Dissertação de mestrado em Genética Molecular | Transcriptions factors are fundamental for gene expression regulation controlling several developmental processes. A new sub-family of transcription factors, called DIV-and-RAD-interacting-factors (DRIFs), was first described in Antirhinum majus and they are involved in the establishment of the asymmetrical shape of the flower. DRIFs, together with DIVARICATA and RADIALIS constitute the antagonistic DIV, DRIF and RAD (DDR) module. Homologues for DDR module genes were already found in Arabidopsis thaliana. However, it is still unknown what molecular and cellular processes this module might be controlling. Previous work has implicated AtDRIFs in developmental processes including flowering time. To better understand how the DDR module might have been recruited in A. thaliana, this thesis will address the functional characterisation of AtDRIFs, with a focus on flowering time. Bioinformatic tools were used to gather information available for this gene family which include gene expression data, promoter cis-regulatory elements, alternative transcripts and protein interactions. The creation of transcriptional and translational fusions of AtDRIFs promoters with green fluorescent protein (GFP) and E. coli uidA gene (GUS) were initiated and will prove valuable for the study of spatial-temporal expression of AtDRIFs. Here the initial steps were taken for the development of knockout mutants for AtDRIFs, using CRISPR-Cas9, that will provide a better understanding of the role of the AtDRIF family in Arabidopsis. Gene expression analysis showed that genes involved in the photoperiod pathway are affected in the atdrifs mutants. Different patterns of AtDRIFs expression during a day were also observed but further studies need to be done to check if they are circadian regulated. Preliminary data on protein turnover, using the AtDRIF3 overexpression line, showed that protein abundance seems unaffected during the day. This analysis will be extended for all AtDRIFs and complemented with the new translational fusion lines. This work provided new insights on the function of AtDRIFs and will be important for further studies of a possible DDR module recruitment in Arabidopsis. | Os fatores de transcrição, são fundamentais para o controlo de vários processos de desenvolvimento. Em Antirhinum majus foi descrita pela primeira vez uma nova subfamília de fatores de transcrição, denominada DIV-and-RAD-interacting-factors (DRIFs). Os DRIFs, juntamente com as proteínas DIVARICATA e RADIALIS desempenham um papel na determinação da assimetria da flor, formando um módulo antagónico de regulação designado por módulo DIV, DRIF e RAD (DDR). Em Arabidopsis thaliana, homólogos para este módulo já foram descobertos, no entanto, ainda é desconhecida a sua função bem como os processos moleculares e celulares nos quais poderá estar envolvido o módulo. Estudos anteriores sugerem que os AtDRIFs desempenham um papel relevante em processos de desenvolvimento incluindo o período de floração. Para melhor elucidar a função do módulo DDR em A. thaliana, esta dissertação tem como objetivo caraterizar funcionalmente os AtDRIFs, principalmente no contexto do período de floração. Ferramentas bioinformáticas foram utilizadas para a recolha de informação sobre a família AtDRIF, incluindo perfis de expressão, elementos regulatórios cis dos promotores, transcritos alternativos e interações proteicas. Neste trabalho foram desenvolvidos construtos de fusões dos promotores dos AtDRIFs a genes repórter, green fluorescent protein (GFP) e o gene E. coli uidA (GUS). O desenvolvimento de mutantes para os AtDRIFs, através da tecnologia CRISPR-Cas9, foi também iniciado, o que irá trazer novas informações sobre a família AtDRIF em Arabidopsis. Estudos de expressão genética mostraram que a expressão de genes envolvidos da via do fotoperíodo se encontra afetada nos mutantes atdrifs. Análises de expressão mostraram que AtDRIFs apresentam diferentes padrões de expressão durante o dia, no entanto, são necessários mais estudos para verificar se são regulados de forma circadiana. Dados preliminares sugerem que o AtDRIF3 não é alvo de degradação a uma parte específica do dia. Estes estudos devem ser aplicados aos outros AtDRIFs e complementados com as linhas de fusão em desenvolvimento. Este trabalho contribuiu para o aumento do conhecimento sobre a função dos AtDRIFs, o que irá ser importante para futuros estudos sobre um possível recrutamento do módulo DDR em Arabidopsis.

Plant sumoylation research has seen significant advances in recent years, particularly since high-throughput proteomic strategies have enabled the discovery of more than one thousand SUMO targets. In the present chapter, we update the previously reported SUMO (small ubiquitin-related modifier) gene network (SGN) to its v4 iteration. SGN is a curated assembly of Arabidopsis thaliana genes that have been functionally associated with sumoylation, from SUMO pathway components to targets and interactors. The enclosed tutorial helps interpret and manage these datasets and details bioinformatic tools that can be used for in silico-based hypothesis generation. The latter include tools for sumoylation site prediction, comparative genomics, and gene network analysis. | FCT -Fundação para a Ciência e a Tecnologia(POCI-01-0145-FEDER-031122)

Several plant species display a temporal separation of the male and female flower organ development to enhance outbreeding; however, little is known regarding the genetic mechanisms controlling this temporal separation. Quercus suber is a monoecious oak tree with accentuated protandry: in late winter, unisexual male flowers emerge adjacent to the swollen buds, whereas unisexual female flowers emerge in the axils of newly formed leaves formed during spring (4-8 weeks after male flowering). Here, a phylogenetic profiling has led to the identification of cork oak homologs of key floral regulatory genes. The role of these cork oak homologs during flower development was identified with functional studies in Arabidopsis thaliana. The expression profile throughout the year of flower regulators (inducers and repressors), in leaves and buds, suggests that the development of male and female flowers may be preceded by separated induction events. Female flowers are most likely induced during the vegetative flush occurring in spring, whereas male flowers may be induced in early summer. Male flowers stay enclosed within the pre-dormant buds, but complete their development before the vegetative flush of the following year, displaying a long period of anthesis that spans the dormant period. Our results portray a genetic mechanism that may explain similar reproductive habits in other monoecious tree species. | - This work was funded by FCT/COMPETE/FEDER with the project grants FCOMP-01-0124-FEDER-019461/PTDC/AGR-GPL/118508/2010 `Characterization of Reproductive Development of Quercus suber'. R.S. was supported by funding from FCT with a PhD grant (ref. SFRH/BD/84365/2012). H.G.S. was supported by funding from FCT with a PhD grant (ref. SFRH/BD/111529/2015). M.M.R.C. was supported by funding from FCT with a grant (ref. SFRH/BSAB/113781/2015). Work supported by UIDP/04046/2020 and UIDB/04046/2020 Centre grants from FCT, Portugal to BioISI.