Genome-wide analysis and expression profile of PIN-formed auxin carrier genes during in vitro IBA-induced adventitious rooting in Olea europaea L.

Olive (Olea europaea subsp. europaea var. europaea L.) comprises several cultivars with reduced capacity to be propagated due its recalcitrant behaviour upon adventitious rooting (AR) stimulus. This prevents their propagation and consequently their availability in the nurseries. By overcoming the difficult-to-root behaviour it will be possible to increase the number of cultivars available for new orchards and consequently taking profit of it on final products differentiation. There are many protocols used in vegetative propagation to induce AR based on auxins, a group of phytohormones largely known as involved in many processes of plant development, including root initiation and development. However, most of these protocols are still based on “trial/error” approaches, where several variables needs to be tested. This happens because genetic control underlying AR is not completely elucidated. Auxins are mainly synthesized in young leaves and apical meristem of shoots and roots. The major auxin distribution is regulated by transport from cell to cell, known as polar auxin transport (PAT). PAT is mediated by three main classes of membrane auxin transporters, the auxin resistant 1/like aux1 (AUX/LAX), the ATP binding cassette subfamily B (ABCB/MDR/PGP) and the pin-formed (PIN) carriers. The PIN gene family encodes a subgroup of auxin efflux carriers shown to be involved in various developmental processes, including lateral/adventitious root formation, in several plant species. To date, PIN genes have been identified in 31 plant species by genome-wide approaches, however, there is still no information regarding its identification in olive. The recent publication of O. europaea genome allowed us to perform the identification of all members belonging to PIN gene family in this species (OePIN). Our work aims to characterize OePIN family, as well as, to investigate the involvement of its members during AR. The expression profile study of OePIN members during AR in IBA-induced in vitro cultured microshoots of cv. ‘Galega vulgar’, attempting to understand if the hard-rooting behaviour of this cultivar might be related with a disturbance in auxin transport. Additionally, reactive oxygen species (ROS) as key signaling molecules that regulate growth and development and coordinate responses to biotic and abiotic stresses in plants are also being analysed in IBA-treated and non-treated explants. Also, cells from phloem, cortex and sub-epidermis are being isolated and gene expression will be performed on these cells in order to find out which cells are responsible for the formation of adventitious roots

Reference Genes Selection and Normalization of Oxidative Stress Responsive Genes upon Different Temperature Stress Conditions in Hypericum perforatum L

Abstract Reverse transcription-quantitative real-time PCR (RT-qPCR) is a widely used technique for gene expression analysis. The reliability of this method depends largely on the suitable selection of stable reference genes for accurate data normalization. Hypericum perforatum L. (St. John’s wort) is a field growing plant that is frequently exposed to a variety of adverse environmental stresses that can negatively affect its productivity. This widely known medicinal plant with broad pharmacological properties (anti-depressant, anti-tumor, anti-inflammatory, antiviral, antioxidant, anti-cancer, and antibacterial) has been overlooked with respect to the identification of reference genes suitable for RT-qPCR data normalization. In this study, 11 candidate reference genes were analyzed in H. perforatum plants subjected to cold and heat stresses. The expression stability of these genes was assessed using GeNorm, NormFinder and BestKeeper algorithms. The results revealed that the ranking of stability among the three algorithms showed only minor differences within each treatment. The best-ranked reference genes differed between cold- and heat-treated samples; nevertheless, TUB was the most stable gene in both experimental conditions. GSA and GAPDH were found to be reliable reference genes in cold-treated samples, while GAPDH showed low expression stability in heat-treated samples. 26SrRNA and H2A had the highest stabilities in the heat assay, whereas H2A was less stable in the cold assay. Finally, AOX1, AOX2, CAT1 and CHS genes, associated with plant stress responses and oxidative stress, were used as target genes to validate the reliability of identified reference genes

Expressão das proteínas da matriz na discondroplasia da tíbia

Doutoramento em BiologiaA discondroplasia da tíbia (TD) em aves consiste numa anomalia do esqueleto onde existe uma falha nos processos normais da ossificação endocondral. Esta patologia é caracterizada pela formação de uma cartilagem não vascularizada e não mineralizada que se estende até à metáfise. Uma vez que existem várias anomalias do esqueleto em mamíferos com lesões semelhantes às apresentadas pela TD, este trabalho teve como objectivo a caracterização desta patologia em termos das moléculas que podem estar envolvidas no seu desenvolvimento. Assim, foi estudada a expressão das macromoléculas da matriz extracelular, das enzimas degradadoras da matriz (metaloproteinases da matriz: MMPs), bem como das moléculas envolvidas na proliferação e diferenciação celular, na angiogénese e apoptose. A expressão génica foi realizada, por PCR quantitativo em tempo real, em placas de crescimento normais e discondroplásicas obtidas a partir de frangos de carne (broilers) da estirpe Cobb. Os níveis proteicos de algumas MMPs foram analisados por immunoblotting e zimografia de gelatina. No presente estudo não se verificou alteração na expressão dos genes dos colagénios do tipo II, IX, X e XI, bem como do agrecano, nas lesões discondroplásicas. Observou-se uma redução acentuada nos níveis de mRNA da gelatinase-B (MMP-9), da colagenase-3 (MMP-13) e das estromalisinas -2 (MMP-10) e -3 (MMP-11), bem como nos níveis proteicos da gelatinase-A (MMP-2) e da MMP-13. Por outro lado, a MMP-7 aumentou drasticamente a expressão do seu gene. As moléculas envolvidas na proliferação e diferenciação dos condrócitos, tais como a PTHrP, o Ihh, o Cbfa-1 e o Sox-9, mantiveram a sua expressão génica nas lesões. Por outro lado, o TGF-β reduziu a sua expressão. A caspase-3 também dimimuiu a sua expressão na patologia. Em relação aos factores angiogénicos, o FGF manteve a sua expressão e o VEGF aumentou significativamente nas lesões. Este aumento do VEGF juntamente com o aumento da MMP-7 sugere um aumento da hipoxia nas lesões. Os nossos resultados sugerem que a acumulação da cartilagem observada na discondroplasia é devida a uma diminuição da proteólise da matriz, resultado de uma sub-expressão das MMPs, e não de um aumento da produção das macromoléculas da matriz. Desta forma, os nossos resultados sugerem que a falha na expressão e/ou activação das MMPs poderá estar associada ao desenvolvimento da discondroplasia da tíbia em aves. Finalmente, os nossos resultados vêm suportar os resultados anteriores que sugerem uma ligação entre a expressão das MMPs e anomalias no processo de ossificação endocondral.Avian tibial dyschondroplasia (TD) is a skeletal disease where the normal events of endochondral bone formation are disrupted. It is characterized by the formation of a lesion composed of nonvascularized and nonmineralized cartilage that can extend into the metaphysis. Because there are several mammalian skeletal diseases with lesions similar to TD, the present work aimed to characterize the disease in terms of the molecules that may be involved in its development. Thus, the expression of extracellular matrix (ECM) macromolecules, ECM-degrading enzymes (matrix metalloproteinases: MMPs), and the regulatory molecules involved in cell proliferation and differentiation, angiogenesis, and apoptosis, were studied. Gene expression was performed by real-time quantitative PCR in normal and TD-affected growth plates from 3- week-old broiler chicks (Cobb strain). The protein levels of some of the MMPs studied were analysed by immunoblotting and gelatin zymography. The collagen types II, IX, X, and XI as well as aggrecan did not change their gene expression in dyschondroplastic lesions. There was a pronounced reduction in the mRNA levels of gelatinase-B (MMP-9), collagenase-3 (MMP-13), and of stromelysins-2 (MMP-10) and -3 (MMP-11), as well as in the protein levels of gelatinase-A (MMP-2) and MMP-13. On the other hand, MMP-7 mRNA has increased significantly. The molecules involved in chondrocyte proliferation and differentiation, such as PTHrP, Ihh, Cbfa-1, and Sox-9 have maintained their mRNA levels in the pathology. On the other hand, TGF-β has decreased its gene expression. Caspase-3 also showed diminished mRNA levels in the pathology. Regarding the angiogenic factors, FGF has maintained its expression and VEGF has increased significantly in the lesions. The increment in VEGF in conjunction with the increased expression of MMP-7 suggests the formation of a hypoxic environment in the lesions. Our results suggest that the accumulated cartilage observed in dyschondroplasia seems to be the result of decreased matrix proteolysis due to the downregulation of MMPs and not to an increased production of the matrix macromolecules. Thus, our results suggest that the failure in the expression or lack in the activation of MMPs might be associated with the development of avian tibial dyschondroplasia. Furthermore, our results strengthen the link between the lack in MMP expression and abnormal endochondral bone formation.FCT - SFRH/BD/6420/200

Expression Profile of PIN-Formed Auxin Efflux Carrier Genes during IBA-Induced In Vitro Adventitious Rooting in Olea europaea L.

Exogenous auxins supplementation plays a central role in the formation of adventitious roots (AR) for several plant species. However, the molecular mechanisms underlying the process of adventitious rooting are still not completely understood and many plants with economic value, including several olive cultivars, exhibit a recalcitrant behavior towards cutting propagation, which limits its availability in plant nurseries. PIN-formed proteins are auxin efflux transporters that have been widely characterized in several plant species due to their involvement in many developmental processes including root formation. The present study profiled the expression of the OePIN1a-c, OePIN2b, OePIN3a-c, OePIN5a-c, OePIN6, and OePIN8 gene members during indole-3-butyric acid (IBA)-induced in vitro adventitious rooting using the olive cultivar ‘Galega vulgar’. Gene expression analysis by quantitative real time PCR (RT-qPCR) showed drastic downregulation of most transcripts, just a few hours after explant inoculation, in both nontreated and IBA-treated microcuttings, albeit gene downregulation was less pronounced in IBA-treated stems. In contrast, OePIN2b showed a distinct expression pattern being upregulated in both conditions, and OePIN5b was highly upregulated in IBA-induced stems. All transcripts, except OePIN8, showed different expression profiles between nontreated and IBA-treated explants throughout the rooting experiment. Additionally, high levels of reactive oxygen species (ROS) were observed soon after explant preparation, decreasing a few hours after inoculation. Altogether, the results suggest that wounding-related ROS production, associated with explant preparation for rooting, may have an impact on auxin transport and distribution via changes in OePIN gene expression. Moreover, the application of exogenous auxin may modulate auxin homeostasis through regulation of those genes, leading to auxin redistribution throughout the stem-base tissue, which may ultimately play an important role in AR formation

Establishment of a reliable protocol for gDNA extraction from olive oil.

With no possibility to reach in quantity the production of countries with large areas of olive orchards, as a small country, Portugal needs to define a strategy for valuing the high quality and specificities of its olive oil. No doubt the focus must be on the valorization of the Portuguese cultivars, the key factor in determining the singularity of the produced olive oils. Fraud detection, as the use of non-Portuguese varieties, is the main aim of varietal and DOP olive oil producers. In this sense, it is mandatory to have tools allowing the control of the varietie(s) giving rise to the olive oil, both in quality and in quantity. One objective of the project Por3O running at University of Évora is the establishment of a molecular tool that identifies the varieties used to produce a given olive oil. Ideally, this tool could be further proposed for screening for frauds and to support olive oil certification. However, as PCR-based tool, it requires the availability of genomic DNA (gDNA) with quality enough to be used on fragment amplification. The step related with extraction of high quality gDNA from olive oil samples, which considers gDNA integrity and absence of inhibitory compounds, is often a limiting step in the development of a PCR-based genotyping tools. The establishment of a robust and efficient extraction protocol is thus crucial. In theory, the use of DNA commercial kits has the advantage of a higher reproductivity greatly removing technician expertise biases. Several DNA commercial kits were tested on its capacity to extract gDNA from a commercial blend olive oil and they were compared with an in-house method based on CTAB-based protocol previously published [1]. The different methods were compared in terms of starting volume of oil sample required for extraction, average gDNA concentration, total gDNA extraction yield and efficiency in Single-Sequence Repeats (SSRs) markers amplification. Considering the results achieved, we propose a workflow regarding gDNA extraction to further molecular analysis

Establishment of a Taqman-based approach to monitor Fusarium spp. Airbone spores.

Aerobiological studies provide important information about the biological particles present in the air. Monitoring the presence of airborne fungal spores can help farmers to prevent the onset of fungal diseases that may affect both quantity and quality of crops. The fungi Fusarium spp. are among the most important phytopathogenic fungal communities with high impact at regional level by affecting important cultures such as almond, tomato, maize and cereals. The establishment of an approach that would enable farmers to early react upon the possibility of a Fusarium spp. infection will lead to a better control of the diseases associated to those fungi. Currently, to monitor Fusarium spp. airborne spores it is followed the Hirst-type methodology, which is based on spore’s identification and quantification by optical microscope; a hard and time consuming process due to the spore’s small size and colorless wall. In this context, the development of an alternative methodology that enable to get accurate and reliable results in a faster way will be of high interest. A Taqman specific assay for Fusarium spp. detection and quantification was previously established for a different purpose [1] and was here applied as a molecular-based tool to detect airborne Fusarium spp. spores. To collect the biological particles from the atmosphere a Burkard 7-Day Volumetric Spore Trap, and the Hirst associated methodology, was used as the methodology recommended by the European Aerobiology Society (EAS) and International Association for Aerobiology (IAA) [2]. As proof-of-concept, the analysis was focused on samples weekly collect, from 1st October - 31st December 2018 (14 weeks) at the station of Portuguese Aerobiology Network (RPA – SPAIC) (38° 34’ N; 7° 54’ W). Genomic DNA (gDNA) was extracted from collected biological particles adhered to the melinex tape following the CTAB protocol [3] with some modifications. Considering the results achieved, we consider the Taqman-specific assay as an alternative methodology for monitoring Fusarium spp. airborne spores

Uso de marcadores moleculares aplicados à rastreabilidade dos Azeites.

A oliveira (Olea europaea L.) é uma das mais antigas culturas arbóreas da bacia do Mediterrânico (Raieta et al. 2015), com uma importância económica, social e cultural inegável na região. A qualidade e características do azeite depende significativamente da escolha da variedade que lhe dá origem. Algumas cultivares de azeite são reconhecidas como de alta qualidade, e derivam de áreas geográficas bem definidas; como consequência dão origem a produtos que impõem preços mais elevados no mercado (Vietina et al. 2011; Raieta et al. 2015). Dado o seu impacto económico, estes produtos podem tornar-se alvo de fraudes e adulterações (Aparicio et al. 2013). Neste contexto, várias Instituições Internacionais têm ativamente elaborado regulamentação de antifraude, exigindo um controlo mais rigoroso nos países produtores e importadores, impondo normas uniformes de rotulagem e técnicas instrumentais precisas na rastreabilidade do azeite (Aparicio et al. 2013). A União Europeia emitiu regras específicas sobre a produção de azeite e seu mercado: Regulamento CE nº 865/2004, a marca de Denominação de Origem Protegida (DOP) e a marca de Indicação Geográfica Protegida (IGP). Em Portugal estão registadas seis regiões DOP relativas a azeites virgens, produzidos a partir de onze variedades Portuguesas e incluindo produtos mistos e monovarietais. Urge o desenvolvimento de métodos que permitam identificar com precisão as variedades de oliveira para proteger a tipicidades destes produtos. As metodologias analíticas que têm sido usadas para análise da composição do azeite [Cromatografia Gasosa (GC), Cromatografia em Coluna de Sílica Gel, Cromatografia Líquida de Alta performance (HPLC) e técnicas espectroscópicas, como ressonância magnética nuclear (RMN) e a espectroscopia no infravermelho (Martins-lopes et al. 2008; Montealegre et al.2010) apresentam algumas limitações devido à alta variabilidade dos compostos presentes no azeite. Adicionalmente, estes métodos são sensíveis às condições ambientais e ao processamento associado à técnica, dificultando a interpretação dos resultados obtidos (Martins-lopes et al. 2008; Raieta et al. 2015). Como alternativa, o uso de marcadores moleculares tem despertado interesse pela sua menor sensibilidade às condições ambientais (Martins-Lopes et al. 2008; Montealegre et al. 2010), tendo o potencial de se tornarem uma ferramenta sólida de diagnóstico para autenticidade dos alimentos e de rastreabilidade. No âmbito do Projeto Por3O a decorrer na Universidade de Évora, pretende-se desenvolver uma ferramenta molecular para identificação das variedades usadas na produção de um determinado azeite. Idealmente, essa ferramenta a demonstrar-se robusta, poderá ser proposta para a deteção de fraudes e no apoio à certificação do azeite. A estratégia seguida teve por base o uso de marcadores Single Sequence Repeats (SSRs) para avaliar a variação genética em cinco variedades Portuguesas representativas dos olivias tradicionais da região do Alentejo ('Galega vulgar', 'Carrasquenha' 'Cordovil de Serpa', 'Cobrançosa' e 'Verdeal Alentejana'). Adicionalmente, foram avaliadas duas variedades não Portuguesas ('Arbequina' e 'Picual'), as quais podem representar risco de contaminação considerando a área de olival que atualmente ocupam nesta região. Tendo em conta a informação existente relativa à utilização de SSRs na genotipagem de variedades de oliveira, recorreu-se à técnica de High-Resolution Melting technique (HRM). Esta técnica, mais económica, rápida e de fácil execução, foi utilizada numa primeira fase para selecionar os SSRs mais polimórficos (com uma maior capacidade discriminatória) de entre um conjunto de 31 SSRs descritos na literatura. Selecionou-se um conjunto de 6 SSRs para identificar as variedades consideradas no estudo, e após uma primeira análise dos mesmos, selecionou-se apenas 3 SSRs (que foram caracterizados por sequenciação Sanger precedida de clonagem) para a análise de fragmentos por electroforese capilar. Como parte da verificação da autenticidade do azeite, foi demonstrada a aplicabilidade dos 3 SSRs selecionados em azeites. A extracção de DNA permanece um passo crítico dada a necessidade de uma quantidade mínima de DNA com integridade suficiente e sem inibidores da actividade enzimática, de modo a permitir a amplificação dos diferentes SSR. A degradação do DNA por nucleases, bem como a presença de compostos fenólicos, podem inibir a atividade da DNA polimerase e consequente não amplificação das regiões pretendidas (Raieta et al. 2015). Contudo, um protocolo de extracção de DNA do azeite, baseado no clássico protocolo CTAB, revelou-se robusto e eficiente na amplificação dos 3 SSRs previamente selecionados