Plants have spread to almost everywhere in the world. As they disperse, they meet many different environments to which they may be able to adapt. For a plant species to adapt to a new environment, genetic variation is needed. The individuals differ from each other in their genetic composition, which often means differences in phenotypes. Those individuals that manage to reproduce will form the next generation. With different conditions in different environments, it will not be the same phenotypes that reproduce everywhere. In that way, plant species will form into a mosaic of locally adapted populations varying genetically as the species disperses. After the last ice age plants have started to disperse away from the equators. With increasing latitudes come increasing challenges to migrating plants. As plant species disperse northwards along this gradient of varying conditions individuals are selected for cold adaptive traits like flowering time and freezing tolerance, acquired by cold acclimation. In this way, genetic variation from the original populations for these traits becomes sorted out along a latitudinal cline. The aim of this thesis was to understand how selection along a latitudinal gradient has shaped natural variation in cold adaptive traits in plants dispersing northwards, and specifically, to investigate what variation can be observed in phenotypes for these traits and how these traits correlate with genetic variation in genes known to be involved in cold acclimation. In this study significant variation was found in a sample of the model plan Arabidopsis thaliana accessions in cold adaptive traits flowering time and freezing tolerance. A clear latitudinal cline in the cold adaptive traits freezing tolerance for A. thaliana was observed. Analysis of nucleotide polymorphism for the cold responsive ICE1 (inducer of CBF expression 1) transcription factor revealed a haplotype structure with two allelic clades as well as unusually high levels of synonymous polymorphism. Nucleotide polymorphism analysis for the transcription factors CBF1, CBF2 and CBF3 (C-repeat binding factors) that play a key role in regulating the expression of a group of target genes known as the “CBF regulon” showed a distinct geographical haplotype structure. One haplotype was dominant in southern accessions while in the other northern accessions overrepresented. There was a significant effect of CBF haplotype on both freezing tolerance and flowering time even after correcting for latitude. Significant differences in CBF expression levels were found between the different CBF genes as well as between different accessions. Sequence variation at CBF was shown to have a significant effect on expression levels of CBF2. No clear correlations were found between CBF gene expression and freezing tolerance or temperature sensitivity for any of the accessions used in the study. This highlights the complex relationship between sequence variation in candidate genes and gene expression, and the problems associated with unraveling the genetic basis of ecologically important traits.

Invasive non-indigenous species are among the principle drivers of global change, altering nutrient cycles, changing disturbance regimes, and generally threatening biodiversity. Climate change is widely expected to exacerbate invasions by relaxing abiotic barriers, such as low temperature, but the mechanistic evidence supporting this is limited. Here, I evaluate the hypothesis that low temperatures determine the northern range limit of kudzu (Pueraria montana var. lobata), an invasive Asiatic vine in North America, by assessing freezing and chilling tolerance of kudzu plants in winter, spring, summer, and fall. Kudzu was widely planted throughout the southeastern U.S. in the early 20th Century to prevent erosion. It is winter-deciduous and reproduces primarily from buds on stem nodes. In the last 40 years, kudzu has migrated northward in concert with a northward shift in the -20oC minimum winter temperature isocline, indicating that less severe winter cold is permitting northward migration. Freezing mortality during winter does not explain this correlation. Electrolyte leakage assays demonstrate that above- and belowground kudzu stems can survive to -27oC and -17oC. Insulation provided by soil and snow protects belowground stems from lethal temperatures to well north of kudzu's current range limit. Severe spring chill stops growth and photosynthesis and causes some shoot mortality, but both growth and photosynthesis recover quickly following the chill. Summer growth rates are rapid (up to 22 cm/d), responding within hours to temperature changes, and are unimpaired by nighttime lows. Photosynthesis is reduced at cool temperatures, but on cool days, kudzu leaves tend to be warmer than air temperature, so photosynthesis rates generally remain close to optimal values. In autumn, growth stops below 15oC, but leaves are retained and maintain modest photosynthetic competence until killed by frost in November. In colder climates that occur far north of kudzu's current range, reductions in the length and quality of the growing season could accumulate over time to reduce kudzu's success. There is, however, no strong evidence that low temperatures in any season will prevent kudzu from migrating throughout southern Ontario, making kudzu a good candidate for invasive species regulations.

Invasive non-indigenous species are among the principle drivers of global change, altering nutrient cycles, changing disturbance regimes, and generally threatening biodiversity. Climate change is widely expected to exacerbate invasions by relaxing abiotic barriers, such as low temperature, but the mechanistic evidence supporting this is limited. Here, I evaluate the hypothesis that low temperatures determine the northern range limit of kudzu (Pueraria montana var. lobata), an invasive Asiatic vine in North America, by assessing freezing and chilling tolerance of kudzu plants in winter, spring, summer, and fall. Kudzu was widely planted throughout the southeastern U.S. in the early 20th Century to prevent erosion. It is winter-deciduous and reproduces primarily from buds on stem nodes. In the last 40 years, kudzu has migrated northward in concert with a northward shift in the -20oC minimum winter temperature isocline, indicating that less severe winter cold is permitting northward migration. Freezing mortality during winter does not explain this correlation. Electrolyte leakage assays demonstrate that above- and belowground kudzu stems can survive to -27oC and -17oC. Insulation provided by soil and snow protects belowground stems from lethal temperatures to well north of kudzu's current range limit. Severe spring chill stops growth and photosynthesis and causes some shoot mortality, but both growth and photosynthesis recover quickly following the chill. Summer growth rates are rapid (up to 22 cm/d), responding within hours to temperature changes, and are unimpaired by nighttime lows. Photosynthesis is reduced at cool temperatures, but on cool days, kudzu leaves tend to be warmer than air temperature, so photosynthesis rates generally remain close to optimal values. In autumn, growth stops below 15oC, but leaves are retained and maintain modest photosynthetic competence until killed by frost in November. In colder climates that occur far north of kudzu's current range, reductions in the length and quality of the growing season could accumulate over time to reduce kudzu's success. There is, however, no strong evidence that low temperatures in any season will prevent kudzu from migrating throughout southern Ontario, making kudzu a good candidate for invasive species regulations.

Die vorliegende Arbeit beschäftigt sich mit Heterosis in der Frosttoleranz von Arabidopsis-Kreuzungen. Die genetische Basis von Heterosis, der Verbesserung heterozygoter F1-Hybriden gegenüber den homozygoten Eltern, ist unbekannt. Vermutet werden Dominanz, Überdominanz oder Epistasie. Die zur Kreuzung verwendeten Parentalakzessionen entstammen unterschiedlichen Klimaten und differieren stark in ihrer Frosttoleranz (Lethaltemperatur, LT50). Von 24 reziproken Kreuzungen mit C24 und Col-0 wurden LT50, Prolin- und Zuckergehalt bestimmt. Die Untersuchungen an nicht akklimatisierten und kälteakklimatisierten Pflanzen zeigen häufiger Heterosis in C24- als in Col-Hybriden mit klarem Anstieg nach dem Akklimatisieren. Es besteht eine klare Abhängigkeit der Frosttoleranz zum Zucker-, Prolin- und Flavonoidgehalt, sowie zwischen der Stärke der Heterosis in Frosttoleranz und Metabolitgehalten. GCMS-Messungen bestätigen diese Ergebnisse. Es wurden 40 Metabolite detektiert, von denen viele signifikant mit der Frosttoleranz korrelieren und/oder deren Heterosis mit der Heterosis der LT50 korreliert. Sechs dieser Stoffe sind wichtige Komponenten des Citratzyklus, was auf eine Rolle von Teilen des Zyklus in der Heterosis der Frosttoleranz und eine Veränderung seiner Flussraten hindeutet. / We investigated heterosis in freezing tolerance of 24 reciprocal Arabidopsis-crosses with C24 and Col-0. The underlying genetic mechanisms of Heterosis, the enhancement of F1-hybrids in comparison to their homozygous parents, are unknown. Different mechanisms such as dominance, overdominance or epistasis are suggested. Parental freezing tolerance (LT50) was shown to correlate with the original habitat temperature. Besides the LT50, proline and sugar contents (glc, fru, suc, raf) have been measured on non-acclimated and cold-acclimated plants. Metabolite profiling and flavonoid measurements revealed significant stronger heterosis in C24- than in Col-crosses. Heterosis increases after cold acclimation. Freezing tolerance clearly correlate with the contents of sugars, proline and several flavonols, as well as the strength of heterosis in freezing tolerance with the metabolite content. GCMS-measurement confirmed these results. Fourty metabolites, of which many significantly correlate with LT50 and/or with heterosis in metabolite content and in LT50, were found. Six of these are important compounds in the TCA-cycle. Changes in flux rates of the TCA-cycle could be connected to Heterosis for the first time. Negative correlation between Heterosis in freezing tolerance and metabolite accumulation, points to a role of parts of the cycle in crosses and to a change of flux rates.

Biological processes in living organisms consist of a vast number of different molecular networks and interactions, which are complex and often hidden from our understanding. This work is focused on recovery of such details for two quite distant examples: acclimation to extreme freezing tolerance in Siberian spruce (Picea obovata) and detection of proteins associated with prostate cancer. The first biological system in the study, upon P. obovata, is interesting by this species ability to adapt and sustain extremely low temperatures, such as -60⁰C or below. Despite decades of investigations, the essential features and mechanisms of the amazing ability of this species still remains unclear. To enhance knowledge about extreme freezing tolerance, the metabolome and proteome of P. obovata’s needles were collected during the tree’s acclimation period, ranging from mid August to January, and have been analyzed. The second system within this study is the plasma proteome analysis of high risk prostate cancer (PCa) patients, with and without bone metastases. PCa is one of the most common cancers among Swedish men, which can abruptly develop into an aggressive, lethal disease. The diagnostic tools, including PSA-tests, are insufficient in predicting the disease’s aggressiveness and novel prognostic markers are urgently required. Both biological systems have been analyzed following similar steps: by two-dimensional difference gel electrophoresis (2D-DIGE) techniques, followed by protein identification using mass spectrometry (MS) analysis and multivariate methods. Data processing has been utilized for searching for proteins that serve as unique indicators for characterizing the status of the systems. In addition, the gas chromatography-mass spectrometry (GC-MS) study of the metabolic content of P.obovata’s needles, from the extended observation period, has been performed. The studies of both systems, combined with thorough statistical analysis of experimental outcomes, have resulted in novel insights and features for both P. obovata and prostate cancer. In particular, it has been shown that dehydrins, Hsp70s, AAA+ ATPases, lipocalin and several proteins involved in cellular metabolism etc., can be uniquely associated with acclimation to extreme freezing in conifers. Metabolomic analysis of P. obovata needles has revealed systematic metabolic changes in carbohydrate and lipid metabolism. Substantial increase of raffinose, accumulation of desaturated fatty acids, sugar acids, sugar alcohols, amino acids and polyamines that may act as compatible solutes or cryoprotectants have all been observed during the acclimation process. Relevant proteins for prostate cancer progression and aggressiveness have been identified in the plasma proteome study, for patients with and without bone metastasis. Proteins associated with lipid transport, coagulation, inflammation and immune response have been found among them.

El óxido nítrico (NO) es una molécula gaseosa altamente reactiva que regula el crecimiento y el desarrollo de las plantas así como sus respuestas de defensa. El NO se produce principalmente a partir de nitrito por las nitrato reductasas (NRs) en balance con las nitrito reductasas (NiRs), y es percibido a través de un mecanismo en el que está involucrada la proteólisis dirigida por la secuencia aminoterminal del grupo VII de los factores de transcripción ERF (ERFVIIs). El NO ejerce especialmente su función señalizadora al causar modificaciones postraduccionales en las proteínas y alterar su función, estructura y/o estabilidad. Por estos medios y en colaboración con distintas rutas de señalización fitohormonales, el NO es capaz de regular un amplio abanico de procesos celulares en plantas, incluyendo aquellos relacionados con la adquisición de tolerancia a la congelación. Utilizando Arabidopsis thaliana como planta modelo, en este trabajo se descubrió que el NO puede regular su propia biosíntesis, puesto que las enzimas NRs y NiRs fueron reguladas por tres factores principales: señalización inducida por nitrato y controlada por la función del factor de transcripción NIN-like protein 7 (NLP7), la proteólisis dirigida por la secuencia aminoterminal, y la degradación mediada por el proteasoma, probablemente ocasionada por modificaciones postraduccionales relacionadas con el NO. Adicionalmente, se descubrió que el factor de transcripción ERFVII RAP2.3 regula negativamente tanto la biosíntesis de NO como las respuestas que desencadena a través de un mecanismo similar a un reóstato en el que están involucradas ramas específicas relacionadas con el NO de las rutas de señalización de jasmonato y ácido abscísico. Por otro lado, una caracterización metabolómica y transcriptómica combinada de plantas mutantes nia1,2noa1-2 deficientes en NO y plantas fumigadas con NO permitió desentrañar una serie de mecanismos que están controlados por NO. En primer lugar, la percepción de NO en los hipocotilos requeriría varias hormonas para ser completada, como fue confirmado por los rastreos de acortamiento de hipocotilo por NO con mutantes relacionados con hormonas y la colección TRANSPLANTA de líneas transgénicas que expresan condicionalmente factores de transcripción de Arabidopsis. En segundo lugar, dosis elevadas de NO causan una reprogramación masiva aunque transitoria de los metabolismos primario y secundario, incluyendo la alteración del estado redox celular, la alteración de la permeabilidad de estructuras lipídicas y el recambio de proteínas y ácidos nucleicos. Por último, se descubrió que el NO previene el desarrollo de la tolerancia a congelación bajo condiciones no estresantes de temperatura, mientras que resulta esencial para la aclimatación a frío desencadenada por bajas temperaturas que conduce a una tolerancia mejorada a congelación. El NO conseguiría esta modulación afinada de la activación de respuestas relacionadas con frío al coordinar la acumulación de diferentes metabolitos y hormonas. En conjunto, este trabajo arroja luz sobre los mecanismos mediante los cuales, al interactuar con varias rutas señalizadoras y metabólicas, el NO puede regular distintos procesos clave de la fisiología vegetal. / L'òxid nítric (NO) és una molècula gasosa altament reactiva que regula el creixement i desenvolupament de les plantes així com les seves respostes de defensa. El NO es produeix principalment a partir de nitrit per les nitrat reductases (NRs) en balanç amb les nitrit reductases (NiRs), i és percebut a traves d'un mecanisme que inclou la proteòlisi dirigida per la seqüència aminoterminal del grup VII dels factors de transcripció ERF (ERFVII). El NO exerceix la seva funció senyalitzadora majoritàriament al provocar modificacions postraduccionals en les proteïnes i alterar la seva funció, estructura i/o estabilitat. Mitjançant aquestes modificacions i en col·laboració amb distintes rutes de senyalització fitohormonals, el NO es capaç de regular un ampli espectre de processos cel·lulars en plantes, inclosos aquells relacionats amb l'adquisició de tolerància a la congelació. Emprant Arabidopsis thaliana com a planta model, en aquest treball es va descobrir que el NO regula la seva pròpia biosíntesi, donat que els enzims NRs i NiRs foren regulades per tres factors principals: senyalització induïda per nitrat i controlada per la funció del factor de transcripció NIN-like protein 7 (NLP7), la proteòlisi dirigida per la seqüència aminoterminal, i la degradació mitjançant el proteasoma, probablement a causa de modificacions postraduccionals relacionades amb el NO. A més, es va descobrir que el factor de transcripció ERFVII RAP2.3 regula negativament tant la biosíntesi de NO com les respostes que desencadena aquest a través d'un mecanisme similar a un reòstat en el que estan involucrades branques específiques de les rutes de senyalització de jasmonat i àcid abscísic relacionades amb el NO. Per altre costat, una caracterització metabolòmica i transcriptòmica combinada de plantes mutants nia1,2noa1-2 deficients en NO i plantes fumigades amb NO va permetre desentranyar una sèrie de mecanismes que estan controlats per NO. En primer lloc, la percepció de NO en els hipocòtils requeriria de varies hormones, com fou confirmat pels rastrejos d'acurtament d'hipocòtil per NO amb mutants relacionats amb hormones i la col·lecció TRANSPLANTA de línies transgèniques d'expressió condicional de factors de transcripció d'Arabidopsis. En segon lloc, dosis elevades de NO causen una reprogramació massiva, encara que transitòria, dels metabolismes primari i secundari, incloent l'alteració de l'estat redox cel·lular, canvis en la permeabilitat de estructures lipídiques i el recanvi de proteïnes i àcids nucleics. Per últim, es va descobrir que el NO prevé el desenvolupament de la tolerància a congelació en condicions no estressants de temperatura, mentre que resulta essencial per a l'aclimatació a fred induïda per baixes temperatures que condueix a una tolerància millorada a congelació. El NO aconseguiria aquesta modulació minuciosa de l'activació de les respostes relacionades amb fred al coordinar l'acumulació de diferents metabòlits i hormones. En conjunt, aquest treball clarifica els mecanismes pels quals el NO pot regular distints processos clau de la fisiologia vegetal al interactuar amb varies rutes senyalitzadores i metabòliques. / Nitric oxide (NO) is a highly reactive gaseous molecule that regulates plant growth and development as well as defense responses. NO is mainly produced from nitrite by nitrate reductases (NRs) in balance with nitrite reductases (NiRs), and is sensed through a mechanism involving the N-end rule pathway-mediated proteolysis of the group VII of ERF transcription factors (ERFVIIs). NO especially exerts its signaling function by triggering post-translational modifications in proteins and altering their function, structure and/or stability. By these means and in collaboration with different phytohormone signaling pathways, NO is capable of regulating a wide array of cell processes in plants, including those related to the acquirement of freezing tolerance. By using Arabidopsis thaliana as model plant, during the development of this work it was found that NO can regulate its own biosynthesis, as NRs and NiR enzymes were regulated by three main factors: nitrate-induced signaling controlled by the function of the NIN-like protein 7 (NLP7) transcription factor, N-end rule proteolytic pathway, and proteasome-mediated degradation, likely triggered by NO-related post-translational modifications. In addition, the ERFVII transcription factor RAP2.3 was found to negatively regulate both the NO biosynthesis and their triggered responses through a rheostat-like mechanism that involves specific NO-related branches of jasmonate and abscisic acid signaling pathways. On the other hand, a combined metabolomic and transcriptomic characterization of NO-deficient nia1,2noa1-2 mutant plants and NO-fumigated plants allowed to unravel a number of mechanisms that are controlled by NO. First, NO perception in hypocotyls would require various hormones to be fulfilled as it was confirmed by NO-triggered hypocotyl shortening screenings with hormone-related mutants and the TRANSPLANTA collection of transgenic lines conditionally expressing Arabidopsis transcription factors. Second, high NO doses caused a massive but transient reprogramming of primary and secondary metabolism, including alteration of the cellular redox status, alteration of the permeability of lipidic structures or turnover of proteins and nucleic acids. Lastly, NO was found to prevent the development of freezing tolerance under non-stress temperature conditions, while being essential for the low temperature stress-triggered cold acclimation that leads to enhanced freezing tolerance. NO would achieve this fine-tuned modulation of the activation of the cold-related responses by coordinating the accumulation of different metabolites and hormones. Altogether, this work sheds light on the mechanisms by which, by interacting with various signaling and metabolic pathways, NO can regulate several key processes of plant physiology. / Costa Broseta, Á. (2018). Regulation of the nitric oxide synthesis and signaling by posttranslational modifications and N-end rule pathway-mediated proteolysis in Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/114825 / TESIS