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Summary:
Primary metabolites like carbohydrates, lipids, proteins, and nucleic acids, work as key components to sustain the plant. Besides those compounds, a wide array of so called ‘secondary metabolites’ are produced by the plant, which have been given much attention in the scientific literature due to their beneficial effect for the plant and the possible positive effect on human health. The number of identified plant secondary metabolites, exceeds 100.000 structures, which briefly can be classified as terpenoids, alkaloids, glucosides, sterols and, last but not least, phenylpropanoids. Fragaria vesca and Rubus idaeus; woodland strawberry and commercial raspberry, respectively, are two of the most popular berries on the market. The characteristic red coloration of their fruits is caused by the presence of anthocyanin pigments, secondary metabolites from the class of polyphenols. Those compounds are mainly produced during the late stages of fruit maturation and are essential phenotypic features; making plant breeders around the world consider these plant metabolites as a trait to follow up. The anthocyanin biosynthesis pathway is well studied in model plants. It is regulated at the transcriptional level by the well-known MBW complex. This complex is formed by the interaction of three different types of transcription factors (TFs): MYB, bHLH, and WD40, which have already been characterized in Arabidopsis thaliana, ornamental plants as Antirrhinum majus and Petunia hybrida, and even in some crops of major economic importance such as corn (Zea mays), soybean (Glycine max), and apple cultivars (Malus domestica). Nowadays, the level of complexity of the regulatory process of the anthocyanin biosynthesis pathway is becoming clear – one gene at a time. This regulation includes TFs, the promoter regions of the genes that are involved and the chromatin modifications necessary to carry out gene activation and consequent translation for the formation of each specific enzyme that will lead to the final anthocyanin formation inside the cells. The recent sequencing and annotation of the genomes of strawberry and raspberry as well as the possibility of transformation and the high amount of health-promoting anthocyanins present in the berries potentially make these plants great model systems to study the regulation of anthocyanin biosynthesis. This study aims to identify the role of bHLH proteins from raspberry and strawberry involved in anthocyanin biosynthesis. Based on the A. thaliana bHLH classification and phylogenetic studies reported, the genomes of F. vesca and R. idaeus were screened, and putative gene candidates were found for both species. Posterior sequence analyses based on protein primary structure and motif conservation were performed, and a total of 98 protein-coding sequences were found in F. vesca genome v1.0 and 90 sequences in the unreleased draft version of the R. idaeus genome. The in silico results obtained in chapter 3.1 provide three and two gene candidates for the woodland strawberry and raspberry, respectively: Fv3-FV2G25270, Fv33-FV7G08120, Fv145-FV5G02910, Ri3 gene36602 and Ri3- gene26116. After the identification of putative bHLH candidate genes, those genes were analyzed during fruit development and their function was studied in vitro and in vivo (chapter 4 and 5). The results of the study presented here forms the beginning of a possibility to breed new berry varieties with better traits, such as higher resistance to various stresses and a with a positive effect on the health of the consumer.

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