Dehydrins are intrinsically disordered plant stress-proteins. Repetitively in their sequence are some highly conserved stretches of 7-17 residues, the so called K-, S-, Y- and lysine rich segments. This thesis aims to give insight into the possible role dehydrins have in the stressed plant cell with main focus on membrane interaction and protection. The work includes four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47 (SK3), Lti29 (SK3), Lti30 (K6) and Rab18 (Y2SK2). Initially, we mimicked crowded cellular environment in vitro to verify that dehydrins are truly disordered proteins. Thereafter, the proposal that the compulsory K-segment determines membrane binding was tested. Experiments show that only Lti30 and Rab18 bind, whereas Cor47 and Lti29 does not. As Lti30 and Rab18 binds they assembles vesicles into clusters in vitro, a feature used to characterize the interaction. From this it was shown that membrane binding of Lti30 is electrostatic and determined by global as well as local charges. Protonation of histidine pairs flanking the K-segments works as an on/off-binding switch. By NMR studies it was shown that the K-segments form a dynamic α-helix upon binding, so called disorder-to-order behaviour. Also, dehydrins electrostatic interaction with lipids can be further tuned by posttranslational phosphorylation or coordination of calcium and zinc ions. Finally, specific binding of Rab18 to inositol lipids, mainly PI(4,5)P2, is reported. The interaction is mainly coordinated by two arginines neighboring one of the K-segments. In conclusion, the K-segments are indeed involved in the binding of dehydrins to membrane but only in combination with extensions (Lti30) or modified (Rab18). / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>

Orientador: Gisela Ferreira / Resumo: Para que as plantas conseguissem conquistar o ambiente terrestre, foi necessário desenvolver habilidades que as tornassem capazes de sobreviver aos desafios de seu habitat, dentre as quais estão o desenvolvimento de mecanismos de tolerância à dessecação das sementes. Espécies que habitam locais com grande disponibilidade de água tendem a produzir sementes recalcitrantes e por outro lado, as espécies que habitam locais secos, como os Cerrados, tendem a produzir sementes ortodoxas, conseguindo tolerar períodos de seca sem acumular danos que prejudiquem a germinação. Dentre os sistemas que atuam no mecanismo de tolerância à dessecação estão a atividade de enzimas antioxidantes, o acúmulo de açúcares e a atividade das proteínas da embriogênese tardia (LEA). A família Annonaceae possui espécies inseridas em habitats contrastantes, como a Annona crassiflora, oriunda do Cerrado e a Annona glabra, oriunda de manguezais o que as torna interessantes para avaliar se suas sementes apresentam diferentes respostas frente à tolerância à dessecação. Para alcançar esse objetivo, sementes das duas espécies foram coletadas e avaliadas após submissão a diferentes níveis de secagem (teor inicial de água, 20%, 10% e 5%) e secagem seguida de reidratação. Foram realizados testes de germinabilidade, quantificação enzimática (superóxido dismutase (SOD) (EC 1.15.1.1), peroxidase (POD) (EC 1.11.1.7) e catalase (CAT) (EC 1.11.1.6)); de açúcares (frutose, galactose, glicose, manose, sacarose e trealose); ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In order for plants to conquer the terrestrial environment, it was necessary to develop skills that would enable them to survive the challenges of their habitat, including the development of seed desiccation tolerance mechanisms. Species that inhabit places with high water availability tend to produce recalcitrant seeds and, on the other hand, species that inhabit dry places, such as Cerrados, tend to produce orthodox seeds, being able to tolerate periods of drought without accumulating germinating damage. Systems that act on the desiccation tolerance mechanism include antioxidant enzyme activity, sugar accumulation, and late embryogenesis (LEA) protein activity. The Annonaceae family has species in contrasting habitats, such as Annona crassiflora, from the Cerrado and Annona glabra, from mangroves, which makes them interesting to evaluate if their seeds have different responses to desiccation tolerance. To achieve this goal, seeds of both species were collected and evaluated after submission to different drying levels (initial water content, 20%, 10% and 5%) and drying followed by rehydration. Germinability, enzymatic quantification (superoxide dismutase (SOD) (EC 1.15.1.1), peroxidase (POD) (EC 1.11.1.7) and catalase (CAT) (EC 1.11.1.6) tests were performed; sugars (fructose, galactose, glucose, mannose, sucrose and trehalose); determination of LEA proteins and lipoperoxide content. The results showed that A. crassiflora seeds were able to tolerate drying up to 10% water co... (Complete abstract click electronic access below) / Mestre

In order to improve our understanding on LEA proteins and their molecular functions in drought tolerance, the present work analyzes in the first place, the composition of LEA subproteomes from Arabidopsis seeds and maize embryos; second, three maize embryo LEA proteins from groups 1, 2, and 3 are analyzed in order to detect functional differences among them and finally, transgenic maize plants over-expressing group 5 “rab28” lea gene are characterized. The following results are presented: - Chapter 1. Proteomic approach to analyze the composition of LEA subproteomes from Arabidopsis seeds by mass spectrometry. The main objective was the development and isolation method to obtain enriched LEA populations from Arabidopsis seeds. LEA subproteomes were obtained using an extraction procedure that combines heat stability and acid solubility of LEA proteins. To identify the protein content, we followed two approaches: first, a classical 1D (SDSPAGE) gel-based procedure associated with MS analysis using an electrospray ionization source coupled on-line to liquid chromatography (LC-ESI-MSMS) and second, a gel-free protocol associated with an off-line HPLC and analysis via matrix assisted laser desorption/ionization (LC-MALDI-MSMS). - Chapter 2. Proteomic analysis of LEA proteins accumulated in maize mature seeds. Identification of LEA protein content by mass spectrometry and selection of three LEA proteins, Emb564, Rab17 and Mlg3, as representatives of groups 1, 2 and 3 for further study. Comparative functional analysis covering different aspects of maize Emb564, Rab17 and Mlg3 proteins, posttranslational modifications, subcellular localization and their properties in in-vitro and in- vivo assays. - Chapter 3. Characterization of transgenic maize plants expressing maize group 5 rab28 LEA gene under the ubiquitin promoter. Evaluation of Rab28 transcripts and protein levels, phenotype and stress tolerance traits of transgenic plants under drought stress. Investigation of the subcellular localization of transgenic and wild-type Rab28 protein using immunocytochemical approaches. / Las proteínas LEA, originalmente fueron descritas en las semillas de algodón; se acumulan en grandes cantidades en estructuras tolerantes a la desecación (semillas, polen) y en tejidos vegetativos sometidos a estrés abiótico, sequía, salinidad y frío. También se hallan en organismos anidrobióticos, en plantas de resurrección, algunos invertebrados y microorganismos. La presencia de proteínas LEA se correlaciona con la adquisición de tolerancia a la desecación. Desde un principio se les atribuyó un papel en las respuestas de las plantas en la adaptación al estrés (revisado en Bartels and Salamini 2001, Tunnacliffe 2007, Shih et al. 2010, Tunnacliffe 2010, Hand et al. 2011). Las proteínas LEA se clasifican en diversos grupos en función de dominios y secuencias de aminoácidos específicos (Wise 2010, Batagglia et al 2008, Bies-Ethève et al 2008). Los grupos 1, 2 y 3 son los más relevantes ya que abarcan la mayoría de las proteínas de la familia LEA. Una característica general de estas proteínas es su elevada hidrofilicidad, alto contenido de aminoácidos cargados y su falta de estructura en estado hidratado. A pesar de encontrarse mayoritariamente en forma de “random coil”, algunas adquieren un cierto grado de estructura durante la deshidratación o en la presencia de agentes promotores de α-hélices (Shih et al. 2010, Hand et al. 2011). A nivel celular se han hallado en todas las localizaciones, citosol, núcleo, nucleolo, mitocondria, cloroplasto, vacuola, retículo endoplásmico, peroxisoma y membrana plasmática, donde se supone ejercen su función protectora frente al estrés (Tunnacliffe and Wise 2007, Hundertmark and Hincha 2008). En relación a las modificaciones post-traduccionales, algunas se hallan fosforiladas (Jiang and Wang 2004; Plana et al. 1991, Heyen et al. 2002, Rohrig et al. 2006). Los efectos protectores de las varias proteínas LEA se han demostrado mediante ensayos in vitro y en aproximaciones transgénicas que han dado lugar a fenotipos resistentes a la sequía, sal y frío. Por lo general, se considera que estas proteínas contribuyen a la protección y a la estabilización de macromoléculas y estructuras celulares en las respuestas de adaptación al estrés en plantas; sin embargo, sus funciones específicas aún no han sido esclarecidas. A nivel molecular se ha propuesto que las funciones de las proteínas LEA pueden ser variadas: estabilización y renaturalización de proteínas, mantenimiento de membranas, en combinación, o no, con azúcares, tampones de hidratación (substitución de moléculas de agua), afinidad por iones y función antioxidante (Tunnacliffe and Wise 2007, Shih et al. 2010, Batagglia et al. 2008). Para finalizar, diremos que los objetivos principales de esta tesis consisten en ampliar los conocimientos sobre las proteínas LEA y sus funciones relativas a la tolerancia a la sequía. Los resultados están presentados en forma de capítulos.