The salicinoids are phenolic glycosides (PGs) characteristic of the Salicaceae family and are known defenses against insect herbivory. Common examples are salicin, salicortin, tremuloidin, and tremulacin, which accumulate to high concentrations in the leaves and bark of willows and poplars. Despite their important role in plant defense, their biosynthetic pathway is not known, although recent work has suggested that benzyl benzoate acts as a possible biosynthetic intermediate. We identified three candidate genes encoding BAHD-type acyltransferases that are predicted to produce benzylated secondary metabolites, named PtACT47, PtACT49, and PtACT54. Expression of PtACT47 and PtACT49 generally correlated with PG content in a variety of tissues and organs of wild type hybrid poplar plants. This correlation was also found in transgenic hybrid poplar where PG content varied with the level of expression of the condensed tannin regulator MYB134 transcript. In these plants, a suppression of PtACT47 and PtACT49 expression was correlated with lower PG content. In contrast, PtACT54 exhibited very low expression in all tissues tested, and this level of expression was not affected in MYB134 plants. In order to better understand their possible biochemical functions, cDNA cloning, heterologous expression, and in vitro functional characterization was performed on these three BAHD acyltransferases. Recombinant PtACT47 exhibited a low substrate selectivity and could utilize acetyl-CoA, benzoyl-CoA, and cinnamoyl-CoA as acyl donors with a variety of alcohols as acyl acceptors. This enzyme showed the greatest Km/Kcat ratio (45.8 nM-1 sec-1) and lowest Km values (45.1 µM) with benzoyl-CoA and salicyl alcohol, and was named benzoyl-CoA:salicyl alcohol O-benzoyltransferase (PtSABT). Recombinant PtACT49 utilized a narrower range of substrates, specifically benzoyl-CoA and acetyl-CoA and a limited number of alcohols. Its highest Km/Kcat (31.8 nM-1 sec-1) and lowest Km (55.3 µM) was observed for benzoyl-CoA and benzyl alcohol, and it was named benzoyl-CoA:benzyl alcohol O-benzoyltransferase (PtBEBT). Both enzymes were also capable of synthesizing plant volatile alcohol esters at trace levels, for example hexenyl benzoate. Recombinant PtACT54 shares low sequence identity with PtSABT (52.3%) and PtBEBT (52.5%) and exhibited only moderate BEBT-like properties. PtSABT and PtBEBT appear to be paralogs based on their high sequence identity (90.6%) and closely related yet distinct biochemical functions. They likely arose from gene duplication and subsequent functional diversification possibly by neofunctionalization. Wounding experiments showed that abiotic damage stimulated the synthesis of specific PGs, notably salicin and salicortin within 24-48hrs. This was accompanied by a proportional increase in the expression of PtSABT and PtBEBT. Furthermore, experiments using transgenic RNAi lines with knock-down suppression of PtBEBT, and PtSABT, and both genes simultaneously, provided the first direct evidence that BAHD acyltransferases are important in PG production. PtSABT suppression, both individually and in the double knock-down suppression, significantly lowered salicortin content, particularly in mature leaves. However, a reduced level of PtBEBT expression did not have a significant effect on the PGs measured. This could indicate that BEBT-like activity may be a shared function among closely related BAHDs. The suppression of multiple BEBT-like genes may be necessary to further delineate their functions. / Graduate / rjchedgy@uvic.ca

Made available in DSpace on 2016-06-02T19:31:49Z (GMT). No. of bitstreams: 1 2941.pdf: 567635 bytes, checksum: 18b3f5d53030b198ba538da511d6b2e3 (MD5) Previous issue date: 2010-03-02 / Universidade Federal de Minas Gerais / Together with fire and climate changes, soil is considered a major determinant in the Braziliancerrado, the richest savanna in the world. Soil can influence plants by filtering species capableof acquiring resources and compete for them, but can also influence plant patterns ofallocation in defense against herbivory. Although many studies focused on plant soilrelationship in cerrado, few focused on the influence of soil at fine scale. We expectedcommunity descriptors, such as floristic composition, richness, evenness, diversity, and totalabundance to be related to soil features at fine scale within a physiognomy. We also expectedplants on nutrient-poor soils to present higher anti-herbivory defenses. In a cerrado site, weplaced 100 contiguous 25 m2 plots, in which we identified all woody individuals, measuredsoil variables and the following leaf traits: specific leaf area, C:N ratio, water content,toughness, trichomes, latex, and presence of tannins, alkaloids, and terpenoids. We did apartial redundancy analysis to test for relationship between soil features and floristiccomposition, controlled for spatial dependence. We also did multiple regression or spatialautoregressive models to test for relationships between soil features and: (1) the abundance ofthe five commonest species, (2) total abundance, (3) richness, (4) evenness, and (5) diversityand to predict defense traits based on soil features. We found no relationship between soil andfloristic composition, probably due to functional redundancy or limited dispersal. Organicmatter was positively related to Myrsine umbellata, the most abundant species, and totalabundance, and negatively related to evenness, what suggests positive feedbacks to cause thedominance by Myrsine umbellata. We also found a positive relationship between sum of basisand species richness, probably reflecting a fertility gradient. Contrary to our expectations, wefound no relationship between total defenses and total soil fertility or soil variables, whatcould result from low variability in soil fertility at fine scale or of high phenotypic variability.Presence of tannins was positively related to organic matter, possibly reflecting a strategytowards lower tolerance due to low reserve allocation or interactions with other resources.However, since tannins decrease leaf decomposition rates, organic matter could beaccumulating in soil. Overall, we suggest that soil is an important factor structuring cerradocommunity even at fine scales and that the dominance of cerrado species could be related topositive plant-soil feedbacks. / O solo, juntamente com o fogo e as variações climáticas, é considerado um dos principaisdeterminantes do cerrado brasileiro, a savana mais rica do mundo. O solo pode influenciar asespécies de planta selecionando aquelas adaptadas a explorar e competir por recursos, mastambém pode influenciar os padrões de alocações em defesas contra herbivoria. Emboramuitos estudos tenham se voltado a entender as relações entre solo e vegetação no cerrado,poucos se focaram efeitos em escala local. De forma geral, nossa expectativa é de quedescritores da comunidade, como composição florística, riqueza, equabilidade, diversidade eabundância total estejam relacionados com o solo, mesmo em escala local, dentro de umadeterminada fisionomia, e que plantas em solos pobres em nutrientes invistam mais emdefesas contra herbivoria devido ao alto custo em repor as folhas perdidas. Em uma área decerrado, lançamos 100 parcelas contíguas de 25 m2 cada, identificamos todos os indivíduosem nível de espécies, coletamos amostras de solo e medimos os seguintes traços foliares dedefesa contra herbivoria: área foliar específica, razão C:N, quantidade de água, dureza,densidade de tricomas, quantidade de látex, e presença de alcaloides, terpenoides e taninos.Para testar a relação entre a composição florística e as variáveis do solo, usamos uma análisede redundância parcial, controlando a autocorrelação espacial. Para testar a relação entre asvariáveis do solo e (1) a abundância das cinco espécies mais abundantes, (2) a abundânciatotal, (3) a riqueza de espécies, (4) a equabilidade e (5) a diversidade de espécies; para prevera distribuição dos traços de defesa contra herbivoria por meio das variáveis do solo,utilizamos regressões múltiplas ou modelos autorregressivos, na presença de autocorrelaçãoespacial. Encontramos uma baixa relação entre o solo e a composição florística,provavelmente devido à presença de espécies funcionalmente redundantes e espécies comdispersão limitada. O conteúdo de matéria orgânica esteve positivamente relacionado àabundância de Myrsine umbellata, a espécie mais abundante na área, e à abundância total, enegativamente relacionada à equabilidade, o que sugere que um mecanismo deretroalimentação positiva pode ser a causa da dominância de Myrsine umbellata. Tambémencontramos uma relação positiva entre soma de bases e a riqueza de espécies provavelmenterefletindo um gradiente de fertilidade. Contrariamente às nossas expectativas, nãoencontramos relação entre o investimento total em defesas e a fertilidade do solo,provavelmente refletindo uma baixa variação nas variáveis do solo em escala local ouvariações fenotípicas entre indivíduos da mesma espécie ou ambas. Entretanto, a presença detaninos esteve relacionada positivamente com o conteúdo de matéria orgânica, o que poderefletir menor tolerância à herbivoria em solos mais pobres ou uma alta acumulação dematéria orgânica no solo devido à lenta taxa de decomposição de folhas com tanino. De formageral, sugerimos que o solo é um importante fator estruturando a comunidade, mesmo emescala local, e que a dominância de espécies de cerrado pode estar relacionada a mecanismosde retroalimentação positiva.