Studies on the potential of alternative oxidase (AOX) as a functional marker candidate for efficient aventitious rooting in olive (Olea europaea L.)

O envolvimento e a importância da oxidase alternativa (AOX) na resposta das plantas a diversas condições de stress têm vindo a ser demonstrado. Tendo em conta esse conhecimento, colocou-se a seguinte hipótese: “o enraizamento adventício (EA) é a uma reação da planta ao stress abiótico no qual a AOX tem um papel determinante”. Com a finalidade de testar a hipótese e avaliar o potencial da AOX para o desenvolvimento de um marcador funcional (MF), relacionado com a eficiência no enraizamento adventício (EA) em Olea europaea L., quatro genes AOX foram isolados (OeAOX1a, OeAOX1b, OeAOX1c e OeAOX2). O envolvimento da AOX na resposta fisiológica ao EA foi estudado em dois sistemas distintos, num utilizando estacas semi-lenhosas e noutro microestacas. As cultivares de oliveira, ‘Galega vulgar’, recalcitrante ao enraizamento por estacaria semilenhosa e ‘Cobrançosa’, de fácil enraizamento por este processo, foram utilizadas nos ensaios. Os resultados obtidos em ambos os sistemas mostraram que o EA é significativamente reduzido pelo tratamento com um inibidor da AOX, o ácido salicilhidroxâmico (SHAM), enquanto que tratamentos com peróxido de hidrogénio ou piruvato, ambos indutores da AOX, resultaram num aumento das taxas de EA. Resultados similares foram obtidos de microestcas de ‘Galega vulgar’. O SHAM, reduziu significativamente o EA, resultado que foi confirmado por análises histológicas, porém não inibiu a formação de calli. As análises de metabólitos mostraram uma redução significativa na quantidade de fenilpropanóides e de lignina após o tratamento com SHAM, indicando que a atividade da AOX durante o EA estará associada ao metabolismo de adaptação desses metabólitos. O estudo da variabilidade nos genes AOX, realizado ao nível do ADN e ARN, centrou-se no gene OeAOX2. A maior frequência de polimorfismos foi identificada na extremidade 3'UTR. Diferenças no nível de acumulação de transcrito durante o EA foram observadas apenas quando se utilizou um primer reverse localizado na 3'UTR, o que indica a ocorrência de uma modificação pos-transcripcional. Em suma, os resultados obtidos permitem considerar a AOX como enzima chave no processo de reprogramação celular associado ao EA. Em suma, os resultados obtidos permitem considerar a AOX como enzima chave no processo de reprogramação celular associado ao EA. Os resultados desta tese são encorajadores e abrem uma linha de investigação promissora com um grande potencial de aplicação que deverá ser considerado a nível regional/nacional/internacional; ‘Studies on the potential of alternative oxidase (AOX) as a functional marker candidate for efficient adventitious rooting in olive (Olea europaea L.)’ Abstract :It has been suggested that alternative oxidase (AOX) plays an important role during plant stress responses. In this thesis the hypothesis is that root induction is a plant cell reaction linked to abiotic stress and that the activity of stress-induced AOX is important for adventitious rooting (AR). In order to investigate AOX as a functional marker (FM) to assist AR efficiency in Olea europaea L., four AOX gene members (OeAOX1a, OeAOX1b, OeAOX1c e OeAOX2) were isolated from ‘Galega vulgar’, a cultivar with low AR efficiency. The involvement of AOX in AR physiological responses was studied in two different system, using a semi-hardwood cuttings and another microcuttings. The olive cultivars 'Galega Vulgar', recalcitrant rooting by semi-hardwood and 'Cobrançosa', easy to root for this process were used in the tests. Rooting was significantly reduced by treatment with salicylhydroxamic acid (SHAM), whereas treatment with hydrogen peroxide or pyruvate, increased the degree of rooting. A similar approach was adopted using microshoots of the cultivar ‘Galega vulgar’. SHAM significantly reduced AR, which was confirmed by histological analysis, but failed to exhibit any effect on the preceding calli stage. Metabolite analyses showed that the amounts of phenylpropanoids and lignin were significantly reduced following SHAM treatment, indicating that the influence of AOX activity on root formation was associated with adaptive phenylpropanoid- and lignin- metabolism. A detailed study of DNA and RNA level was carried out on OeAOX2. 3’-UTR was the most important source for polymorphisms. Differential transcript accumulation of OeAOX2 during AR induction was observed when using primers in the 3’UTR indicating posttranscriptional modification. In summary, the results obtained allow to consider AOX as a key enzyme in cellular reprogramming associated with the EA process. The results of this thesis are encouraging and open a promising line of research with a great potential for application to be considered at regional / national / international level

Intron polymorphism pattern in AOX1b of wild St John's wort (Hypericum perforatum) allows discrimination between individual plants

The present paper deals with the analysis of natural polymorphism in a selected alternative oxidase (AOX) gene of the medicinal plant, St John's wort. Four partial AOX gene sequences were isolated from the genomic DNA of a wild plant of Hypericum perforatum L. Three genes belong to the subfamily AOX1 (HpAOX1a, b and c) and one to the subfamily AOX2 (HpAOX2). The partial sequence of HpAOX1b showed polymerase chain reaction (PCR) fragment size variation as a result of variable lengths in two introns. PCR performed by Exon Primed Intron Crossing (EPIC)-PCR displayed the same two-band pattern in six plants from a collection. Both fragments showed identical sequences for all exons. However, each of the two introns showed an insertion/deletion (InDel) in identical positions for all plants that counted for the difference in the two fragment sizes. The InDel in intron 1 influenced the predictability of a pre-microRNA site. The almost identical PCR fragment pattern was characterized by a high variability in the sequences. The InDels in both introns were linked to repetitive intron single nucleotide polymorphisms (ISNP)s. The polymorphic pattern obtained by InDels and ISNPs from both fragments together was appropriate to discriminate between all individual plants. We suggest that AOX sequence polymorphism in H. perforatum can be used for studies on gene diversity and biodiversity. Further, we conclude that AOX sequence polymorphism of individual plants should be considered in biological studies on AOX activity to exclude the influence of genetic diversity. The identified polymorphic fragments are available to be explored in future experiments as a potential source for functional marker development related to the characterization of origins/accessions and agronomic traits such as plant growth, development and yield stability

Physiologic responses and gene diversity indicate olive alternative oxidase as a potential source for markers involved in efficient adventitious root induction

Olive (Olea europaea L.) trees are mainly propagated by adventitious rooting of semi-hardwood cuttings.However, efficient commercial propagation of valuable olive tree cultivars or landraces by semi-hardwood cuttings can often be restricted by a low rooting capacity. We hypothesize that root induction is a plant cell reaction linked to oxidative stress and that activity of stress-induced alternative oxidase (AOX) is importantly involved in adventitious rooting. To identify AOX as a source for potential functional marker sequences that may assist tree breeding, genetic variability has to be demonstrated that can affect gene regulation. The paper presents an applied, multidisciplinary research approach demonstrating first indications of an important relationship between AOX activity and differential adventitious rooting in semi-hardwood cuttings. Root induction in the easy-to-root Portuguese cultivar 'Cobrançosa' could be significantly reduced by treatment with salicyl-hydroxamic acid, an inhibitor of AOX activity. On the contrary, treatment with H2O2 or pyruvate, both known to induce AOX activity, increased the degree of rooting. Recently, identification of several O. europaea (Oe) AOX gene sequences has been reported from our group. Here we present for the first time partial sequences of OeAOX2. To search for polymorphisms inside of OeAOX genes, partial OeAOX2 sequences from the cultivars 'Galega vulgar', 'Cobrançosa' and 'Picual' were cloned from genomic DNA and cDNA, including exon, intron and 3'-untranslated regions (3'-UTRs) sequences. The data revealed polymorphic sites in several regions of OeAOX2. The 3'-UTR was the most important source for polymorphisms showing 5.7% of variability. Variability in the exon region accounted 3.4 and 2% in the intron. Further, analysis performed at the cDNA from microshoots of 'Galega vulgar' revealed transcript length variation for the 3'-UTR of OeAOX2 ranging between 76 and 301 bp. The identified polymorphisms and 3'-UTR length variation can be explored in future studies for effects on gene regulation and a potential linkage to olive rooting phenotypes in view of marker-assisted plant selection

AOX – A potential marker for efficient rooting of olive shoot cuttings.

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’

Alternative Oxidase (AOX) Senses Stress Levels to Coordinate Auxin-Induced Reprogramming From Seed Germination to Somatic Embryogenesis—A Role Relevant for Seed Vigor Prediction and Plant Robustness

Somatic embryogenesis (SE) is the most striking and prominent example of plant plasticity upon severe stress. Inducing immature carrot seeds perform SE as substitute to germination by auxin treatment can be seen as switch between stress levels associated to morphophysiological plasticity. This experimental system is highly powerful to explore stress response factors that mediate the metabolic switch between cell and tissue identities. Developmental plasticity per se is an emerging trait for in vitro systems and crop improvement. It is supposed to underlie multi-stress tolerance. High plasticity can protect plants throughout life cycles against variable abiotic and biotic conditions. We provide proof of concepts for the existing hypothesis that alternative oxidase (AOX) can be relevant for developmental plasticity and be associated to yield stability. Our perspective on AOX as relevant coordinator of cell reprogramming is supported by real-time polymerase chain reaction (PCR) analyses and gross metabolism data from calorespirometry complemented by SHAM-inhibitor studies on primed, elevated partial pressure of oxygen (EPPO)–stressed, and endophyte-treated seeds. In silico studies on public experimental data from diverse species strengthen generality of our insights. Finally, we highlight readyto- use concepts for plant selection and optimizing in vivo and in vitro propagation that do not require further details on molecular physiology and metabolism. This is demonstrated by applying our research & technology concepts to pea genotypes with differential yield performance in multilocation fields and chickpea types known for differential robustness in the field. By using these concepts and tools appropriately, also other marker candidates than AOX and complex genomics data can be efficiently validated for prebreeding and seed vigor prediction

Alternative Oxidase (AOX) Senses Stress Levels to Coordinate Auxin-Induced Reprogramming From Seed Germination to Somatic Embryogenesis—A Role Relevant for Seed Vigor Prediction and Plant Robustness

Somatic embryogenesis (SE) is the most striking and prominent example of plant plasticity upon severe stress. Inducing immature carrot seeds perform SE as substitute to germination by auxin treatment can be seen as switch between stress levels associated to morphophysiological plasticity. This experimental system is highly powerful to explore stress response factors that mediate the metabolic switch between cell and tissue identities. Developmental plasticity per se is an emerging trait for in vitro systems and crop improvement. It is supposed to underlie multi-stress tolerance. High plasticity can protect plants throughout life cycles against variable abiotic and biotic conditions. We provide proof of concepts for the existing hypothesis that alternative oxidase (AOX) can be relevant for developmental plasticity and be associated to yield stability. Our perspective on AOX as relevant coordinator of cell reprogramming is supported by real-time polymerase chain reaction (PCR) analyses and gross metabolism data from calorespirometry complemented by SHAM-inhibitor studies on primed, elevated partial pressure of oxygen (EPPO)–stressed, and endophyte-treated seeds. In silico studies on public experimental data from diverse species strengthen generality of our insights. Finally, we highlight ready-to-use concepts for plant selection and optimizing in vivo and in vitro propagation that do not require further details on molecular physiology and metabolism. This is demonstrated by applying our research & technology concepts to pea genotypes with differential yield performance in multilocation fields and chickpea types known for differential robustness in the field. By using these concepts and tools appropriately, also other marker candidates than AOX and complex genomics data can be efficiently validated for prebreeding and seed vigor prediction.</p

Utilização de Asclepias Curassavica (Asclepiadaceae) por adultos de Heliconius erato Phyllis (Lepidoptera, Nymphalidae)

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Utilização de Asclepias Curassavica (Asclepiadaceae) por adultos de Heliconius erato Phyllis (Lepidoptera, Nymphalidae)

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Genetic diversity assessment in Portuguese Olea europea L. cultivars using the combinatorial β-Tubulin-based polymorphism (cTBP) marker system

In Olea europaea L. subsp. europaea var. sativa one of the most outstanding issues for commercial production and product certification lies in clarifying genetic relationships among the highly diverse, cultivated olive material. This report validates the applicability of the combinatorial β-tubulin based polymorphism (cTBP) molecular marker system for Olea europaea L. genotypes. The method is based on the analysis of length polymorphisms resulting from combined amplification of introns 1 and 2 of the β-tubulin genes, followed by fragment resolution in polyacrylamide electrophoresis gel. When compared with other molecular tools the technique is advantageous in terms of low costs and time consumption. The cTBP method was applied on a collection of morphologically classified olive trees (Coleção Nacional de Referência de Cultivares de Oliveira) that are established in the ‘Instituto Nacional de Investigação Agrária e Veterinária’ (INIAV, Elvas, Portugal). The results show that the cTBP marker system is useful to trace genetic variability and phylogenetic relationships in olive material thus able to help clarifying misleading classifications based on morphological characterization. Therefore, the method here presented can be recommended as an efficient tool for commercial applications and scientific studies that aim to verify genetic relationships in Olea europaea L