events. This repression is in part due to the direct binding of PpeBPC1, a BASIC PENTACYSTEINE PROTEIN, to the GAGA motifs present in an intronic regulatory region of PpeDAM6 gene that becomes enriched in H3K27me3 chromatin modification after dormancy release. In addition, the ectopic expression of PpeDAM6 in Arabidopsis shows abnormal flower phenotypes resembling 35S::SVP plants. On the other hand, overexpression in plum causes stunted growth in the transgenic lines due to an altered hormonal homeostasis. The changes in hormone content are mediated by the modulation of genes involved in jasmonic acid, cytokinins and gibberellic acid metabolism and signalling pathways. These results suggest that PpeDAM6 works as a master growth repressor maintaining dormancy, stress tolerance response and flowering inhibition by mainly modulating hormone homeostasis. Therefore, this thesis provides a dynamic snapshot of different molecular mechanism that take place inside the bud. The studied genes have a crucial role regulating dormancy processes, stress tolerance response and flowering pathways and all of them are potential candidate genes for breeding new plants more adapted to the climate change. / [ES] El objetivo general de esta tesis es el estudio de la latencia desde un punto de vista molecular, identificando mecanismos y genes diana que la controlen. Para ello, nos hemos centrado en el estudio de tres genes que se expresan de manera diferencial durante el desarrollo de una yema reproductiva en melocotón, bajo el marco conceptual de los tres procesos que convergen espacialmente y temporalmente en una yema reproductiva: latencia, tolerancia a estrés y desarrollo floral. El primer gen que se estudió codificó para una STRESS ASSOCIATED PROTEIN (PpSAP1) con dos dominios tipo Zn-finger, A20 y AN1 que disminuye su expresión durante la latencia. Las proteínas tipo SAP se han relacionado con resistencias a distintos tipos de estrés tanto en plantas como en animales. De hecho, se ha visto que PpSAP1 aumentó su expresión en yemas de melocotón bajo condiciones de estrés por sequía, de forma similar a como lo hacen otras SAP en distintas plantas. Además, la expresión ectópica de PpSAP1 en ciruelos transgénicos ha permitido aumentar la tolerancia a estrés hídrico en estas líneas al incrementar la cantidad de agua retenida. Asimismo, estas plantas transgénicas también mostraron alteraciones en el tamaño de las hojas, provocadas principalmente por una menor área celular de las células que formaban parte de ellas y relacionadas con una represión de distintos genes implicados en crecimiento celular. Todo ello sugiere que PpSAP1 probablemente tenga una doble función relacionada tanto con resistencia a estrés como con crecimiento celular durante la latencia de melocotonero. El segundo gen de estudio fue PpeS6PDH, el cual codifica para una enzima con actividad sorbitol-6-fosfato deshidrogenasa. PpeS6PDH está diferencialmente regulado durante el desarrollo de la yema, aumentando su expresión en yemas latentes de manera consistente a la acumulación de sorbitol. Simultáneamente a la disminución de PpeS6pDH en las yemas no latentes, alrededor del sitio de inicio de la traducción del gen se mostraron cambios a nivel de cromatina en el estado de metilación de los residuos específicos de la histona H3 (H3K4 y H3K27). Estos datos apuntan a la existencia de una regulación transcripcional de PpeS6PDH a nivel de modificaciones de la cromatina. Además, también se ha visto que distintos tipos de estrés abiótico afectan a la expresión de PpeS6PDH. Tratamientos con bajas temperaturas inducieron su expresión tanto en yemas como en hojas, mientras que la desecación aumentó la expresión en yemas pero no en hojas. Estos estudios sugieren que la función de PpeS6PDH durante la latencia de melocotonero es dar tolerancia a estrés por frío y sequía. Finalmente, el tercer gen de estudio fue PpeDAM6, uno de los mayores reguladores de la latencia en yemas de melocotonero. PpeDAM6 está fuertemente reprimido durante el desarrollo de la yema con una relación directa con los eventos de salida de latencia. Esta represión se debe en parte a la unión directa de PpeBPC1, una BASIC PENTACYSTEINE PROTEIN, a dos motivos GAGA presentes en la región intrónica reguladora de PpeDAM6. Justamente esta región se encuentra modificada a nivel de cromatina con un enriquecimiento en H3K27me3 después de la salida de latencia. Además, la expresión ectópica de PpeDAM6 en Arabidopsis mostró fenotipos de floración anormal parecidos a los producidos en plantas 35S::SVP. Por otro lado, la sobreexpresión en ciruelos provocó retrasos en el crecimiento de las líneas transgénicas, debido a una alteración en los niveles hormonales. Así mismo, se determinó que estos cambios en la homeostasis hormonal estaban producidos por la regulación diferencial de genes involucrados en las rutas del ácido jasmónico, las citoquininas y del ácido giberélico en las plantas transgénicas. Estos resultados sugieren que PpeDAM6 actúa como un represor máster del crecimiento, manteniendo la latencia, la respuesta de tolerancia a estrés y la inhibición floral a través de la regulación del equilibrio hormonal. Con todo ello, esta tesis proporciona una instantánea dinámica de los diferentes mecanismos moleculares que tienen lugar dentro de la yema. Los genes estudiados tienen una función crucial regulando tanto el proceso de latencia como la respuesta de tolerancia a estrés y las rutas de floración, y todos ellos son potenciales candidatos para mejorar nuevas plantas más adaptadas al cambio climático. / [CA] L'objectiu general d'aquesta tesi és l'estudi de la latència des d'un punt de vista molecular, identificant mecanismes i gens diana que la controlen. Per això, ens hem centrat en l'estudi de tres gens que s'expressen d'una manera diferencial durant el desenvolupament d'una gemma reproductiva en el préssec, sota el marc conceptual dels tres processos que convergeixen espacialment i temporalment en una gemma reproductiva: latència, tolerància a estrés i desenvolupament floral. El primer gen d'estudi codifica per a una STRESS ASSOCIATED PROTEIN (PpSAP1) amb dos dominis tipus Zn-finger, A20 i AN1, i disminueix la seua expressió durant la latència. Les proteïnes tipus SAP s'han relacionat amb resistències a diferents tipus d'estrés tant en plantes com en animals. De fet, s'ha vist que PpSAP1 va augmentar la seua expressió en gemmes de préssec sota condiciones d'estrés per sequia, de manera similar a com ho fan altres SAPs en diferents plantes. A més, l'expressió ectòpica de PpSAP1 en pruneres transgèniques ha permés augmentar la tolerància a estrés en aquestes línies en incrementar la quantitat d'aigua retinguda. Així mateix, aquestes plantes trnasgèniques també mostraren alteracions en la mida de les fulles, causades principalmente per una menor àrea cel¿lular de les cèl¿lules que formen part d'elles i relacionades amb una repressió de diferents gens implicats en el creixement cel¿lular. Tot aço, suggereix que PpSAP1 probablement tinga una doble funció relacionada tant amb resistència a estrés com amb creixement cel¿lular durant la latència del préssec. El segon gen d'estudi va ser una PpeS6PDH, la qual codificava per a un enzim amb activitat sorbitol-6-fosfato dehidrogenasa. PpeS6PDH està diferencialment regulada durant el desenvolupament de la gemma, augmentant la seua expressió en gemmes latents de manera consistent a l'acumulació de sorbitol. Simultàniament a la disminució de PpeS6PDH en les gemmes no latents, al voltant del lloc d'iniciació de la traducció del gen es van mostrar canvis a nivell de cromatina en l'estat de metilació dels residus específics de la històna H3 (H3K4 i H3K27). Aquestes dades assenyalen l'existència d'una regulació transcripcional de PpeS6PDH a nivell de modificacions de la cromatina. A més, també s'ha vist que diferents tipus d'estrés abiòtic afecten a l'expressió de PpeS6PDH. Tractaments amb baixes temperatures van induir la seua expressió tant en gemmes com en fulles, mentres que la desecació va augmentar l'expressió en gemmes però no en fulles. Aquests estudis suggereixen que la funció de PpeS6PDH durant la latència del préssec és donar tolerància a estrés per fred i sequia. Finalment, el tercer gen d'estudi va ser PpeDAM6, un dels majors reguladors de la latència en gemmes de préssec. PpeDAM6 està fortament représ durant el desenvolupament de la gemma amb una relació directa amb els events d'eixida de la latència. Aquesta repressió és deguda en part a la unió directa de PpeBPC1, una BASIC PENTACUSTEINE PROTEIN, a dos motius GAGA presents en la regió intrònica reguladora de PpeDAM6. Justament aquesta regió es troba modificada a nivell de cromatina amb un enriquiment en H3K27me3 després de l'eixida de latència. A més, l'expressió ectòpica de PpeDAM6 en Arabidopsis va mostrar fenotips de floració anormal semblants als produïts en plantes 35S::SVP. Per un altra banda, la sobreexpressió en pruneres va provocar retards en el creixement de les línies transgèniques a causa d'una alteració en els nivells hormonals. Aixi mateix, es va determinar que aquests canvis en l'homeostasi hormonal estaven produïts per la regulació diferencial de gens involucrats en les rutes d'àcid jasmònic, citoquinines i àcid gibberèl·lic en les plantes transgèniques. Aquests resultats suggereixen que PpeDAM6 actua com un repressor master del creixement, mantenint la latència, la resposta de tolerància a estrés i la inhibició floral a través de la regulació de l’equilibri hormonal. Com a conclusió, aquesta tesi proporciona una instantània dinàmica dels diferents mecanismes moleculars que tenen lloc dins de la gemma. Els gens estudiats tenen una funció fonamental, regulant tant el mateix procés de la latència com la resposta de tolerància a estrés i les rutes de floració i tots ells són potencials candidats per a millorar noves plantes més adaptades al canvi climàtic. / Lloret Compañ, A. (2020). A dynamic snapshot of bud dormancy in peach [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153795 / TESIS

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20817号 / 農博第2257号 / 新制||農||1056(附属図書館) / 学位論文||H30||N5099(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 北島 薫, 教授 神﨑 護, 教授 小杉 緑子 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

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In dioecious plants, selection due to sex function differences has produced sex-specific life histories, morphologies, and physiologies. In many dioecious seed plants, dimorphisms and population sex ratios have been plausibly linked, but similar links are not yet apparent in dioecious bryophytes. Population sex ratio bias is often expected to favor the sex with lower investment in sexual reproduction, especially in resource-poor environments. Unlike in seed plants, bryophyte males may have higher average reproductive investment than females, which typically have low offspring production rates due to sperm limitation. However, traits aside from reproductive investment such as shoot and leaf arrangement may be differentially selected and could influence life history and sex ratio, but these are rarely tested. My questions concentrated on the dimorphic traits responsible for sex ratio bias and their links to sex function. My studies, using the moss Bryum argenteum, included field and greenhouse experiments investigating sex ratio bias and morphological plasticity along a light/canopy openness (exposure) gradient, a greenhouse comparison of clump morphology and water-holding capacity, and a field and growth chamber study on sex-specific responses to stress (high temperature and desiccation). The sex ratio of urban Lexington, KY was highly female-biased, did not correlate with exposure, and was not linked with pre-zygotic reproductive investment. Leaf characteristics of B. argenteum plastically responded to exposure but were not sex-specific. However, juvenile females produced shoots at a faster rate and grew taller in high light. Juvenile male shoots held more external water than female shoots, but this did not predict mature clump water-holding capacity. Male clumps were shorter, denser, and held less water than females likely to shed sperm-laden water for sexual reproduction. Clump height did not trade off with reproductive investment, adding evidence that sex-specific size is linked with other aspects of sex function. Although chlorophyll fluorescence data (a measure of the status of photosystem II) from both field and growth chamber experiments indicated subtle sex-specific stress recovery responses among sexually immature and mature plants, differences were weaker than predicted and sexually mature shoots did not fare worse than vegetative shoots. The sex differences in size, clump morphology, and clump water-holding capacity very likely affect survival, growth, competitive ability, and ultimately adult sex ratio bias.

O estresse de calor prejudica o crescimento e reprodução dos organismos vegetais, ao alterar a permeabilidade de membranas biológicas e desnaturar proteínas, limitando o metabolismo primário. Dentre as respostas ao estresse abiótico, está a síntese de metabólitos secundários. Braquicerina é um alcaloide monoterpeno indólico com ação antioxidante, protetora contra UV e antimutagênica sintetizado por partes aéreas de Psychotria brachyceras. O objetivo deste trabalho é investigar o metabolismo de braquicerina sob estresse de calor. Assim, espera-se contribuir para o conhecimento acerca do metabolismo secundário nas respostas ao estresse de calor, descrever a função in planta do composto, e fornecer ferramentas para obtenção do alcaloide para fins farmacêuticos. O acúmulo de braquicerina foi induzido em discos foliares mantidos a 40ºC por três dias, tanto em regime de elevação abrupta como gradual da temperatura. Baixa temperatura (10ºC) não afetou o acúmulo do alcaloide. Discos foliares de Psychotria carthagenensis, uma espécie que não sintetiza alcaloides monoterpeno indólicos, foram também desafiados por estresse de calor. Clorofila total, teor de peróxido de hidrogênio e peroxidação lipídica foram quantificados em ambas as espécies. P. carthagenensis foi relativamente tolerante ao calor, o que pode estar relacionado à sua elevada concentração de antocianinas, fortemente induzidas por choque térmico de 50ºC por 6h. Peroxidação lipídica foi reduzida nas amostras de P. brachyceras sob estresse de calor agudo ou gradual em comparação à condição controle, sendo este parâmetro inalterado nas duas condições em P. carthagenensis. O teor de peróxido de hidrogênio foi menor em P. brachyceras submetida a choque de térmico em relação ao controle, enquanto o mesmo parâmetro não foi alterado em P. carthagenensis. Discos foliares das espécies sensíveis ao calor Brugmansia suaveolens e Brassica oleracea, pré-tratadas com braquicerina em concentrações similares às encontradas em P. brachyceras, adquiriram fenótipo tolerante ao choque térmico. A expressão do gene que codifica a enzima triptofano descarboxilase (TDC), envolvida na biossíntese de braquicerina em P. brachyceras, foi fortemente inibida em discos foliares submetidos à 40ºC por 6h, 12h e 24h, sugerindo que o efeito da temperatura na estimulação de acúmulo de alcaloide ocorra em nível pós-transcricional. Em conjunto, os dados indicam que a exposição ao calor é um meio efetivo de aumentar o rendimento de braquicerina, cuja acumulação contribui para proteção contra os danos oxidativos associados. / Heat stress impairs plant growth and reproduction by altering membrane permeability and promoting protein denaturation, which limits primary metabolism. Secondary metabolites often take part in protection against abiotic stress responses. Brachycerine is a monoterpene indole alkaloid with antioxidant, UV protectant, and antimutagenic activity synthesized by Psychotria brachyceras shoots. Brachycerine metabolism was analyzed under heat stress, in order to shed light on brachycerine‘s in planta function and to provide potential tools to improve alkaloid yields for pharmaceutical analysis. Accumulation was induced in leaf disks kept at 40ºC for three days, both by abrupt and stepwise temperature increase. Brachycerine concentration was not affected by low temperature (10ºC) exposure. Leaf disks of Psychotria carthagenensis, a species devoided of alkaloids, were also challenged by heat. Total chlorophyll, hydrogen peroxide and lipid peroxidation concentrations were determined in both species. P. carthagenensis was relatively tolerant to heat treatments which may be explained by its high anthocyanin concentration, strongly induced by heat shock of 50ºC for 6h. Brugmansia suaveolens and Brassica oleracea, pre-treated with brachycerine in concentrations equivalent to those found in P. brachyceras, had a heat shock tolerant phenotype, based on chlorophyll content. Expression of the TRYPTOPHAN DECARBOXYLASE gene, which encodes for an enzyme involved in alkaloid biosynthesis (TDC) was strongly repressed in leaf disks exposed to 40ºC for 6h, 12h e 24h, suggesting that temperature effect may occur at post-transcriptional level. Taken together, data indicate that heat exposure is an effective means to increase yields of brachycerine, whose accumulation contributes to protect against associated oxidative damage.

Abiotic and biotic stresses affect tree growth and play a major role in determining the geographic distribution of species. The objective of this study is to elucidate the following questions: (1) are GABA aminoacid and stomatal control good indicators of tolerance to water stress in Eucalyptus clones? In addition, what are the anatomical differences between drought-tolerant and drought-sensitive clones of Eucalyptus? (2) Are there differences of xylem vulnerability to cavitation in Pinus flexilis families susceptible and resistant to white pine blister rust (WPBR) and with different origins (high and low altitudes)? Two studies were carried out to elucidate the issues above. On chapters 1, eight Eucalyptus clones from different geographical and climatological conditions, three drought-sensitive (CNB, FIB and JAR), three drought-tolerant (GG, SUZ and VM), and two plastics (VER and COP), were studied in normal water supply (control treatment) and in water stress conditions (stress treatment). The first chapter concluded that GABA is an aminoacid very sensitive to water stress, but there was no relation between GABA concentration and tolerance to water stress of the clones. In addition, all clones decreased stomatal conductance with increasing vapor pressure deficit, and plastics and drought-tolerant clones (except GG) presented lower stomatal sensitivity to vapor pressure deficit under stress conditions than drought-sensitive clones. Besides, all clones showed differences on the anatomical parameters between, and only COP (plastic) and SUZ (drought-tolerant) showed homogeneous mesophyll and amphi-hipostomatic leaves. All clones increased the number of stomata and reduced leaf thickness of the leaves formed after water stress period. On the chapter 2, we studied 12 families of Pinus flexilis originating from high and lower altitudes, in which six families previously shown to contain the dominant C4 allele (resistant to WPBR) and six families without C4 allele (susceptible to WPBR). This study showed that the mean cavitation pressure (MCP) of Pinus flexilis varying between 3.63 a -4.84 Mpa, although there was a significant difference in vulnerability to cavitation comparing all families, this variable was not related to WPBR and origin region. These studies highlight that the physiological responses of plants under water stress conditions are important tools that can be used to complement the strategies of genotype selection in forest breeding programs. / Estresses abióticos e bióticos podem afetar o crescimento das árvores e desempenham um papel importante na determinação da distribuição geográfica das espécies. O objetivo deste estudo, foi elucidar as seguintes questões: (1) o aminoácido GABA e o controle estomático são bons indicadores da tolerância ao estresse hídrico em clones de Eucalyptus? E quais são as diferenças anatômicas entre clones de Eucalyptus tolerantes e sensíveis ao estresse hídrico? (2) existem diferenças de vulnerabilidade a cavitação do xilema entre famílias de Pinus flexilis suscetíveis e resistentes à ferrugem do pinho branco (WPBR) e com diferentes procedências (elevada e baixa altitudes)? Dois estudos foram desenvolvidos para elucidar as questões acima descritas. No capítulo 1, oito clones de Eucalyptus de diferentes procedências e condições climáticas, sendo três clones sensíveis ao estresse hídrico (CNB, FIB e JAR), três clones tolerantes ao estresse hídrico (GG, SUZ e VM) e dois clones plásticos (VER e COP), foram estudados sob duas condições distintas: sob adequado suprimento de água (tratamento controle) e sob condições de estresse hídrico (tratamento estresse). Do primeiro capítulo concluiu-se que o GABA é um aminoácido que possui alta sensibilidade ao estresse hídrico, no entanto, não houve relação entre a concentração de GABA e os níveis de tolerância ao estresse hídrico dos clones. Além disso, todos os clones reduziram a condutância estomática em relação ao aumento do déficit de pressão de vapor (DPV), sendo que, sob condições de estresse hídrico, os clones plásticos e tolerantes à seca (exceto o clone GG) apresentaram menor sensibilidade estomática ao DPV do que os clones sensíveis ao estresse hídrico. Além disso, todos os clones apresentaram diferenças anatômicas, sendo que, diferentemente dos demais, os clones COP (plástico) e SUZ (tolerante) apresentaram mesofilo homogêneo e folhas anfi-hipoestomáticas. Todos os clones aumentaram a quantidade de estômatos e reduziram a espessura foliar das folhas formadas após períodos de estresse hídrico. No segundo capítulo foram avaliadas 12 famílias de Pinus flexilis procedentes de regiões de baixa e alta altitudes, sendo seis famílias contendo um alelo dominante C4 (resistente à WPBR) e seis famílias sem o alelo C4 (suscetíveis à WPBR). Este estudo apresentou uma variação da pressão média da cavitação (MCP) para Pinus flexilis de -3,63 a -4,84 Mpa, e embora tenha havido uma diferença significativa da susceptibilidade a cavitação entre todas as famílias estudadas, esta variável não relacionou-se com a susceptibilidade a doença WPBR e com a região de procedência das famílias. Estes estudos comprovam que a avaliação das respostas fisiológicas das plantas sob condições de estresse hídrico são importantes ferramentas que podem ser utilizadas para complementar as estratégias da seleção de genótipos em programas de melhoramento florestal.

Le climat alpin à plus de 2400 mètres d’altitude montre des fortes variations de température, des intensités lumineuses très élevées (3000 µmol photons m-2 s-1) qui sont connues pour générer un état de réduction importante de la chaine de transport des électrons photosynthétique. Le bon fonctionnement du processus photosynthétique est primordial pour les quelques espèces de plantes vasculaires qui sont présentes à l’étage alpin et qui doivent terminer leur cycle de vie lors d’une très courte période de végétation.Soldanella alpina et Ranunculus glacialis sont deux espèces inféodées aux étages alpin et nival. Dans leur site naturel de croissance nous avons mesuré des températures faibles (0.7°C) et fortes (37°C) sous des lumières supérieures à 2500 µmol photons m-2 s-1. Chez les espèces non-alpines ces conditions induisent la photoinhibition du PSII, ce qui est évité chez S. alpina et R. glacialis, par des mécanismes très différents. Les systèmes antioxydants et le quenching non photochimique sont particulièrement importants chez S. alpina. Chez Ranunculus glacialis, la photorespiration reste très importante et un contenu élevé en PTOX est décrit. Le rôle des antioxydants et de la PTOX dans la photoprotection des deux espèces ont été étudiés. Dans une partie de thèse, nous avons montré qu’une diminution de la capacité antioxydante par une diminution de la concentration en glutathion n’affecte pas la tolérance vis-à-vis de la photoinhibition à basse température. Dans une deuxième partie les résultats supposent qu’une surexpression de la PTOX chez le tabac augmente la photoinhibition à lumière forte par production des espèces réactives d’oxygène. En utilisant différentes conditions environnementales de croissance pour Ranunculus glacialis, nous avons pu montrer que l’expression de la PTOX est induite par des fortes lumières et non par des basses températures. Grâce à une approche associant mesures d’échanges gazeux et mesures de la fluorescence de la chlorophylle, nous avons montré qu’un flux d’électrons conséquent vers l’oxygène, indépendant de la photorespiration, corrélait avec la présence de la PTOX mais que l’activité de la PTOX sous des conditions qui permettent l’assimilation du CO2 et la photorespiration n’est pas maximale. Grâce à des mesures de fluorescence chlorophyllienne en présence de différents inhibiteurs photosynthétiques, nous avons pu montrer que l’importance de ce flux d’électrons vers l’oxygène corrèle avec la quantité de PTOX présente dans les feuilles, dans des conditions réductrices. Ces résultats nous ont amenés à conclure que chez Ranunculus glacialis, la PTOX peut prendre en charge un flux significatif d’électrons, éviter ainsi l’apparition d’un état réduit de la chaine de transfert photosynthétique, et protéger la plante vis-à-vis de la photoinhibition en agissant comme une valve de sécurité. Ces travaux permettent d’apporter des précisions sur un modèle original de photoprotection, qui a été l’objet de nombreuses controverses. / The alpine climate above 2400 meters altitude shows large variations in temperature and very important light intensity (3000 µmol photons m-2 s-1), which are known to generate a state of significant reduction in the photosynthetic electron transport chain. The proper functioning of the photosynthetic process is essential for vascular plants species that are present in this alpine environment and must complete their life cycle within a very short growing season.Soldanella alpina and Ranunculus glacialis are two species restricted to alpine and snow floors. In their natural growth environment we measured very low (0.7 ° C) and high temperature (37 ° C) under lights above 2500 µmol photons m-2 s-1. Among non-alpine species such conditions induce photoinhibition of PSII, which is avoided in S. alpina and R. glacialis, by very different mechanisms. Antioxidant systems and non-photochemical quenching are particularly important in S. alpina. In Ranunculus glacialis, photorespiration remains very important and a high content of PTOX is described. The roles of antioxidants and PTOX in photoprotection of both species were studied.In one part of the thesis, we showed that a decrease in antioxidant capacity by reducing the concentration of glutathione does not affect tolerance to low-temperature photoinhibition. In the second part the results imply that overexpression of PTOX in tobacco enhances photoinhibition by strong light to produce reactive oxygen species.Using different environmental conditions for Ranunculus glacialis growth, we showed that expression of the PTOX is induced by strong light, but not by low temperatures. With an approach combining gas exchange measurements and chlorophyll fluorescence measurements, we showed that an electron flow to oxygen, independent of photorespiration, correlated with the presence of PTOX. Through measures of chlorophyll fluorescence in the presence of various inhibitors photosynthetic, we could show that the importance of this electron flow to oxygen correlates with the amount of PTOX in the leaves, under reducing conditions. These results led us to conclude that in Ranunculus glacialis, the PTOX may support a significant flow of electrons, thus avoiding the appearance of a reduced state of the photosynthetic chain transfer, and protect the plant from photoinhibition, acting as a safety valve. These studies are discussed to help clarify a new pathway of photoprotection, which was the subject of much controversy.