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.

Histone post-translational modifications (PTMs) and their recognition by histone readers exert crucial functions in eukaryotes. Despite extensive studies, conservation and diversity of histone PTM regulation between animals and plants remain less explored because of a lack of systematic knowledge of histone readers in plants. Based on a high-throughput surface plasmon resonance imaging (SPRi) platform, we report the lab-on-chip profiling of interactions between 204 putative reader domains and 11 types of histone peptides in Arabidopsis thaliana. Eleven reader hits were then chosen for histone combinatorial readout pattern profiling. Systematic analysis of histone PTM recognition in Arabidopsis thaliana reveals that plant and human histone readers share conservation in domain types and recognition mechanisms. The differences in particular histone mark recognition by transcription regulator EML1 and DNA damage repair factor MSH6 indicate plant-specific histone PTMs function in Arabidopsis thaliana acquired during evolution.

Chromatin Affinity Purification (ChAP) is widely used to study chromatin architecture and protein complexes interacting with DNA. Here we present an efficient method for ChAP from Arabidopsis thaliana rosette leaves, in which in vivo biotinylation system is used. The chromatin is digested by Micrococcal Nuclease (MNase), hence the distribution of nucleosomes is also achieved. The in vivo biotinylation system was initially developed for Drosophila melanogaster (Mito et al., 2005), but the presented protocol has been developed specifically for Arabidopsis thaliana (Sura et al., 2017).

We recently demonstrated the biosynthesis of 24- ethylidene brassinosteroids in Arabidopsis thaliana. To determine the physiological role of biosynthesis of 24-ethylidene brassinosteroids, metabolism of 28-homodolichosterone as the end product of 24-ethylidene brassinosteroids biosynthesis was examined by a crude enzyme solution prepared from A. thaliana. In wild-type plants, dolichosterone and castasterone were identified as enzyme products on GC-MS analysis. In a mutant where DWARF1 was overexpressed (35S-DWF1), the conversion rate of 28-homodolichosterone to castasterone was significantly increased. These results indicate that conversion of 28-homodolichosterone to castasterone is mediated by dolichosterone in Arabidopsis. In the root growth assay, inhibitory activity was enhanced in the order of castasterone greater than dolichosterone greater than 28-homodolichosterone, demonstrating that conversion of 28-homodolichosterone to castasterone via dolichosterone is a biosynthetic reaction that increases BR activity in Arabidopsis. Compared to Arabidopsis grown under dark conditions, light-grown Arabidopsis showed up-regulated DWARF1 expression, resulting in an increased conversion rate of 28-homodolichosterone to castasterone, suggesting that light is an important regulatory factor for the biosynthetic connection of 24-ethylidene brassinosteroids and 24-methyl brassinosteroids in A. thaliana. Consequently, 24-ethylidene brassinosteroids biosynthesis to generate 28-homodolichosterone is a lightregulated alternative route for synthesis of the biologically-active BRs, castasterone and brassinolide in Arabidopsis plants.

Auxin is an important regulator of plant ontogenies including embryo development and the exogenous application of this phytohormone has been found to be necessary for the induction of the embryogenic response in plant explants that have been cultured in vitro. However, in the present study, we show that treatment of Arabidopsis explants with trichostatin A (TSA), which is a chemical inhibitor of histone deacetylases, induces somatic embryogenesis (SE) without the exogenous application of auxin. We found that the TSA-treated explants generated somatic embryos that developed efficiently on the adaxial side of the cotyledons, which are the parts of an explant that are involved in auxin-induced SE. A substantial reduction in the activity of histone deacetylase (HDAC) was observed in the TSA-treated explants, thus confirming a histone acetylation-related mechanism of the TSA-promoted embryogenic response. Unexpectedly, the embryogenic effect of TSA was lower on the auxin-supplemented media and this finding further suggests an auxin-related mechanism of TSA-induced SE. Congruently, we found a significantly increased content of indolic compounds, which is indicative of IAA and an enhanced DR5::GUS signal in the TSA-treated explants. In line with these results, two of the YUCCA genes (YUC1 and YUC10), which are involved in auxin biosynthesis, were found to be distinctly up-regulated during TSA-induced SE and their expression was colocalised with the explant sites that are involved in SE. Beside auxin, ROS were extensively accumulated in response to TSA, thereby indicating that a stress-response is involved in TSA-triggered SE. Relevantly, we showed that the genes encoding the transcription factors (TFs) that have a regulatory function in auxin biosynthesis including LEC1, LEC2, BBM, and stress responses (MYB118) were highly up-regulated in the TSA-treated explants. Collectively, the results provide several pieces of evidence about the similarities between the molecular pathways of SE induction that are triggered by TSA and 2,4-D that involve the activation of the auxin-responsive TF genes that have a regulatory function in auxin biosynthesis and stress responses. The study suggests the involvement of histone acetylation in the auxin-mediated release of the embryogenic program of development in the somatic cells of Arabidopsis.

PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) is a basic ​helix–loop–helix transcription factor with critical roles in light signaling. Recent work identified PIF3 as a negative regulator of arabidopsis (Arabidopsis thaliana) freezing tolerance.

Arabidopsis (Arabidopsis thaliana) AtBIK1 and rice (Oryza sativa) OsRLCK176 are orthologous receptor-like cytoplasmic kinases involved in immune signaling. Recent studies indicate that proteasomal turnover of these kinases is regulated by orthologous Ca2+-dependent protein kinases AtCPK28 and OsCPK4, revealing conserved interplay between phosphorylation and ubiquitination in immune homeostasis.

During secondary growth in most eudicots and gymnosperms, the periderm replaces the epidermis as the frontier tissue protecting the vasculature from biotic and abiotic stresses. Despite its importance, the mechanisms underlying periderm establishment and formation are largely unknown. The herbaceous Arabidopsis thaliana undergoes secondary growth, including periderm formation in the root and hypocotyl. Thus, we focused on these two organs to establish a framework to study periderm development in a model organism. We identified a set of characteristic developmental stages describing periderm growth from the first cell division in the pericycle to the shedding of the cortex and epidermis. We highlight that two independent mechanisms are involved in the loosening of the outer tissues as the endodermis undergoes programmed cell death, whereas the epidermis and the cortex are abscised. Moreover, the phellem of Arabidopsis, as in trees, is suberized, lignified and peels off. In addition, putative regulators from oak and potato are also expressed in the Arabidopsis periderm. Collectively, the periderm of Arabidopsis shares many characteristics/features of woody and tuberous periderms, rendering Arabidopsis thaliana an attractive model for cork biology.

Plants must constantly adjust their growth and defense responses to deal with the wide variety of stresses they encounter in their environment. Among phytohormones, brassinosteroids (BRs) are an important group of plant steroid hormones involved in numerous aspects of the plant lifecycle including growth, development and responses to various stresses including insect attacks. Here, we show that BRs regulate glucosinolate (GS) biosynthesis and function in insect herbivory. Preference tests and larval feeding experiments using the generalist herbivore, diamondback moth (Plutella xylostella), revealed that the larvae prefer to feed on Arabidopsis thaliana brassinosteroid insensitive 1 (bri1‐5) plants over wild‐type Ws‐2 or BRI1‐Flag (bri1‐5 background) transgenic plants, which results in an increase in larval weight. Analysis of GS contents showed that 3‐(methylsulfinyl) propyl GS (C3) levels were higher in bri1‐5 than in Ws2 and BRI1‐Flag transgenic plants, whereas sinigrin (2‐propenylglucosinolate), glucoerucin (4‐methylthiobutylglucosinolate) and glucobrassicin (indol‐3‐ylmethylglucosinolate) levels were lower in this mutant. We investigated the effect of brassinolide (BL) on GS biosynthesis in Arabidopsis and radish (Raphanus sativus L.) by monitoring the expression levels of GS biosynthetic genes, including MAM1, MAM3, BCAT4 and AOP2, which increased in a BL‐dependent manner. These results suggest that BRs regulate GS profiles in higher plants, which function in defense responses against insects.