Abstract Flavonoids are a class of secondary metabolites in plants that are involved in many important functions. Various flavonoid compounds have also been reported to be beneficial for human health. Bilberry (Vaccinium myrtillus L.) is the characteristic field layer species in boreal forests and the fruits of bilberry are rich in anthocyanin pigments, a subclass of flavonoids. In the present work, flavonoid biosynthesis was examined in different tissues of bilberry. The focus was on the developing fruits of wild type and natural color mutants of bilberry, and on effect of solar radiation on flavonoid biosynthesis in bilberry leaves. For the isolation of RNA for gene expression analysis, a method was optimized for different tissues of bilberry. The cDNA fragments of five genes from the flavonoid pathway, coding phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavanol 4-reductase and anthocyanidin synthase, were isolated from bilberry using polymerase chain reaction technique, sequenced, and labelled with dioxigenin-dUTP label. These homologous, bilberry-specific probes were used for determining the expression of the flavonoid pathway genes in bilberry fruits, flowers and leaves with a modified non-radioactive method developed in the course of the study. The anthocyanins, catechins, proanthocyanidins, flavonols and hydroxycinnamic acids in fruits, leaves and different fractions of bilberry were identified and quantified with high-performance liquid chromatography combined with a diode array detector and mass spectrometer. The results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. A correlation between flavonol and anthocyanin biosynthesis was detected in bilberry and also in previous literature collected from flavonol and anthocyanin analyses from other fruits. Accordingly, models for the connection between flavonol and anthocyanin synthesis in fruit species were suggested. Activation of the expression of flavonoid pathway genes and accumulation of flavonoids and hydroxycinnamic acids was detected in leaves growing under direct solar radiation, compared to the shadow leaves of the same plants. Based on the results, it is suggested that cyanidin of anthocyanins and flavonol quercetin play a predominant role in the defence against high solar radiation in Vaccinium leaves. The results give new information about the biosynthesis of flavonoids in bilberry at the gene level, in addition to the information of the composition and content of flavonoids during fruit development and in different parts of the bilberry plant. Also, new information was obtained of the roles of flavonoids in protecting plants from excess solar radiation.

O bagaço de cana-de-açúcar é um material rico em celulose, molécula que pode ser convertida enzimaticamente em glicose pela ação de celulases. No entanto, no bagaço, a celulose está associada à hemicelulose e à lignina, componentes que limitam a sua digestibilidade enzimática. A lignina é o principal limitante da hidrólise da celulose, reduzindo a acessibilidade das enzimas celulolíticas. A hemicelulose também limita a ação das celulases e a presença de ácidos hidroxicinâmicos, como ferúlico e cumárico contribuem para reduzir os níveis de hidrólise. Nesse trabalho, o bagaço de cana-de-açúcar foi tratado com clorito de sódio em meio ácido com o objetivo de remover seletivamente a lignina e produzir modelos com teores reduzidos desse componente. O bagaço de cana controle continha 22,8% de lignina e após a deslignificação por 4 horas, materiais com até 6,8% de lignina foram obtidos, mantendo quase inalteradas as frações celulósicas e hemicelulósicas. Os bagaços foram submetidos à hidrólise enzimática com celulases comerciais. Apenas 26% da celulose de bagaço controle foram convertidos em glicose e 11% da xilana foram convertidos em xilose, com 72 horas de hidrólise. Nos bagaços com menores teores de lignina, os valores de conversão de celulose e de xilana alcançaram 85% e 64% no mesmo tempo de hidrólise, respectivamente. Os resultados mostraram que existe uma correlação inversa entre o teor de lignina e a conversão de polissacarídeos e que a remoção de 60% da lignina confere níveis elevados de hidrólise. Para reduzir a inibição pelos produtos de hidrólise da celulose, foi adicionada β-glicosidase ao meio reacional. Com a adição dessa enzima, os bagaços mais deslignificados alcançaram 100% de conversão de celulose e 82% de xilana, com 48 horas de hidrólise. No presente trabalho, o efeito da lignina não ligada aos polissacarídeos também foi avaliado. Para isso, lignina de bagaço de cana foi adicionada ao cartão de celulose, e este foi submetido à hidrólise enzimática. Não houve diferença entre os níveis de hidrólise na presença e ausência de lignina, indicando que a lignina limita a hidrólise dos polissacarídeos quando ligada a eles. Foi utilizada a técnica de microespectrofotometria ultravioleta para detectar a lignina e os ácidos hidroxicinâmicos em fibras, vasos e células de parênquima das regiões de córtex e de medula, deslignificados e controle. Os espectros UV dos três tipos celulares mostraram bandas em 278 nm, associada à lignina, e em 315 nm, associada aos ácidos hidroxicinâmicos. Os vasos foram os tipos celulares mais lignificados, seguidos pelas fibras e células parenquimatosas. O tratamento com clorito causou uma rápida remoção dos ácidos hidroxicinâmicos das células de parênquima, enquanto que as fibras foram deslignificadas apenas com 4 horas de tratamento. As amostras de córtex e de medula também foram submetidas à hidrólise com celulases. A região de medula não tratada foi prontamente hidrolizada, alcançando 63% de conversão de celulose com 72 horas, enquanto que o córtex não tratado atingiu apenas 20%. O tratamento com clorito de sódio não aumentou os níveis de hidrólise da medula. No entanto, a remoção de lignina do córtex aumentou consideravelmente a conversão. / The sugarcane bagasse is a material rich in cellulose, wich can be enzymatically converted into glucose by the action of cellulases. However, in the bagasse, it is associated to hemicellulose and lignin, components that limit its enzymatic digestibility. Lignin is the main limitant of the enzymatic hydrolysis of cellulose, reducing the accessibility of cellulases. Hemicellulose also limits the cellulase action and the presence of the hydroxycinnamic acids, as ferulic and coumaric, contribute to reduce the hydrolysis levels. In this work, the sugarcane bagasse was treated with acid sodium chlorite, aiming to selective remove the lignin and produce models with lower contents of this component. The control bagasse had 22.8% of lignin and after a 4-hour delignification, materials with up to 6.8% of lignin were obtained, retaining the cellulosic and hemicellulosic fractions almost unchanged. The bagasses were submitted to the enzymatic hydrolysis with comercial cellulases. Only 26% of the cellulose of the control bagasse were converted into glucose and 11% of the xylan were converted into xylose, with 72 hours of hydrolysis. In the bagasses with lower lignin content, the cellulose and xylan conversion values reached 85% and 64% in the same time of hysylysis, respectively. The results showed that there is an inverse correlation between the lignin content and the enzymatic conversion of polysaccharides and that the removal of 60% of the lignin results in high levels of hydrolysis. To reduce the inhibition by the products of the cellulose hydrolysis, β- glucosidase was added to the reaction medium. With this enzyme, the most delignified bagasses reached 100% of cellulose and 82% of xylan convertion, with 48 hours of hydrolysis. In this work, the effect of non-linked lignin was also analysed. For this, sugarcane bagasse lignin was added to cellulose card, and it was submitted to the enzymatic hydrolysis. There were no differences between the hydrolysis levels in the presence or absence of lignin, suggesting that lignin only limits hydrolysis when linked to the polysaccharides. The ultraviolet microspectrophotometry technique was used to detect lignin and hydroxycinnamic acids in fiber, vessels and parenchyma cells in rind and pith regions, delignified and control. The UV spectra of the three cell types showed bands in 278 nm, related to lignin, and 315 nm, related to the hydroxycynamic acids. The vessels were the most lignified cell type, followed by fibers and parenchyma cells. The chlorite treatment caused a rapid removal of hydroxycynnamic acids from parenchyma cell walls, while fibers where delignified only with a 4-hour treatment. The rind and pith samples were also submitted to enzymatic hydrolysis. The untreated pith region was promptly hydrolysed, reaching 63% of cellulose convertion in 72 hours, while untreated rind reached only 20%. The sodium chlorite treatment did not enhance the pith hydrolysis levels. However, the lignin removal in rind samples substantially enhanced the conversion.

O bagaço de cana-de-açúcar é um material rico em celulose, molécula que pode ser convertida enzimaticamente em glicose pela ação de celulases. No entanto, no bagaço, a celulose está associada à hemicelulose e à lignina, componentes que limitam a sua digestibilidade enzimática. A lignina é o principal limitante da hidrólise da celulose, reduzindo a acessibilidade das enzimas celulolíticas. A hemicelulose também limita a ação das celulases e a presença de ácidos hidroxicinâmicos, como ferúlico e cumárico contribuem para reduzir os níveis de hidrólise. Nesse trabalho, o bagaço de cana-de-açúcar foi tratado com clorito de sódio em meio ácido com o objetivo de remover seletivamente a lignina e produzir modelos com teores reduzidos desse componente. O bagaço de cana controle continha 22,8% de lignina e após a deslignificação por 4 horas, materiais com até 6,8% de lignina foram obtidos, mantendo quase inalteradas as frações celulósicas e hemicelulósicas. Os bagaços foram submetidos à hidrólise enzimática com celulases comerciais. Apenas 26% da celulose de bagaço controle foram convertidos em glicose e 11% da xilana foram convertidos em xilose, com 72 horas de hidrólise. Nos bagaços com menores teores de lignina, os valores de conversão de celulose e de xilana alcançaram 85% e 64% no mesmo tempo de hidrólise, respectivamente. Os resultados mostraram que existe uma correlação inversa entre o teor de lignina e a conversão de polissacarídeos e que a remoção de 60% da lignina confere níveis elevados de hidrólise. Para reduzir a inibição pelos produtos de hidrólise da celulose, foi adicionada β-glicosidase ao meio reacional. Com a adição dessa enzima, os bagaços mais deslignificados alcançaram 100% de conversão de celulose e 82% de xilana, com 48 horas de hidrólise. No presente trabalho, o efeito da lignina não ligada aos polissacarídeos também foi avaliado. Para isso, lignina de bagaço de cana foi adicionada ao cartão de celulose, e este foi submetido à hidrólise enzimática. Não houve diferença entre os níveis de hidrólise na presença e ausência de lignina, indicando que a lignina limita a hidrólise dos polissacarídeos quando ligada a eles. Foi utilizada a técnica de microespectrofotometria ultravioleta para detectar a lignina e os ácidos hidroxicinâmicos em fibras, vasos e células de parênquima das regiões de córtex e de medula, deslignificados e controle. Os espectros UV dos três tipos celulares mostraram bandas em 278 nm, associada à lignina, e em 315 nm, associada aos ácidos hidroxicinâmicos. Os vasos foram os tipos celulares mais lignificados, seguidos pelas fibras e células parenquimatosas. O tratamento com clorito causou uma rápida remoção dos ácidos hidroxicinâmicos das células de parênquima, enquanto que as fibras foram deslignificadas apenas com 4 horas de tratamento. As amostras de córtex e de medula também foram submetidas à hidrólise com celulases. A região de medula não tratada foi prontamente hidrolizada, alcançando 63% de conversão de celulose com 72 horas, enquanto que o córtex não tratado atingiu apenas 20%. O tratamento com clorito de sódio não aumentou os níveis de hidrólise da medula. No entanto, a remoção de lignina do córtex aumentou consideravelmente a conversão. / The sugarcane bagasse is a material rich in cellulose, wich can be enzymatically converted into glucose by the action of cellulases. However, in the bagasse, it is associated to hemicellulose and lignin, components that limit its enzymatic digestibility. Lignin is the main limitant of the enzymatic hydrolysis of cellulose, reducing the accessibility of cellulases. Hemicellulose also limits the cellulase action and the presence of the hydroxycinnamic acids, as ferulic and coumaric, contribute to reduce the hydrolysis levels. In this work, the sugarcane bagasse was treated with acid sodium chlorite, aiming to selective remove the lignin and produce models with lower contents of this component. The control bagasse had 22.8% of lignin and after a 4-hour delignification, materials with up to 6.8% of lignin were obtained, retaining the cellulosic and hemicellulosic fractions almost unchanged. The bagasses were submitted to the enzymatic hydrolysis with comercial cellulases. Only 26% of the cellulose of the control bagasse were converted into glucose and 11% of the xylan were converted into xylose, with 72 hours of hydrolysis. In the bagasses with lower lignin content, the cellulose and xylan conversion values reached 85% and 64% in the same time of hysylysis, respectively. The results showed that there is an inverse correlation between the lignin content and the enzymatic conversion of polysaccharides and that the removal of 60% of the lignin results in high levels of hydrolysis. To reduce the inhibition by the products of the cellulose hydrolysis, β- glucosidase was added to the reaction medium. With this enzyme, the most delignified bagasses reached 100% of cellulose and 82% of xylan convertion, with 48 hours of hydrolysis. In this work, the effect of non-linked lignin was also analysed. For this, sugarcane bagasse lignin was added to cellulose card, and it was submitted to the enzymatic hydrolysis. There were no differences between the hydrolysis levels in the presence or absence of lignin, suggesting that lignin only limits hydrolysis when linked to the polysaccharides. The ultraviolet microspectrophotometry technique was used to detect lignin and hydroxycinnamic acids in fiber, vessels and parenchyma cells in rind and pith regions, delignified and control. The UV spectra of the three cell types showed bands in 278 nm, related to lignin, and 315 nm, related to the hydroxycynamic acids. The vessels were the most lignified cell type, followed by fibers and parenchyma cells. The chlorite treatment caused a rapid removal of hydroxycynnamic acids from parenchyma cell walls, while fibers where delignified only with a 4-hour treatment. The rind and pith samples were also submitted to enzymatic hydrolysis. The untreated pith region was promptly hydrolysed, reaching 63% of cellulose convertion in 72 hours, while untreated rind reached only 20%. The sodium chlorite treatment did not enhance the pith hydrolysis levels. However, the lignin removal in rind samples substantially enhanced the conversion.