Fruit cell culture as a model system to study cell wall changes during strawberry fruit ripening

Strawberry (Fragaria x ananassa, Duch.) fruit is characterized by its fast ripening and soft texture at the ripen stage, resulting in a short postharvest shelf life and high economic losses. It is generally believed that the disassembly of cell walls, the dissolution of the middle lamella and the reduction of cell turgor are the main factors determining the softening of fleshy fruits. In strawberry, several studies indicate that the solubilisation and depolymerisation of pectins, as well as the depolymerisation of xyloglucans, are the main processes occurring during ripening. Functional analyses of genes encoding pectinases such as polygalacturonase and pectate lyase also point out to the pectin fraction as a key factor involved in textural changes. All these studies have been performed with whole fruits, a complex organ containing different tissues that differ in their cell wall composition and undergo ripening at different rates. Cell cultures derived from fruits have been proposed as model systems for the study of several processes occurring during fruit ripening, such as the production of anthocyanin and its regulation by plant hormones. The main objective of this research was to obtain and characterize strawberry cell cultures to evaluate their potential use as a model for the study of the cell wall disassembly process associate with fruit ripening. Cell cultures were obtained from cortical tissue of strawberry fruits, cv. Chandler, at the stages of unripe-green, white and mature-red. Additionally, a cell culture line derived from strawberry leaves was obtained. All cultures were maintained in solid medium supplemented with 2.5 mg.l-1 2,4-D and incubated in the dark. Cell walls from the different callus lines were extracted and fractionated to obtain CDTA and sodium carbonate soluble pectin fractions, which represent polyuronides located in the middle lamella or the primary cell wall, respectively. The amounts of homogalacturonan in both fractions were estimated by ELISA using LM19 and LM20 antibodies, specific against demethylated and methyl-esterified homogalacturonan, respectively. In the CDTA fraction, the cell line from ripe fruit showed a significant lower amount of demethylated pectins than the rest of lines. By contrast, the content of methylated pectins was similar in green- and red-fruit lines, and lower than in white-fruit and leaf lines. In the sodium carbonate pectin fraction, the line from red fruit also showed the lowest amount of pectins. These preliminary results indicate that cell cultures obtained from fruits at different developmental stages differ in their cell wall composition and these differences resemble to some extent the changes that occur during strawberry softening. Experiments are in progress to further characterize cell wall extracts with monoclonal antibodies against other cell wall epitopes.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Down-regulation of a pectin acetylesterase gene modifies strawberry fruit cell wall pectin stracture and increases fruit firmness

Antisense-mediated down-regulation of several fruit-specific genes has previously demonstrated how the cell wall disassembly in strawberry fruit is mediated by a series of enzymes that act sequentially (Posé et al. 2011). An interesting example, the silencing of the polygalacturonase gene FaPG1, was also related with a significant increase of the post-harvest strawberry fruit firmness (Posé et al. 2013). Our research group has isolated a pectin acetylesterase gene, FaPAE1, which expression is enhanced during strawberry ripening. The main goal of this work was to elucidate the role of the degree of acetylation in cell wall integrity and fruit firmness through the antisense-mediated down-regulation of FaPAE1 in strawberry plants. Several transgenics lines were generated and 5 of them produced fruits 5-15% firmer than controls. Cell wall from ripe fruits was isolated from two independent transgenic lines and a control line, and sequentially extracted with different solvents (PAW, H2O, CDTA, Na2CO3). Modifications in fraction yield, its sugar composition and the degree of acetylation in each fraction were determined. Higher amounts of CDTA and Na2CO3 fractions were obtained in transgenic fruits, suggesting a decreased pectin solubilization as results of FaPAE1 silencing. Accordingly, the degree of acetylation of the Na2CO3-soluble pectins was greater in the transgenic lines than the control, but the opposite result was found in pectins from the CDTA fraction. These results suggest that PAE is preferentially active in pectis that are tightly bound to the cellulose-hemicellulose network and its activity could reduce the complexity of the cell wall structure, allowing that other hydrolytic enzymes could access the pectin chains. Thus, the increased fruit firmness observed in the transgenic FaPAE1 lines could be attributed to the direct effect of the silencing of the PAE enzyme and also to the indirect effect that the increase of the degree of acetylation of pectins has on the activity of other enzymes involved in the cell wall degradation. * Posé et al. (2011). Genes, Genomes and Genomics, 5 (Special Issue 1):40-48 * Posé et al. (2013). Plant Physiology, 150: 1022-1032 We acknowledge support from the Spanish Ministry of Economy and competitivity and Feder EU Funds (grant reference AGL2011-24814), FPI fellowships support for SP (BES-2006-13626) and CP (BES-2009027985), and grant "Ramón y Cajal" support for AJMA (RYC-2011-08839).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Rhamnogalacturonase lyase gene downregulation in strawberry and its potential on mechanical fruit properties

Strawberry softening is one of the main factors that reduces fruit quality and leads to economically important losses. Textural changes during fruit ripening are mainly due to the dissolution of middle lamellae, a reduction in cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Functional studies of genes encoding pectinase enzymes (polygalacturonase, pectate lyase and -galactosidase) support a key role of pectin disassembly in strawberry softening. Evidence that RG-I may play an important role in strawberry texture has been obtained from the transient silencing of a RG-lyase gene. Pectins are major components of fruit cell walls and highly dynamic polysaccharides, but due to their heterogeneity the precise relation between the structures and functions is incomplete. In this work, stable transgenic strawberry lines with a rhamnogalacturonate lyase gene (FaRGLyase1) down-regulated have been analyzed. Several transgenic lines showing more than 95% silencing of FaRGLyase1 displayed fruit firmness values higher than control. Cell walls from these lines were extracted and analyzed by ELISA and Epitope Detection Chromatography (EDC). This last technique is based on the detection of specific cell wall oligosaccharide epitopes and provides information on sub-populations of pectins containing homogalacturonan and RG-I domains, but also reveals potential links with other cell wall polysaccharides such as xyloglucan. The results obtained indicate that the silencing of FaRGLyase1 reduces degradation of RG-I backbones, but also homogalacturonan, in cell walls, especially in pectin fractions covalently bound to the cell wall. These changes contribute to the increased firmness of transgenic fruits.This research was supported by FEDER EU Funds and the Ministerio de Economía y Competitividad of Spain (grant reference AGL2014-55784-C2), a Marie Curie IEF within the 7th European Community Framework Programme (reference: PIEF-2013-625270) for SP and a FPI fellowship (BES-2015-073616) to support PR-V. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Maximum photosynthetic assimilation in quinoa varieties (Chenopodium quinoa) from different geographic origins, and its relation to leaf morphology

Este trabajo brinda datos sobre las diferentes respuestas fisiológicas y morfológicas de seis variedades de Chenopodium quinoa Willd, de diferentes orígenes geográ- ficos, cultivadas bajo las mismas condiciones ambientales. Se analizaron el intercambio de gases (asimilación fotosintética máxima, Amax; conductancia estomática, gs, transpiración, Tr, concentración interna de CO2, Ci y eficiencia intrínseca en el uso del agua EUAi, anatomía foliar (densidad estomática, DE, tamaño estomático, TE, espesor de los tejidos, ET), área foliar específica, AFE y contenido de pigmentos fotosintéticos y protectores. Las experiencias se realizaron en una región árida del Noroeste Argentino (Amaicha del Valle, 22º31’S, 65º 59’ W, 1.985 m) bajo condiciones de sequía. Las variedades utilizadas fueron: Kancolla (Perú) y Sayaña (Altiplano Boliviano, 3.850 m), Quinoa Blanca (Cochabamba, Bolivia, 2.570 m), Quinoa Roja (Cangrejillos, Argentina, 3.700 m), CICA (originalmente obtenida en Perú pero ahora cultivada en Argentina a 2.000 m) y CO-407, una variedad de Chile, de zonas bajas (140 m). En general, los datos muestran que gs es una variable importante en el control de Amax pero la magnitud de este control depende de la variedad considerada. El valor más alto de Amax fue registrado para la variedad CICA (31 µmol m-2 s-1) y el más bajo para la variedad CO-407 con 19 µmol m-2 s-1. En relación a la capacidad de carboxilación y EUAi, los datos obtenidos permiten inferir que las variedades de altura (Sayaña y Kancolla) poseen valores altos de EUAi y capacidad de carboxilación en relación a las variedades provenientes de media y baja altitud (CO-407 y CICA). El valor de AFE no mostró variaciones de significación entre las variedades estudiadas. Todas resultaron ser anfiestomáticas con DE mayor en la epidermis inferior. Se encontró una correlación lineal positiva entre Amax y DE. Los resultados obtenidos aportan datos de valor biológico que pueden ser utilizados para los estudios relacionados al mejoramiento y/o selección varietal en ambientes de media y alta montaña. Palabras clave: Fotosíntesis, quinoa, tejidos foliares.Leaf gas exchange (Maxima photosynthetic assimilation, Amax; stomatal conductance, gs; leaf transpiration, Tr; internal CO2, and Ci concentration, and anatomical leaf data (stomatal density and size, tissue thickness), specific leaf area (SLA), photosynthetic and protection pigments in six quinoa varieties from dif ferent origins were analysed. Trials were carried out in an arid region of Argentina’s Nor thwest (Amaicha del Valle, 22º31’S, 65º59’ W, 1,985 m asl) under drought conditions. The varieties used were: Kancolla (Perú) and Sayaña (from Bolivian Altiplano, 3,850 m asl), Quinoa Blanca (from Cochabamaba, Bolivia, 2,570 m asl), Quinoa Roja (from Cangrejillos, Argentina, 3,700 m asl), CICA (originally form Perú but currently cultivated at 2,000 m asl in Argentina) and CO-407 a lowland Chilean variety (140 m asl). In general, our data showed that gs is an important variable in the net CO2 assimilation (Amax) but this control magnitude depends on the considered variety. Highest (31 µmol m-2 s-1 ) and lowest (19 µmol m-2 s-1) Amax values were recorded for CICA and CO-407 varieties. Regarding the carboxylic capacity and intrinsic water use efficiency (iWUE), it may be inferred from the obtained data, that the high altitude varieties (Sayaña and Kancolla) have both, the highest iWUE and carboxylation capacity in relation to other varieties from middle and low altitude (CO-407 and CICA). SLA value did not vary significantly among the studied varieties. Every analysed variety was amphiestomatic with higher stomatal density (SD) on the lower epidermis. A positive linear correlation between Amax and SD was found. The obtained results provide data of unquestionable biological value, which can be useful to varietal breeding studies in middle and high mountain environments.Fil: González, Juan A.. Fundación Miguel Lillo; ArgentinaFil: Ponessa, Graciela I.. Fundación Miguel Lillo; ArgentinaFil: Buedo, Sebastia. E.. Fundación Miguel Lillo; ArgentinaFil: Mercado, Maria Ines. Fundación Miguel Lillo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Prado, Fernando Eduardo. Universidad Nacional de Tucuman. Facultad de Ciencias Naturales e Instituto Miguel Lillo. Catedra de Fisiologia Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Stereoacuity Improvement using Random-Dot Video Games

Conventional amblyopia therapy involves occlusion or penalization of the dominant eye, though these methods enhance stereoscopic visual acuity in fewer than 30% of cases. To improve these results, we propose a treatment in the form of a video game, using random-dot stimuli and perceptual learning techniques to stimulate stereoacuity. The protocol is defined for stereo-deficient patients between 7-14 years of age who have already received treatment for amblyopia and have a monocular best corrected distance visual acuity of at least 0.1 logMAR. Patients are required to complete a perceptual learning program at home using the video game. While compliance is stored automatically in the cloud, periodic optometry center visits are used to track patient evolution and adjust the game's stereoscopic demand until the smallest detectable disparity is achieved. The protocol has proved to be successful, and effectiveness is gauged in terms of a two-level gain on a random stereoacuity test (global stereoacuity or cyclopean stereoacuity reference test). Moreover, the random-dot stimuli learning transfers to medial lateral stereoscopic acuity according to a Wirt Circles test, in which success criteria is a final stereoacuity of over 140", and the attained enhancement corresponds to no less than two levels of stereoscopic acuity. Six months later, a random-dot stereoacuity test recorded no reduction in the stereoacuity that was achieved

Efecto del silenciamiento de genes que codifican poligalacturonasas sobre el reblandecimiento del fruto de fresa asociado a la maduración

La degradación de las pectinas de la pared celular mediada por poligalacturonasas juega un papel clave en el reblandecimiento de la fresa. Así, el silenciamiento del gen FaPG1 incrementa la firmeza del fruto maduro y alarga su vida postcosecha. Además de FaPG1, en fresa se ha descrito otro gen que codifica una poligalacturonasa específica de maduración, FaPG2. Con el fin de profundizar en el papel de estos genes, se han obtenido plantas transgénicas con el gen FaPG2 silenciado (líneas BPG), así como plantas con FaPG1 y FaPG2 silenciados (líneas ABPG), obtenidas mediante retransformación de una línea antiFaPG1 (APG29) que mostraba un fuerte silenciamiento del gen y un incremento en la firmeza de fruto. Se obtuvieron 24 líneas BPG y 15 ABPG. Estas plantas, junto con la línea APG29 y controles sin transformar, fueron analizadas durante 3 años consecutivos. El 50% de las líneas BPG mostraron mayor firmeza de fruto rojo que el control sin transformar, aunque el incremento en firmeza fue similar al obtenido en la línea APG29. Todas las líneas dobles transformantes dieron frutos de mayor firmeza que el control, siendo los valores ligeramente superiores a los de la línea APG29 en alguna de ellas. A nivel de expresión, las líneas BPG seleccionadas mostraron un silenciamiento del gen FaPG2 que varió entre el 60-70%, e inesperadamente, un silenciamiento significativo de FaPG1, a pesar de la baja homología entre ambos genes. El silenciamiento de FaPG1 en las líneas ABPG fue superior al 95%; sin embargo, el silenciamiento de FaPG2 fue similar al obtenido en las plantas BPG. Estos resultados confirman el papel clave de las poligalacturonasas en el reblandecimiento de la fresa y sugieren la existencia de una regulación compleja en la expresión de ambos genes. Este trabajo ha sido financiado por el proyecto AGL2011-24814 y Fondos FEDERUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The ecology of peace : preparing Colombia for new political and planetary climates

ABSTRACT: Colombia, one of the world’s most species-rich nations, is currently undergoing a profound social transition: the end of a decadeslong conflict with the Revolutionary Armed Forces of Colombia, known as FARC. The peace agreement process will likely transform the country’s physical and socioeconomic landscapes at a time when humans are altering Earth’s atmosphere and climate in unprecedented ways. We discuss ways in which these transformative events will act in combination to shape the ecological and environmental future of Colombia. We also highlight the risks of creating perverse development incentives in these critical times, along with the potential benefits – for the country and the world – if Colombia can navigate through the peace process in a way that protects its own environment and ecosystems

Evaluation of Safety of a Newly Formulated Pirfenidone in Chronic Kidney Disease: A Non-Randomized Pilot Study in Mexican Patients

The aim of this pilot clinical trial was to evaluate the safety of a new formulation of prolonged-release Pirfenidone (PR-PFD) in chronic kidney disease (CKD), specifically focal and segmental glomerular hyalinization (FSGH). Open-label, pilot, nonrandomized trial. Eighteen patients previously diagnosed with CKD stages 1– 5 according to “Kidney Disease: Improving Global Outcomes” were enrolled in the study. Target dos-age of PFD was 1200 mg twice a day in the form of prolonged-release tablets to reach a full dosage of 2400 mg daily. Clinical trial was carried out for 60 months to evaluate the safety and efficacy of a newly formulated PR-PFD in patients with CKD. After the treatment for 60 months, it was found that PR-PFD kept renal function from declining significantly in CKD patients, as the glomerular filtration rate (GFR) showed only minimal variations throughout the study. Estimated glomerular filtration rate (eGFR) showed no differences at both baseline and the end points. Proteinuria improved, and creatinine, cystatin C, urea, hemoglobin and hepatic transaminases remained constant without any considerable changes across the study. Minor side effects were noticed when compared with those found in previous studies, indicating an increased tolerance to this pharmaceutical formulation of PFD. Prolonged-released PFD could be safely used as an adjuvant therapy in patients with CKD.Registry number was obtained from ClinicalTrials.gov (NCT02408744)