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.

Simplificação do processo de multiplicação in vitro da oliveira "Olea europaea L."

A oliveira tem sido multiplicada ao longo dos tempos por métodos convencionais de propagação vegetativa como a enxertia e a estacaria lenhosa e semilenhosa. No entanto, estes métodos revelam-se lentos ou ineficientes para determinadas cultivares. No caso da cv. ‘Galega Vulgar’, ainda com grande expressão no olival português, e de difícil enraizamento por estacaria semilenhosa, tem sido usada a micropropagação de modo a contornar essas limitações e assim obter um elevado número de plantas em curto período de tempo. O custo final de produção por este processo ainda é elevado, podendo comprometer a sua aplicação a nível comercial. Grande parte dos custos estão relacionados com a fase de enraizamento in vitro que carece de ambiente estéril e condições de assépsia para a sua execução. Com vista a uma redução de custos associados a esta fase de produção, pretendeu-se com este trabalho testar a viabilidade do enraizamento ex vitro, na ausência de condições de assépsia. Este método poderá permitir uma significativa redução da mão-de-obra, ao mesmo tempo que facilitará a aclimatização das plantas e a obtenção de um sistema radicular de melhor qualidade. Compararam-se as taxas de enraizamento in vitro (controlo), com as obtidas ex vitro. Foram utilizados explantes provenientes de dois clones da cv. ‘Galega Vulgar’, (cl. 1441 e cl. 2022) cultivados e mantidos in vitro há vários anos no Laboratório de Melhoramento e Biotecnologia da Universidade de Évora. Para além do clone foi avaliada a influência do tipo de estaca (basal e apical), da hormona de enraizamento (AIB e ANA), da sua concentração (540 e 3000 ppm) e ainda de dois substratos, Preformas Jiffy® e pastilhas de fibra coco. Os melhores resultados foram obtidos com o clone 1441 em pastilhas de fibra de coco prensada, com o uso de estacas basais. Quanto à auxina, não se observaram diferenças significativas entre a utilização de ANA na concentração de 540 ppm e AIB na concentração de 3000 ppm. A aclimatização das plantas foi conseguida com taxas elevadas de sucesso, independentemente do tratamento utilizado. Conclui-se que a aplicação do método de enraizamento ex vitro simplifica procedimentos e mantém taxas de enraizamento elevadas, conduzindo assim a uma efetiva redução de tempo e custos associados; Simplifying procedures for in vitro propagation of olive “Olea europaea L.” Abstract: The olive tree has been multiplied throughout the ages by conventional methods of vegetative propagation such as grafting and wood or softwood cuttings. These propagation methods are somehow inefficient for certain cultivars. For the CV. ‘Galega Vulgar‘, still with great expression in Portuguese olive orchards, propagation has been attempted by in vitro culture in order to circumvent these limitations and so obtain a large number of plants in short time period. The final production fees associated to this process are still high which may compromise its application to a commercial level. Most of this process fees are related to the in vitro rooting phase which lacks sterile and aseptic conditions for its implementation. Aiming to reduce the costs associated with this production phase, this work tested the feasibility of the ex vitro rooting in the absence of aseptic conditions, which can allow a significant reduction of the manpower involved and an easier plant acclimatization due to its transplant with a balled-root system. In vitro rooting rates (control) were compared with those obtained with the ex vitro experiments. Explants from two clones of the cv. ‘Galega Vulgar‘ (cl. 1441 and cl. 2022), grown and maintained in vitro for several years in the Laboratory of Biotechnology and Plant Breeding of the University of Évora, were used in the trials. In addition to the clone, the effect of the cutting type (basal and apical), the rooting hormone (AIB and ANA), their concentration (540 and 3000 ppm) and two substrates, Preformas Jiffy ® and pressed coco fiber pellets, were also evaluated. The best results were obtained with the clone 1441, when rooted in pressed coco fiber pellets, using basal cuttings. Under this conditions no significant differences were observed between the use of ANA at 540 ppm or AIB in the 3000 ppm. Acclimatization of plants was achieved with high rates of success, regardless of the treatment used. It can be concluded that the application of the ex vitro rooting method allows to maintain high rooting rates, contributing for an effective reduction of time and fees of the rooting process.

Seasonal dynamics and operational monitoring of hedgerow olive tree transpiration in response to applied water

We used 2012 sap flow measurements to assess the seasonal dynamics of daily plant transpiration (ETc) in a high-density olive orchard (Olea europaea L. cv. ‘Arbequina’) with a well-watered (HI) control treatment A to supply 100 % of the crop water needs, and a moderately (MI) watered treatment B that replaced 70% of crop needs. To assure that treatment A was well-watered, we compared field daily ETc values against ETc obtained with the Penman-Monteith (PM) combination equation incorporating the Orgaz et al. (2007) bulk daily canopy conductance (gc) model, validated for our non-limiting conditions. We then tested the hypothesis of indirectly monitoring olive ETc from readily available vegetation index (VI) and ground-based plant water stress indicator. In the process we used the FAO56 dual crop coefficient (Kc) approach. For the HI olive trees we defined Kcb as the basal transpiration coefficient, and we related Kcb to remotely sensed Soil Adjusted Vegetation Index (SAVI) through a Kcb-SAVI functional relationship. For the MI treatment, we defined the actual transpiration ETc as the product of Kcb and the stress reduction coefficient Ks obtained as the ratio of actual to crop ETc, and we correlated Ks with MI midday stem water potential (ψst) values through a Ks-ψ functional relationship. Operational monitoring of ETc was then implemented with the ETc = Kcb(SAVI)Ks(ψ)ETo relationship stemmed from the FAO56 approach and validated taking as inputs collected SAVI and ψst data reporting to year 2011. Low validation error (6%) and high goodness-of-fit of prediction were observed (R2 = 0.94, RSME = 0.2 mm day-1, P = 0.0015), allowing to consider that under field conditions it is possible to predict ETc values for our hedgerow olive orchards if SAVI and water potential (ψst) values are known.

Quantification of free auxins in semi-hardwood plant cuttings and microshoots by dispersive liquid–liquid microextraction/microwave derivatization and GC/MS analysis

Several studies have suggested that differences in the natural rooting ability of plant cuttings could be attributed to differences in endogenous auxin levels. Hence, during rooting experiments, it is important to be able to routinely monitor the evolution of endogenous levels of plant hormones. This work reports the development of a new method for the quantification of free auxins in auxin-treated Olea europaea (L.) explants, using dispersive liquid–liquid microextraction (DLLME) and microwave assisted derivatization (MAD) followed by gas chromatography/mass spectrometry (GC/MS) analysis. Linear ranges of 0.5–500 ng mL 1 and 1–500 mg mL 1 were used for the quantification of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), respectively. Determined by serial dilutions, the limits of detection (LOD) and quantification (LOQ) were 0.05 ng mL 1 and 0.25 ng mL 1, respectively for both compounds. When using the calibration curve for determination, the LOQ corresponded to 0.5 ng mL 1 (IAA) and 0.5 mg mL 1 (IBA). The proposed method proved to be substantially faster than other alternatives, and allowed free auxin quantification in real samples of semi-hardwood cuttings and microshoots of two olive cultivars. The concentrations found in the analyzed samples are in the range of 0.131–0.342 mg g 1 (IAA) and 20–264 mg g 1 (IBA).

RELAZIONE TRA IL CONSUMO DI OLIO EXTRAVERGINE DI OLIVA E I CASI DI DECESSO PER MALATTIA DI ALZHEIMER IN SPAGNA

In the chemical composition of olive oil (Olea europaea L.) it is emphasized the massive presence of oleic acid (over 70%), monounsaturated fatty acid part of the family of omega 9, a 7-8% linoleic acid (omega 6) and a small presence (0.5-1%) of linolenic acid (omega 3). For its high content of monounsaturated fatty acids, olive oil is the most stable and therefore the most suitable for heating, compared to oils with a dominance of polyunsaturated fatty acids. Interest in vitamin E has increased in recent years, thanks to its high antioxidant power and its role against related diseases with age-related, visual, dermatological, cardiovascular disorders Alzheimer’s disease and more. Vegetable oils are a major source of vitamin E through diet (Sayago et al., 2007), especially with the variety of olives “Hojiblanca”. Thanks to unsaturated fatty acids cell oxidation can be prevented: this helps prevent many illness, and even premature aging. So far, the advantages acknowledged to olive oil are those of lowering cholesterol, preventing cardiovascular disease, diabetes and cancer. Among the most recent researches it is important to distinguish the studies carried out on their contribution to the prevention and treatment of breast cancer and Alzheimer’s disease. Researchers found that in addition to the benefi ts that give monounsaturated fats, in extra virgin olive oil, there is a substance called “oleocanthal”, which helps protect nerve cells damaged in Alzheimer’s disease. The importance of this discovery is enormous when one considers that only Alzheimer’s disease affects 30 million people around the world, with a different distribution depending on the type of oil in the diet (Olguín Cordero, 2012). The latest research endorses that oleocanthal works by destroying cancer cells without affecting the healthy ones, as it is stated in the Molecular and Cellular Oncology Journal. Studies carried out in different Spanish universities have concluded that thanks to the antioxidant power of olive oil, a disease such as Alzheimer can be prevented. In conclusion, we can say that the Mediterranean diet rich in extra virgin olive oil greatly infl uences on human health, reducing, delaying or even eliminating several diseases.

Allergenic profile of Quercus rotundifolia pollen in Alentejo, Portugal

Background and Aim: Grasses and olive are the most relevant allergenic species in the Alentejo region. However, aggravation of allergic symptoms has been reported in the early spring, before grass and olive pollen seasons. Quercus pollen is the most abundant pollen type in the early spring in Alentejo, nonetheless its allergen profile has not yet been evaluated. The aim of this work was to characterize the allergen profile of pollen from Quercus rotundifolia among the most representative species showing pollination in April, prior to the main pollen season in Alentejo. Methods: Pollen from Quercus rotundifolia, Olea europaea and Dactylis glomerata was extracted with ammonium bicarbonate buffer, lyophilized and stored at -80ºC until analysis. Extract from Quercus ilex pollen was kindly offered by Bial. Protein content was determined by the Bradford method. SDS-PAGE followed by western blot, using allergic patient sera (obtained from the Hospital do Espírito Santo de Évora – HESE), were performed to evaluate the allergen profile of the pollen. Sensitization and cross-reactivity was assessed by solid phase immunoblot. Results: Most of the patient evidenced sensitization to pollen extracts of Q. rotundifolia. Protein profile of Q. rotundifolia has shown several bands in the Mr 10-90 kDa, mostly overlapping with Q. ilex. Western blot have shown several immunoreactive bands. Immunoreactive bands were also observed in the protein profile according to the pI in the range 4.0-6.1. Cross-reactivity between Q. rotundifolia with O. europaea and D. glomerata was found. Conclusion: These results evidenced allergens found in Q. rotundifolia pollen. It also shows that protein profile of Q. rotundifolia and Q. ilex are mostly alike suggesting that similarities in allergen profile are expected. Moreover, cross–reactivity between Q. rotundifolia and highly allergenic species such as O. europaea and D. glomerata was found which probably contributes to the aggravation of pollinosis in the early spring. Acknowledgments: This work was supported by FEDER through the “Programa Operacional Fatores de Competitividade – COMPETE” (Strategic projects of ICAAM and ICT 2013-2015). We also aknowledge Bial-Aristegui for supplying pollen and extract samples of Q. ilex.

Perfil em alergénios do pólen de Quercus rotundifólia

Background and Aim: Grasses and olive are the most relevant allergenic species in the Alentejo region. However, aggravation of allergic symptoms has been reported in the early spring, before grass and olive pollen seasons. Quercus pollen is the most abundant pollen type in the early spring in Alentejo, nonetheless its allergen profile has not yet been evaluated. The aim of this work was to characterize the allergen profile of pollen from Quercus rotundifolia the most representative species showing pollination in April, prior to the main pollen season in Alentejo. Methods: Pollen from Quercus rotundifolia, Olea europaea and Dactylis glomerata was extracted with ammonium bicarbonate buffer, lyophilized and stored at -80ºC until analysis. Extract from Quercus ilex pollen was kindly offered by Bial. Protein content was determined by the Bradford method. SDS-PAGE followed by western blot, using allergic patient sera (obtained from the Hospital do Espírito Santo de Évora – HESE), were performed to evaluate the allergen profile of the pollen. Results: Protein profile of Q. rotundifolia has shown several bands in the Mr 10-90 kDa, mostly overlapping with Q. ilex. Western blot have shown several immunoreactive bands. Protein profile according to the pI showed immunoreactive bands in the pI range 4.0-6.1. Cross-reactivity of Q. rotundifolia with O. europaea and D. glomerata was found. Conclusion: These results evidenced allergens found in Q. rotundifolia pollen. It also shows that protein profile of Q. rotundifolia and Q. ilex are mostly alike suggesting that similarities in allergen profile are expected. Moreover, cross–reactivity between Q. rotundifolia and highly allergenic species such as O. europaea and D. glomerata was found which probably contributes for aggravation of pollinosis in the early spring. Acknowledgments: This work was supported by “FEDER - Programa Operacional Factores de Competitividade – COMPETE”. A special acknowledgment to our colleague Prof. Rui Brandão, deceased, for his dedication to the present work, to the implantation and development of Aerobiology in the University of Évora. We have the honour of dedicating this work to the memory of Prof. Rui Brandão.

Séries de vegetação prioritárias para a conservação no centro e sul de Portugal continental

Como corolário de vários estudos geobotânicos realizados, no sudoeste Ibérico, no âmbito do seminário internacional Gestão e Conservação da Biodiversidade VIII, apresentou-se uma conferência sobre as principais séries de vegetação prioritárias para a conservação no centro e sul de Portugal continental. Assim, neste trabalho, após uma breve introdução apresenta-se uma súmula da caracterização biofísica deste território e um conjunto de séries singulares destas superfícies. Assim, selecionaram-se as séries de vegetação potencial climatófilas, edafoxerófilas e edafo-higrófilas que possuem, do ponto de vista corológico ou florístico, maior originalidade. Dentro destas, com elevado valor conservacionista, destacam-se quatro séries de sobreirais de Quercus suber, seis carvalhais de folha marcescente (três de Quercus broteroi, uma de Quercus canariensis, uma de Quercus marianaica e uma de Quercus pyrenaica), seis de zimbrais (quatro de Juniperus turbinata, uma de Juniperus navicularis e uma de Juniperus oxycedrus subsp. lagunae), um carrascal arbóreo (Quercus rivasmartinezii), um zambujal (Olea europaea var. sylvestris), um salgueiral (Salix salviifolia subsp. australis) e duas comunidades arborescentes de cursos de água torrenciais (Nerium oleander e Flueggea tinctoria). Porém, algumas destas séries encerram o seu volar, particularmente nas suas etapas seriais, essencialmente devido à presença de plantas endémicas e/ou com estatuto de proteção legal. Neste sentido, salienta-se ainda ao nível da série, a sinecologia, a sincorologia e a sinestrutura. Contudo, a revisão da dinâmica serial e os trabalhos de campo efectuados recentemente, permitiram a identificação de uma nova associação vegetal descrita no presente trabalho, no âmbito das séries de Aro neglecti-Querco suberis sigmetum. Finalmente expõem-se alguns dados relativos ao estado de conservação das etapas seriais mais próximas do clímax, apresentando-se sugestões que visam a recuperação e valorização destes habitats boscosos, tendo em vista a criação e consolidação de uma estratégia nacional de conservação.