Como os outros organismos, as plantas têm a habilidade de se defenderem através do reconhecimento de patógenos (resposta de hipersensibilidade - RH), causando a morte imediata das células no sítio primário da infecção, desta maneira oferecendo resistência ao seu crescimento. A RH é caracterizada pela necrose dos tecidos neste local, através de muitos sinais ainda não completamente elucidados, como a formação de radicais livres, incluindo o peróxido de hidrogênio (H2O2), e o reforço da parede celular. O objetivo deste estudo foi estabelecer a relação entre esses sinais em cultura de células de amora-preta (Rubus fruticosus). As condições experimentais para a análise da parede celular, das espécies reativas de oxigênio (EROs) e do H2O2 foram padronizadas. O polissacarídeo ácido (ramnoglucuronogalactana, F-I), o ácido salicílico (AS), e o metil jasmonato (MeJA), bem estabelecidos efetores da resposta da defesa, foram usados como elicitores. A produção das EROs e do H2O2 foram ativadas por F-I e pelo AS, seguidos da liberação de fragmentos de dissacarídeos da parede celular, aparentemente devido a sua degradação. Por outro lado, uma produção pequena de EROs e de H2O2 foram observadas na presença de MeJA, assim como um aumento de fragmentos de massa molecular mais elevada, que podem funcionar como sinais para o reforço da parede celular, indução de enzimas e para a produção de outra moléculas de defesa. Quando da elicitação, concomitante, com dois elicitores, AS + MeJA, houve a inibição da produção de EROs causada pelo MeJA e foi mantida a liberação de compostos extracelulares de massa molecular mais elevada. / Like the other organisms, plants have the ability to self-defend through recognition of pathogens (hypersensitive response - HR), causing immediate cell death at the primary infection site, thus offering resistance to their grown. The HR is characterized by necrosis of tissues in this site via many signals still not completely elucidated, like formation of free radicals including H2O2 and reinforcement of cell wall. The aim of this study was to establish the relationship between these signals in blackberry-black cell culture (Rubus fruticosus). The experimental conditions for analysis of cell wall, reactive oxygen species (ROS) and H2O2, were established. Acid polysaccharide (rhamnoglucuronogalactan, F-I), salicylic acid (SA), and methyl jasmonate (MeJA), well established effectors of the defense response, were used as elicitors. ROS and H2O2 production was activated by F-I and SA, followed by release of fragments like disaccharides from the cell wall, apparently due to its degradation. By contrast, a small production of ROS and H2O2 was observed in presence of MeJA, as well as an increase of high molecular weight fragments, that may function as signals for reinforcement of cell wall, enzyme induction and production of others defense molecules. Together, the two elicitors SA and MeJA inhibited the ROS production, caused by MeJA, while sustaining release of the extra cellular compounds of high molecular weight.

Secondary metabolism in plants produces numerous compounds with wide-ranging activities, including the antineoplastic compound taxol and related taxanes. The biotechnological production of taxol has so far been based on empirical studies. The aim of the present work has been to study how the factors that enhance taxane production affect the metabolic profiles and gene expression in productive cells. As a consequence of this work, new potential candidates have been obtained for unknown taxane biosynthetic genes, some bottle-neck steps of taxane biosynthesis (in vitro and in silico) have been identified and a master regulator, not only for taxane biosynthesis, but also for other secondary metabolism routes, has been characterized. Coronatine, a powerful and less harmful elicitor than methyl jasmonate, has been successfully assayed and found to increase taxane production. In the different studies of this work, the expression level of genes that participate in taxol biosynthesis has been determined, clarifying their involvement in the production of this anti-cancer agent. / El metabolismo secundario de las plantas produce numerosos compuestos con un amplio rango de actividades, entre los que se encuentra el compuesto antineoplásico taxol y los taxanos relacionados, la producción biotecnológica del cual se basa en estudios empíricos. El objetivo de este trabajo ha sido estudiar como los factores que incrementan la producción de taxanos afectan los perfiles metabólicos y la expresión génica en los cultivos. De esta manera se han identificado nuevos genes candidatos que codifican para los genes desconocidos de la biosíntesis, algunos pasos limitantes de ésta y se ha caracterizado un regulador relacionado con el metabolismo secundario. Se ha ensayado la coronatina, un elicitor más eficiente para mejorar la producción de taxanos y menos dañino que el jasmonato de metilo. En los diferentes ensayos de este trabajo han sido determinados los niveles de expresión de genes que participan en la biosíntesis de taxol, ayudando a comprender su papel en la producción de este anticancerígeno.

The chemical elicitor methyl jasmonate (MeJA) could replace insecticides in Europe and Asia for protection of young conifers against the pine weevil (Hylobius abietis). This thesis mainly focuses on the effects of MeJA treatment on chemical defenses of conifers from seedling batches with documented field resistance. Tissues of three pine species and one spruce species, with various treatments, were here extracted in hexane, whereafter volatile contents of tissues were separated and analyzed by 2D GC-MS. Induced responses of seedlings of Maritime pine (Pinus pinaster) and Monterey pine (Pinus radiata) to the folivore pine processionary (Thaumetopoea pityocampa), and the phloem-feeder H. abietis, have been studied. Amounts of mono- and sesqui-terpenes (and also non-volatile resin) in conifer tissues (needles and phloem) were less induced by T. pityocampa than by H. abietis. The MeJA-treated seedlings of Scots pine (Pinus sylvestris) changed their composition of phloem monoterpenes (induction of (-)-β-pinene), and were better protected in field than the seedlings of Norway spruce (Picea abies), which increased their total amounts of monoterpenes. Orientation bioassays with H. abietis showed deterrent effects of (-)-β-pinene, (+)-3-carene, (-)-bornyl acetate and 1,8-cineole. Conversely, (-)-α-pinene (induced in P. abies but not in P. sylvestris) was non-deterrent. MeJA-treated seedlings fed on by H. abietis contained higher amounts of the H. abietis antifeedant 2-phenylethanol, in tissues and emissions. Phloem of control seedlings instead induced the (+)-α-pinene, which is one of the enantiomers of the H. abietis attractant α-pinene. In response to MeJA treatment, the relative amounts of the (+)-α-pinene increased in the phloem of  P. radiata, while it decreased in P. pinaster phloem. The preference of H. abietis in the field for P. radiata before P. pinaster may be explained by these changes in enantiomers of the H. abietis attractant α-pinene. / Barrträd är viktiga för skogsindustrin. I Sverige består 57% av ytan av produktiv skogsmark, och av dessa träd utgörs 80% av gran och tall. Som försvar mot insekter och sjukdomar, producerar barrträden kådämnen. För unga barrträd i Europa och Asien är snytbaggen (Hylobius abietis) en allvarlig skadegörare, som orsakar skogsnäringen stora kostnader. För att skydda små gran- och tallplantor före utplanteringen, skulle växthormonet metyljasmonat (MeJA) kunna användas. I denna avhandling har effekter av MeJA-behandling och insektsangrepp undersökts på de två viktigaste barrträden i Sverige, tall och gran (Pinus sylvestris och Picea abies) och på två arter i medelhavsområdet (Pinus radiata och Pinus pinaster). De kemiska försvarsämnena i barrträd har analyserats för att utvärdera effekterna av MeJA-behandling. Vävnader från barrträd med olika behandling har extraherats i organiska lösningsmedel och dofterna från plantorna har insamlats genom fastfas-mikroextraktion. De flyktiga ämnena har separerats och identifierats med hjälp av gaskromatografi och masspektrometri (GC-MS). Dessutom har optiskt aktiva doftämnen separerats med hjälp av en tvådimensionell GC-MS. Plantornas olika försvarsreaktioner har studerats vid angrepp av snytbaggen, som är en floemätare, och av tallfjärilslarv (Thaumetopoea pityocampa), som huvudsakligen äter barr. De lättflyktiga ämnena (mono- och seskviterpener) och de icke-flyktiga ämnena (kådsyror) ökade mer i stam och barr från angrepp av H. abietis än av T. pityocampa. Sammansättning av monoterpener ändrades i de MeJA-behandlade tallplantorna i Sverige, specifikt ökade (-)-β-pinene i stamfloemet. Tallplantorna var bättre skyddade i fält än granplantorna, där den totala mängden monoterpener ökade. (-)-β-Pinene, (+)-3-carene, (-)-bornyl acetate och 1,8-cineole visade sig ha en negativ effekt på snytbaggens orientering mot talldoft i orienteringstester. En av monoterpenerna, (-)-α-pinene, som av MeJA-behandlingen inducerats i granen, men inte i tallen, hade ingen negativ effekt på snytbaggen. MeJA-behandlade tallplantor som angripits av snytbaggen, innehöll större mängder av snytbagge-äthämmaren 2-fenyletanol, både i vävnader och i emissioner. Stam från kontrollplantor inducerade istället (+)-α-pinene, vilken är en av de optiska isomererna av snytbagge-attrahenten α-pinene. För de spanska arterna, som svar på MeJA-behandling, ökade de lättflyktigaste terpenerna i barren hos P. radiata men inte hos P. pinaster, vilken redan innehöll en hög terpenhalt. De relativa mängderna av (+)-α-pinene ökade också i stammen hos P. radiata, medan de minskade i P. pinaster. I fält väljer snytbaggen att gå till P. radiata- framför P. pinaster-plantor, vilket kan vara kopplat till de skillnader i α-pinene-isomerer, vilka här har observerats. / <p>QC 20160304</p>