Understanding the role of auxins and oxidative enzymes on adventitious root formation in olive (olea europaea L.) cultivars

Olive (Olea europaea L.), one of the main crops in the Mediterranean basin, is mainly propagated by cuttings, a classical propagation method that relies on the ability of the cuttings to form adventitious roots. While some cultivars are easily propagated by this technique, some of the most interesting olive cultivars are considered difficult-to-root which poses a challenge for their preservation and commercialization. Therefore, increasing the current knowledge on adventitious root formation is extremely important for species like olive. This research focuses on evaluating the role of free auxins and oxidative enzymes on adventitious root formation of two olive cultivars with different rooting ability - ‘Galega vulgar’ (difficult-to-root) and ‘Cobrançosa’ (easy-to-root). In this context, free auxin levels and enzyme activities were determined in in vitro-cultured ‘Galega vulgar’ microshoots and in semi-hardwood cuttings of cvs. ‘Galega vulgar’ and ‘Cobrançosa’. To attain this goal, an analytical method for the quantification of free indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) was developed, which is based on dispersive liquid-liquid microextraction followed by microwave derivatization (DLLME-MAD) and gas chromatography-mass spectrometry (GC/MS) analysis. The developed method was validated in terms of linearity, recovery, limit of detection (LOD) and limit of quantification (LOQ) and proved to be useful in the analysis of two very different types of plant tissues. The results from auxin quantification in olive samples point at a relationship between free auxin levels and rooting ability of both microshoots and semihardwood cuttings. A defective IBA-IAA conversion, resulting in a peak of free IAA during initiation phase, seems to be associated with low rooting ability. Likewise, differences in the activity of oxidative enzymes also appear to be related with rooting ability. Higher polyphenol oxidases (PPO) activity is likely related with an easyto- root behavior, while the opposite is true for peroxidases (POX) (including IAA oxidase (IAAox)) activity. A possible hypothesis for adventitious root formation in olive microcuttings is presented herein for the first time. Free auxins, oxidative enzymes, alternative oxidase (AOX) and reactive oxygen species (ROS) are some of the factors that may be involved in this highly complex physiological process. Interestingly, while temporal changes in auxin levels were similar between microshoots and semihardwood cuttings, the conclusions obtained from enzyme activity results in microshoots didn’t translate to semi-hardwood tissues, showing the emerging need for adaptation of classical agronomical research studies to modern techniques; Resumo: Procurando compreender o papel das auxinas e enzimas oxidativas na formação de raízes adventícias em cultivares de oliveira (Olea europaea L.) A oliveira (Olea europaea L.) é uma das principais culturas da bacia Mediterrânica e é propagada maioritariamente por estacaria, um processo altamente dependente da capacidade das estacas para formar raízes adventícias. Enquanto algumas cultivares são fáceis de propagar desta forma, algumas das cultivares de oliveira mais interessantes são consideradas difíceis de enraizar, o que dificulta a sua preservação e comercialização e torna extremamente importante aprofundar o conhecimento sobre o enraizamento adventício desta espécie. Este trabalho foca-se na avaliação do papel das auxinas livres e das enzimas oxidativas na formação de raízes adventícias em duas cultivares de oliveira com diferente capacidade de enraizamento - ‘Galega vulgar’ (difícil de enraizar) e ‘Cobrançosa’ (fácil de enraizar). Neste contexto, determinaram-se os níveis de auxinas livres e as actividades de enzimas oxidativas em microestacas de ‘Galega vulgar’ cultivadas in vitro bem como em estacas semi-lenhosas das cvs. ‘Galega vulgar’ e ‘Cobrançosa’. Para tal foi necessário desenvolver uma metodologia analítica para a quantificação de ácido indol-3-acético (IAA) e ácido indol-3-butírico (IBA), baseada em microextracção dispersiva líquido-líquido (DLLME) seguida de derivatização em microondas (MAD) e análise por cromatografia gasosa acoplada a espectrometria de massa (GC/MS). O método desenvolvido foi validado em termos de linearidade, recuperação, limite de detecção (LOD) e limite de quantificação (LOQ), e mostrou-se eficaz na análise de dois tipos de tecidos vegetais bastante diferentes. Os resultados da análise de auxinas em amostras de oliveira apontam para uma possível relação entre os níveis de auxinas livres e a capacidade de enraizamento, tanto em microestacas como em estacas semi-lenhosas. Uma conversão IBA-IAA deficiente, que resulta num pico de IAA durante a fase de iniciação, parece estar associada à baixa capacidade de enraizamento. Por outro lado, a capacidade de enraizamento também parece estar relacionada com diferenças na actividade de enzimas oxidativas. Comportamentos fáceis de enraizar estão associados a actividade mais elevada das polifenoloxidases (PPO), enquanto o oposto é verdade para a actividade das peroxidases (POX) (incluindo a IAA oxidase (IAAox)). Neste trabalho propõe-se pela primeira vez uma possível explicação para o enraizamento adventício em microestacas de oliveira. Auxinas livres, enzimas oxidativas, oxidase alternativa (AOX) e espécies reactivas de oxigénio (ROS) são alguns dos factores envolvidos neste processo fisiológico altamente complexo. Curiosamente, enquanto as alterações temporais nos níveis de auxinas foram semelhantes entre microestacas e estacas semi-lenhosas, o mesmo não se observou relativamente à actividade enzimática, o que mostra a necessidade de adaptação dos estudos agronómicos tradicionais às técnicas correntes.

Tracking biochemical changes during adventitious root formation in olive (Olea europaea L.)

The activity of oxidative enzymes and the levels of free auxins were determined during adventitious root formation in olive explants. Rooting trials were performed both with in vitro-cultured micro shoots of the cultivar ‘Galega Vulgar’, treated with indole-3-butyric acid (IBA) and with salicylhydroxamic acid(SHAM) + IBA, as well as with semi-hardwood cuttings of the cultivars ‘Galega Vulgar’ (difficult-to-root)and ‘Cobrançosa’ (easy-to-root), treated with IBA. The auxin (IBA) was used in all experiments as a rooting promoter, while SHAM was used in micropropagation trials as rooting inhibitor, providing a negative control. Free indole-3-acetic acid (IAA) and IBA concentrations were determined in microshoots, as well as in semi-hardwood cuttings, throughout the rooting period at pre-established time-points. At the sametime-points, the enzymatic activity of polyphenol oxidases (PPO), peroxidases (POX), and IAA oxidase(IAAox) was evaluated in the microshoots. Microshoots treated with SHAM + IBA revealed higher POX and IAAox activity, as well as lower PPO activity, than those treated only with IBA. IAA levels were higher in IBA-treated microshoots during induction phase, but lower during early initiation phase. Incontrast, free IBA levels were higher in microshoots treated with SHAM + IBA during induction, but lower during initiation. A similar pattern of free auxin levels was observed in semi-hardwood cuttings of the two contrasting cultivars under evaluation. The similarities found on the auxin patterns of microshoots treated with SHAM and those of semi-hardwood cuttings of the difficult-to-root olive cultivar allow considering SHAM a reliable control for when simulation of a difficult-to-root behavior is necessary. The inhibitory effect of SHAM in root formation could be related with 1) the inhibition of alternative oxidase(AOX), leading to a down regulation of phenylpropanoid biosynthetic pathways, which would decrease the concentration of phenolic substrates for PPO; 2) an increase in IAAox activity resulting in lower free IAA levels or; 3) a defective conversion of IBA into IAA.

Lipoxygenase activities during development of root and nodule of soybean

The objective of this work was to evaluate root and nodule soybean lipoxygenases in Doko cultivar and in a near isogenic line lacking seed lipoxygenases, inoculated and uninoculated with Bradyrhizobium elkanii. The lipoxygenase activities from roots collected at 3, 5, 9, 13, 18 and 28 days post-inoculation and from nodules collected at 13, 18 and 28 days post-inoculation were measured. The pH-activity profiles from root and nodules suggested that the lipoxygenases pool expressed in these organs from Doko cultivar and triple-null near isogenic lines are similar. The root lipoxygenase activity of Doko and triple-null lines, inoculated and uninoculated, reduced over time. The highest lipoxygenase activity observed at the beginning of root formation suggests the involvement of this enzyme in growth and development of this organ. However, for nodules an expressive increase of lipoxygenase activity was noticed 28 days post-inoculation. Root and nodule showed, at least, two mobility groups for lipoxygenases in immunoblottings, with approximately 94 and 97 kDa.

In vitro somatic embryogenesis and adventitious root initiation have a common origin in eggplant (Solanum melongena L.)

Somatic embryogenesis was induced from cotyledon explants of eggplant cultured on MS medium supplemented with 54 µM NAA. Anatomical analysis of somatic embryo initiation and development was performed during the first four weeks. Proembryo formation was observed after the second day of culture, directly from perivascular cells or via pro-embryogenic masses derived from indeterminate meristematic masses (IMMs) originated in the vascular tissue. Those IMMs also gave rise to root primordia after 10 days of culture. The origin of embryos is discussed as well as the similarities between somatic embryogenesis and adventitious root formation.

Xyloglucans from Hymenaea courbaril var. stilbocarpa seeds affect Arabidopsis thaliana seedling growth by enhancing lateral root development

The effect of crude xyloglucan (XG) preparations from jatobá (Hymenaea courbaril var. stilbocarpa (Hayne) Y. T. Lee & Langenh.) seeds on Arabidopsis thaliana (L.) Heynh. root system development was investigated. The XG extracts exerted a dual effect on root system development by slowing down root growth and improving lateral root formation. These observed morphological changes were not due to oligosaccharides that could be generated following hydrolysis of the XG polymers, since XG hydrolysate induced a drastic inhibition of the overall growth process of the Arabidopsis thaliana seedlings. Histochemical test of GUS gene expression assay performed on seven and 14-days-old transgenic Arabidopsis thaliana plants carrying the CycB1;1-GUS fusion indicated that the improvement of the lateral root development by jatobá XG extracts was not correlated with the expression of this cell cycle marker gene in the root system. A potential agricultural application of jatobá seeds XG extract is discussed.

Influence of a Parafunctional Oral Habit on Root Fracture Development After Trauma to an Immature Tooth

Root fractures in immature teeth are rare because the resilience of the alveolar bone is more favorable to the occurrence of luxation. This article reports a case of traumatic injury in an immature permanent tooth that progressed to root fracture, having a parafunctional oral habit as the possible modifying factor of case evolution. A 12-year-old boy presented for treatment complaining of a defective restoration and mild pain on the maxillary right central incisor. The patient had a history of crown fracture in this tooth due to trauma 2 years before. The clinical examination showed healthy gingival tissues and no abnormal tooth mobility, whereas radiographic projections revealed healthy periradicular tissues, incomplete root formation, and no visible root fracture. As pulp necrosis was diagnosed, calcium hydroxide therapy was started for canal disinfection and subsequent obturation. However, after 4 weeks of treatment, a horizontal fracture line was observed radiographically in the root's middle third. The patient denied a new traumatic injury, but revealed the habit of chewing on a pencil. Refraining from the deleterious oral habit was strongly advised, and root canal filling with mineral trioxide aggregate was performed to treat the root fracture. After 4 years of follow-up, the tooth has normal function and no abnormal mobility. Images suggestive of remodeling at the apical end of the coronal segment and replacement resorption of the apical segment are seen radiographically. This case demonstrates the need of following cases of dental trauma and the possible influence of parafunctional oral habits as modifying factors of case progression.

Orthodontic extrusion as treatment option for crown-root fracture: literature review with systematic criteria

To review the literature searching for a consensus for the choice of orthodontic extrusion as treatment for crown-root fracture. An electronic search was performed in the databases PubMed, Cochrane Central Register of Controlled Trials and Scopus and a manual search of the Journal Dental Traumatology. Forty articles were found in PubMed and 38 in Scopus and after removal of duplicate sample 51 contained articles. Of these, 48 were excluded for not having orthodontic treatment, no follow-up or follow-up less than 6 months, or not report the presence of crown-root fracture. In manual search in Dental Traumatology 20 articles were found, but none of them met the prerequisites established. So, three articles formed the basis of the study. The choice of how to treat orthodontic extrusion of crown-root fracture was effective and stable, without root and periodontal changes. Factors, such as root formation and presence of pulp vitality were decisive for determining the stages of treatment, however, there is no consensus based on scientific evidence about these protocols.

Improvement of root architecture under abiotic stress through control of auxin homeostasis in Arabidopsis and Brassica crops

Auxin plays an important role in many aspects of plant development including stress responses. Here we briefly summarize how auxin is involved in salt stress, drought (i.e. mostly osmotic stress), waterlogging and nutrient deficiency in Brassica plants. In addition, some mechanisms to control auxin levels and signaling in relation to root formation (under stress) will be reviewed. Molecular studies are mainly described for the model plant Arabidopsis thaliana, but we also like to demonstrate how this knowledge can be transferred to agriculturally important Brassica species, such as Brassica rapa, Brassica napus and Brassica campestris. Moreover, beneficial fungi could play a role in the adaptation response of Brassica roots to abiotic stresses. Therefore, the possible influence of Piriformospora indica will also be covered since the growth promoting response of plants colonized by P. indica is also linked to plant hormones, among them auxin.

Mortal: A Mutant of White Clover Defective in Nodal Root Development

A monogenic dominant mutant of white clover (Trifolium repens L.), designated Mortal, which is defective in the formation of adventitious nodal roots, is described. Mortal plants grown at temperatures ranging from 10 to 25°C do not initiate nodal root primordium development. However, all other aspects of plant development are normal, including the formation of lateral roots and wound-induced adventitious roots. In some genetic backgrounds, the Mortal mutation has a temperature-sensitive conditional phenotype. Mortal plants shifted from growing conditions of 20 to 30°C for 2 to 3 d form nodal root meristems. However, new nodes that develop after plants are returned to 20°C exhibit the mutant phenotype. The capacity to form nodal roots on cuttings placed in water is also influenced by the genetic background of the Mortal mutation. Genetic analysis established that the physiological reversion of Mortal to nodal root formation is controlled by at least two separate dominant genetic loci, one for Nodal water response (Now) and one for Nodal temperature response (Not); the Now locus has a dominant epistatic interaction with the Not locus. The conditional nature of Mortal should provide opportunities for the identification of genetic and physiological mechanisms that influence the development of nodal roots.

Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype

Legume plants carefully control the extent of nodulation in response to rhizobial infection. To examine the mechanism underlying this process we conducted a detailed analysis of the Lotus japonicus hypernodulating mutants, har1-1, 2 and 3 that define a new locus, HYPERNODULATION ABERRANT ROOT FORMATION (Har1), involved in root and symbiotic development. Mutations in the Har1 locus alter root architecture by inhibiting root elongation, diminishing root diameter and stimulating lateral root initiation. At the cellular level these developmental alterations are associated with changes in the position and duration of root cell growth and result in a premature differentiation of har1-1 mutant root. No significant differences between har1-1 mutant and wild-type plants were detected with respect to root growth responses to 1-aminocyclopropane1-carboxylic acid, the immediate precursor of ethylene, and auxin; however, cytokinin in the presence of AVG (aminoetoxyvinylglycine) was found to stimulate root elongation of the har1-1 mutant but not the wild-type. After inoculation with Mesorhizobium loti, the har1 mutant lines display an unusual hypernodulation (HNR) response, characterized by unrestricted nodulation (hypernodulation), and a concomitant drastic inhibition of root and shoot growth. These observations implicate a role for the Har1 locus in both symbiotic and non-symbiotic development of L. japonicus, and suggest that regulatory processes controlling nodule organogenesis and nodule number are integrated in an overall mechanism governing root growth and development.

Medidas lineares dos estágios de desenvolvimento da dentição permanente humana usando Tomografia Computadorizada de Feixe Cônico: um estudo preliminar

OBJETIVO: determinar as medidas lineares dos estágios de desenvolvimento da dentição permanente humana, usando tomografia computadorizada de feixe cônico (TCFC). MÉTODOS: este estudo foi desenvolvido a partir de bancos de dados de clínicas radiológicas privadas, envolvendo 18 pacientes (13 do sexo masculino, 5 do sexo feminino, com idades variando entre 3 e 20 anos). As imagens das TCFC foram obtidas por meio do sistema i-CAT e medidas com uma função específica do programa desse mesmo sistema. Duzentos e trinta e oito dentes foram analisados, em diferentes estágios de desenvolvimento, nos planos coronal e sagital. O método foi baseado na delimitação e mensuração das distâncias entre pontos anatômicos correspondentes ao desenvolvimento das coroas e raízes dentárias. A partir dos valores obtidos, pôde-se desenvolver um modelo quantitativo para se avaliar os estágios inicial e final de desenvolvimento para todos os grupos dentários. RESULTADOS E CONCLUSÕES: as medidas obtidas dos diferentes grupos dentários estão de acordo com as estimativas das investigações publicadas previamente. As imagens por TCFC dos diferentes estágios de desenvolvimento podem contribuir no diagnóstico, planejamento e resultado dos tratamentos em diversas especialidades odontológicas. As dimensões das coroas e das raízes dentárias podem ter importantes aplicações clínicas e em pesquisas, constituindo uma técnica não invasiva que contribui com estudos in vivo. Entretanto, mais estudos são recomendados a fim de minimizar possíveis variáveis metodológicas.

Management of a permanent tooth after trauma to deciduous predecessor: an evaluation by cone-beam computed tomography

Impaction of permanent teeth represents a clinical challenge with regard to diagnosis, treatment plan, and prognosis. There is a close relationship between deciduous teeth and permanent teeth germ, and any injury in the deciduous dentition may influence the permanent teeth eruption. The extent of the damage caused to the permanent teeth germ depends on the patient age at the time of injury, type of trauma, severity, and direction of the impact. Conventional radiographic images are frequently used for diagnosis; however, recent developments in three-dimensional (3D) imaging systems have enabled dentistry to visualize structural changes effectively, with better contrast and more details, close to the reality. The cone-beam computed tomography (CBCT) has been used in the diagnosis and treatment plan of these impacted teeth. The purpose of the present case report is to describe a successful conservative management of a retained permanent maxillary lateral incisor with delayed root development after a trauma through the deciduous predecessor in a 9 year-old patient. After clinical and radiographic examination, a CBCT examination of the maxilla was requested to complement the diagnosis, providing an accurate 3D position of the retained tooth and its relationship to adjacent structures. The proposed treatment plan was the surgical exposure and orthodontic traction of the retained tooth. The lateral incisor spontaneously erupted after 6 months. Therefore, this case report suggests that permanent teeth with incomplete root formation have a great potential for spontaneous eruption because no tooth malposition or mechanical obstacles are observed.

Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium

The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days, The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11), The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH, Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.

Rapid isolation of total RNA and genomic DNA from Hakea actities

Tissues of the Australian native plant species Hakea actities (Proteaceae) contain numerous metabolites and structural compounds that hinder the isolation of nucleic acids. Separate RNA and genomic DNA extraction procedures were developed to isolate high quality nucleic acids from H. actities. Total RNA was extracted from leaves, roots and cluster roots of H. actities grown in low nutrient levels. Cluster root formation in H. actities only occurs when the plants are grown in low nutrient concentrations. However, under these conditions, nucleic acid extraction becomes increasingly difficult. The new procedures are faster than many of the published nucleic acid extraction protocols, and avoid the use of hazardous chemicals. The RNA extraction method was used successfully on another Australian species and a crop species, suggesting that the procedure is useful for molecular studies of a broad range of plants.

Estacas de amoreira tratadas com hormônios vegetais em dois sistemas de plantio e referentes coberturas do estufim

Four experiments on root formation on cuttings of mulberry trees of the variety Catania 1 were carried out. In each case the hormones Dieradix "M D", Dieradix "D", indol 3-yl-acetic acid, and I-naphthyl acetic acid were used, besides the control, without hormone. In all cases "normal" and "upside-down" planting were tried. The percentage x of cuttings with roots, after 54 days, were computed and transformed by the formula y = arc sin √P/100 for use in statistical analysis. The combined analysis of variance of the 4 trials led to the following results: "Upside-down" planting showed significantly higher percentage of rooting; Indol 3-yl-acetic acid was significantly better than control or other hormones. The percentages of rooted cuttings were as follows: Normal planting Upside-dow planting Indol 3-yl acetic acid 43.5% 90.9% I-naphthyl acetic acid 1.9% 69.3% Control 4.7% 22.2% Dieradix «M D» 2.4% 63.8% Dieradix «D» 1.3% 36.0%