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

Somatic Embryogenesis in Iberian Grapevine (Vitis vinifera) Cultivars Using Carpels as Initial Explants: Protocol Establishment and Histological Evaluation

Trincadeira and Aragonez are two important grapevine cultivars in the Iberian Peninsula, used for high quality red wines production. Both cultivars are strongly affected by fungal diseases, with consequent high loses on plant productivity and fruit quality. A successful protocol for plant regeneration via somatic embryogenesis (SE) was established for both cultivars allowing further plant improvement based on gene transfer technology. Several factors were evaluated during the three different phases which characterize an SE plant regeneration protocol. The culture room temperature during the induction phase, a parameter usually accepted as standard by most researchers, proved in these trials to significantly affect the embryo induction rates. Concerning embryo conversion, it was specially affected by the embryo developmental stage, by the intensity and duration of the chilling treatment and by the supplementation of conversion culture medium with activated charcoal. The responses obtained, both for induction and conversion, proved to be highly genotype dependent. Calli structure, as well as embryo integrity, was histologically observed, allowing to characterize embryonic and non-embryonic masses and to identify abnormalities on embryo development

Establishment of a somatic embryogenesis protocol for in vitro olive plant propagation – a focus on the valorization of the Portuguese cv. 'Galega vulgar'.

Due to its capacity to regenerate plants from a single cell, in vitro somatic embryogenesis, apart from being an efficient plant propagation system is also fundamental to assist plant breeding by genetic transformation, and fundamental research for validation of data coming from different omics. The establishment of efficient protocols for plant regeneration through somatic embryogenesis must be taken as the first achievement on the successful regeneration of a transgenic plant. Focused on the validation of transcriptomic data, achieved in the frame of a running project (OLEAVALOR) and in the potential use of genetic engineering methodologies to further assist olive breeding programs, a research work has been carried out to establish an efficient somatic embryogenesis protocol for the Portuguese olive cv. 'Galega vulgar'. This cultivar is characterized by high quality olive oils and is still today the most representative of the traditional Portuguese olive orchards. However, it also presents several agronomic problems that highly limit its use in modern high density olive orchards, being mandatory the establishment of a breeding line for its valorisation. This study aimed on taking a first step in this direction, seeking to obtain an efficient somatic embryogenesis protocol that could later be used in breeding programs using genetic engineering approaches (e.g. cisgenesis, CRISPR/Cas9). Leaf petioles, taken from in vitro growing plantlets, as well as radicles and cotyledons, taken from mature seeds, were used as initial explants. The culture media composition and culture conditions were adapted from available reports (Rugini and Caricato 1995; Rugini and Silvestri 2016; Cerezo et al. 2011). A protocol for plant regeneration via somatic embryogenesis was achieved when mature zygotic embryos were used as initial explants. From the same initial explants, repetitive somatic embryogenesis was also successfully established, making available plant material for genetic transformation experiments. Regarding the use of leaf petioles from mature tissues, despite the formation of calli and the neoformation of leaf structures, it was not possible to obtain somatic embryos. Nevertheless, the results allow to foresee encouraging perspectives for further development of a protocol based on the use of adult plant material as initial explant

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

Editorial: [Advances on the biological mechanisms involved in adventitious root formation: from signaling to morphogenesis]

The neoformation of adventitious roots in cuttings is the key in the success of plant vegetative propagation systems. Two main steps are recognized in adventitious root (AR) formation. The first is known as the induction phase, in which high auxin levels are required for reprogramming of competent cells toward root development, which is further linked to other biochemical changes (Ahkami et al., 2013; Druege et al., 2019). This stage is followed by the formation phase, when cell division, differentiation and growth occur, leading to AR primordium development and, finally, to AR emergence/expression (Da Costa et al., Druege et al., 2019). Based on fossil records and phylogenetic data, Mhimdi and Pérez-Pérez proposed AR formation as the default state of root development in plants, highlighting that root initiation from non-root tissues might be considered as an ancestral trait, as ARs are formed by default in lycophytes and ferns. According to the same authors, in many angiosperms, AR formation seems to be repressed through mechanisms that maintain competent cells in a quiescent state. Activation of competent cells is triggered by environmental conditions to which the cuttings or intact plants are exposed, like exogenous auxin treatment, wounding, waterlogging, or light exposure and mineral nutrition determining the activation of a complex network of signaling, metabolic, and transport pathways (Bannoud and Bellini, Mhimdi and Pérez-Pérez, Yang et al., 2019). All these network pathways are under the control of distinct genes, being at the end responsible for efficient cell reprogramming, acquisition of a new evolutionary pattern and de novo organ differentiation and development

O itinerário da viagem reflexiva de uma professora-estagiária: À descoberta do Mundo do Ensino

O presente documento corporiza um relato pessoal da transição da formação académica para o Mundo do Ensino profissional. Demonstrando a perspetiva da autora relativamente ao primeiro contacto com a sua profissão de sonho, revelando as dificuldades com que se deparou no contexto real, colmatando com uma evolução pessoal que, por sua vez, permitiu criar uma identidade profissional. O principal realce incide sobretudo no vasto leque de experiências e vivências que colocaram à prova os conhecimentos até então adquiridos, proporcionando a reflexão crítica acerca dos mesmos, compreendendo quais os modelos e estratégias de ensino prediletos, tendo sempre em especial atenção a faixa etária e as características da turma. Faz alusão à partilha do conhecimento e ideologias com os colegas do núcleo de estágio, assim como a orientação e supervisão do Professor Orientador e da Professora Cooperante, que fizeram com que esta viagem tivesse ainda mais valor. A prática pedagógica decorreu em dois destinos distintos, mas com ambições semelhantes, estando ambos situados em Vila Nova de Gaia, proporcionando a aquisição de competências reais de ensino. Como forma de guiar o leitor pelo cenário de viagem apresentando, revelou-se fulcral subdividir o documento em capítulos, designadamente o "Enquadramento Biográfico: Conhecer a viajante" espaço onde se revela a filosofia de vida da autora, "Enquadramento Institucional: O Mundo do Ensino" contextualiza as escolas e os intervenientes nesta viagem, "Enquadramento Operacional: definir o itinerário" narra os acontecimentos marcantes desta viagem, traçando um percurso sobre as ideologias, estratégias e modelos aplicados, "Participação na Escola e Relação com a Comunidade: Descoberta da povoação e novas paragens" refere-se a atividades de caráter facultativo, mas essenciais para conceber uma imagem das várias funções a assumir na profissão de docente e por fim, as "Perspetivas para a próxima viagem". Pressupõe-se que este documento consiga refletir e transmitir o leque de emoções, evoluções e aprendizagens decorrentes deste ano tão atípico, mas enriquecedor.This document presents a personal account of the transition from academic education to the world of professional education. It demonstrates the author's perspective regarding her first contact with her dream profession, exposing the difficulties she faced in the context of real life, and finally, addressing the personal evolution that, in turn, allowed her to create her professional identity. The main highlight mostly insides on the wide range of experiences that put to the test the knowledge acquired so far, providing a critical reflection about them and allowing an understanding about which teaching strategies and techniques are preferred, always keeping in mind the age group and the characteristics of the class. It also alludes to the sharing of knowledge and ideologies with colleagues within the internship nucleus, as well as the guidance and supervision of the Internship Guiding Teacher and the Collaborate Teacher, who made this experience even more valuable. The teaching practice took place in two different destinations, both with similar ambitions and located in Vila Nova de Gaia, allowing for the acquisition of real teaching skills. As a way of guiding the reader through the present travel scenario, it was crucial to subdivide the document into chapters, namely "Enquadramento Biográfico: Conhecer a viajante" [Biographical Framework: Getting to know the traveler], a space where the author's philosophy of life is explained, "Enquadramento Institucional: O Mundo do Ensino" [Institutional Framework: The Teaching World] which contextualizes the schools and the participants of this trip, "Enquadramento Operacional: definir o itinerário" [Operational Framework: defining the itinerary], which narrates the remarkable events of this trip, navigating the ideologies, strategies and models applied, "Participação na Escola e Relação com a Comunidade: Descoberta da povoação e novas paragens" [Engaging with the School and community relationships: Meeting the community and new stops] refers to activities that are optional but essential to understand the various functions of the teaching profession, and finally, "Perspetivas para a próxima viagem" [Expectations for the next trip]. It is expected that this document will be able to reflect and transmit the range of emotions, progress and learning resulting from this atypical but enriching year

Carrot AOX2a transcript profile responds to growth and chilling exposure

Alternative oxidase (AOX) is a key enzyme of the alternative respiration, known to be involved in plant development and in response to various stresses. To verify the role of DcAOX1 and DcAOX2a genes in carrot tap root growth and in response to cold stress, their expression was analyzed in two experiments: during root growth for 13 weeks and in response to a cold challenge trial of 7 days, in both cases using different carrot cultivars. Carrot root growth is initially characterized by an increase in length, followed by a strong increase in weight. DcAOX2a presented the highest expression levels during the initial stages of root growth for all cultivars, but DcAOX1 showed no particular trend in expression. Cold stress had a negative impact on root growth, and generally up-regulated DcAOX2a with no consistent effect on DcAOX1. The identification of cis-acting regulatory elements (CAREs) located at the promoters of both genes showed putative sequences involved in cold stress responsiveness, as well as growth. However, DcAOX2a promoter presented more CAREs related to hormonal pathways, including abscisic acid and gibberellins synthesis, than DcAOX1. These results point to a dual role of DcAOX2a on carrot tap root secondary growth and cold stress responseinfo:eu-repo/semantics/publishedVersio

Isolation of alternative oxidase (AOX) genes of Olea europaea L.

Alternative oxidase (AOX) is recently suggested to be a potential candidate as functional marker for efficient cell reprogramming under stress (Arnholdt-Schmitt et al., 2006a). The presented work is part of a Marie Curie Chair project, that was established to investigate the potential role of the multigene AOX to assist breeding on efficient rooting of olive shoot cuttings (Arnholdt-Schmitt et al. 2006b). Plant mito-chondrial AOX is a small nuclear-encoded multigene family consisting of the two subfamilies AOX1 and AOX2. The intron-exon structure of AOX has been well characterized in several species, revealing a large degree of conservation. Here we report for the first time about the isolation of AOX multigene se-quences of olive (Olea europaea L.). The genes were isolated from a portuguese clone of the landrace ‘Galega vulgar’

Estratégias de ensino-aprendizagem na promoção de implicação e bem-estar emocional em crianças do 1º CEB

Mestrado em Educação Pré-Escolar e Ensino do 1º Ciclo do Ensino BásicoA época que vivemos apresenta-se em constante mudança a nível social e educativo. As crianças do presente século necessitam de aprender a enfrentar as dificuldades com perseverança, almejar novas aprendizagens e desenvolver um pensamento crítico e reflexivo para que possam ser adultos realizados e integrados na sociedade. No entanto, ainda, observamos escolas do século XIX e crianças que a encaram como uma rotina. Desta forma, é fulcral que o professor seja capaz de promover contextos de qualidade e aproximar a escola do século XIX da criança do século XXI. Neste sentido, conforme Laevers (1994) e Vygotsky (1996), o professor atual, além de conferir liberdade às crianças na tomada de decisões estimula o pensamento e respeita as necessidades básicas de cada uma delas. De acordo com um critério de classificação de estratégias de ensino-aprendizagem indicado por Vieira (2005), este professor planifica a sua ação centrada na criança assumindo um papel mais passivo comparado ao da criança. Assim sendo, este estudo teve como objetivo avaliar os níveis de implicação e de bem-estar emocional durante a implementação de diferentes estratégias de ensino-aprendizagem, tendo em conta o que cada estratégia oferece. O presente estudo, realizado numa escola do 1.ºCEB do concelho de Aveiro, inseriu-se num estudo qualitativo com características de investigação-ação e teve como participantes três crianças do 4.º ano do 1.ºCEB. Os resultados permitiram perceber que, atendendo às características das crianças, a implementação de estratégias de discussão proporcionou níveis de bem-estar e implicação elevados.The period in which we live in is constantly changing at a social and educational level. Students of today need to learn how to face difficulties with perseverance, aim for new learning and develop a critical and reflexive mind for the creation of realized and integrated individuals in society. However, we observe nineteenth-century schools of though and students who are viewed as a routine. That established, it is crucial that the teacher is able to promote quality contexts and bring a nineteenth century school closer to the student of the 21st century. In this sense, the current teacher, according to Laevers and Vygotsky, needs to confer freedom to the students in the decision making process and stimulate the thought, while respecting the basic needs of each student. According to a classification criterion of teaching-learning strategy indicated by Vieira, the teacher plans his action centered on the student, assuming a more passive role compared to the later. Therefore, this study aimed to evaluate the levels of implication and emotional well-being during the implementation of different teaching-learning strategies, taking into account what each strategy offers. The present study, carried out in the 1st CEB school of Aveiro, is inserted in the qualitative research with characteristics of action-research and had as object of study three students of the 4th year of the 1st CEB. Data analysis allowed us to perceive that, taking into account the characteristics of the students, the implementation of a discussion strategy provided levels of well-being and a high level of implication

Alternative oxidase involvement in Daucus carota somatic embryogenesis

Plant alternative oxidase (AOX) is a mitochondrial inner membrane enzyme involved in alternative respiration.The critical importance of the enzyme during acclimation upon stress of plant cells is not fully understood and is still an issue of intensive research and discussion. Recently, a role of AOX was suggested for the ability of plant cells to change easily its fate upon stress. In order to get new insights about AOX involvement in cell reprogramming, quantitative real-time polymerase chain reaction (PCR) and inhibitor studies were performed during cell redifferentiation and developmental stages of Daucus carota L. somatic embryogenesis. Transcript level analysis shows that D. carota AOX genes (DcAOX1a and DcAOX2a) are differentially expressed during somatic embryogenesis. DcAOX1a shows lower expression levels, being mainly down- regulated, whereas DcAOX2a presented a large up-regulation during initiation of the realization phase of somatic embryogenesis. However, when globular embryos start to develop, both genes are down-regulated, being this state transient for DcAOX2a. In addition, parallel studies were performed using salicylhydroxamic acid (SHAM) in order to inhibit AOX activity during the realization phase of somatic embryogenesis. Embryogenic cells growing in the presence of the inhibitor were unable to develop embryogenic structures and its growth rate was diminished. This effect was reversible and concentration dependent. The results obtained contribute to the hypothesis that AOX activity supports metabolic reorganization as an essential part of cell reprogramming and, thus, enables restructuring and de novo cell differentiation