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

Isolation and transfection of strawverry protoplasts for gene editing

Strawberry is the most economically important soft fruit. The improvement of the organoleptic qualities of ripe fruit and the postharvest shelf life are main objectives of strawberry breeding programs. Fruit softening is mainly due to the disassembly of cell walls and the dissolution of middle lamella. In strawberry, functional analyses of genes encoding polygalacturonases (PGs) indicate that these enzymes play a key role in fruit softening, i.e. the antisense downregulation of PG genes FaPG1 or FaPG2 increased fruit firmness and postharvest shelf life (Paniagua et al., 2020). These results suggest that PG encoding genes are excellent targets for gene editing to improve strawberry fruit quality. Transfection of protoplasts with CRISPR/Cas9 ribonucleoprotein complexes is currently being explored in many species to produce DNA-free edited plants. In this research, a protocol for strawberry protoplasts transfection has been optimized with the final goal of producing non-transgenic strawberry plants with the FaPG1 gene edited. Protoplasts were isolated from 9 weeks old in vitro grown plants of Fragaria x ananassa, cv. ‘Chandler’, micropropagated in Murashige and Skoog (MS) medium supplemented with 2 mg/L of BA. Protoplast extraction and purification was performed as described by Barceló et al. (2019). Using this protocol, a yield of 1 x 105 protoplast/g fresh tissue was obtained and nearly 50-70% of them were viable. Protoplasts were transfected with the plasmid pHBT-sGFP(S65T)-NOS using a PEG-mediated transformation system, as reported by Yoo et al. (2007). To improve the efficiency of protoplast transfection, different variables were evaluated: PEG concentration, time of incubation on PEG and DNA concentration. At 48 h after transfection, the highest percentage of protoplasts showing GFP expression, 18%, was obtained with 15 minutes incubation in 20% of PEG and 5 µg of DNA

Caracterización de indicadores de la calidad del fruto en líneas de fresa transgénicas con genes silenciados que codifican para enzimas pectinolíticas

Se han evaluado algunos indicadores de calidad del fruto en líneas transgénicas de fresa con los genes de poligalacturonasa FaPG1 (líneas PG) o pectato liasa FaplC (líneas APEL) silenciados. Se analizaron dos líneas independientes por genotipo transgénico. No se observaron diferencias en el contenido de sólidos solubles entre las líneas transgénicas y el control. De igual forma, la acidez total y el pH fueron similares en las líneas PG29, APEL21 y el control; sin embargo, la acidez de los frutos de las líneas PG62 y APEL39 fue superior al control. Los parámetros de color L*, a* y b* fueron similares en todos los genotipos; sin embargo, el contenido en antocianos fue menor en la línea APEL21. Los valores más altos de firmeza de fruto, estimada mediante un ensayo de extrusión, se observaron en las dos líneas transgénicas PG y en la línea APEL39. En cuanto a las pérdidas por goteo (drip loss), la línea APEL39 presentó un valor mayor que el control, pero la línea APEL21 registró valores menores. El contenido de compuestos fenólicos se analizó en la línea PG29, no encontrándose diferencias estadísticas con respecto al control. Finalmente, la capacidad del fruto para captar radicales libres fue ligeramente menor en la línea PG29 que en el control. Los resultados indican que el silenciamiento de los genes de pectinasas incrementa significativamente la firmeza de la fresa sin modificar sustancialmente parámetros de calidad del fruto maduro como color, acidez, sólidos solubles o contenido en antocianos.Some quality traits of transgenic strawberry fruits with low levels of expression of the pectinase genes FaPG1 (PG lines) or FaplC gene (APEL lines) were evaluated. Two independent lines per transgenic genotype were analyzed. Soluble solids were similar in control and transgenic lines. Similarly, pH and titratable acidity was similar in lines PG29, APEL21 and control; however, lines PG62 and APEL39 showed acidity values higher than the control. The color parameters L*, a* and b* were similar in control and transgenic fruits; however, line APEL21 displayed a lower value of anthocyanin content. The highest values of fruit firmness, measured with an extrusion test, were observed in both PG transgenic lines and in the APEL39 line. Regarding the drip loss, APEL39 line showed a higher value than the control, but the APEL21 line displayed lower values. The content of phenolic compounds was analyzed in line PG29, not observing significant differences with the control. Finally, the antiradical activity of the fruit was slightly lower in the line PG29 than in the control. The results obtained indicate that the silencing of the pectinase genes increases the firmness of the fruit without substantially modifying other quality parameters such as color, acidity, soluble solids or anthocyanin content

Failure analysis of Co–Cr hip resurfacing prosthesis during solidification

In this study a failure originated during solidification process into the femoral stem component of Hip Resurfacing prosthesis was investigated. Visual inspection, optical microscopy, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and a commercial software simulation ProCAST were carried out in order to determine the cause and solution of this failure. The results exhibited hot tearing, shrinkage porosity and metal oxide films due to inadequate heat dissipation during solidification process, as a consequence of poor investment casting ceramic mold configuration. Also in this paper was improved the casting design solving this kind of defects

Obtención de poliploides en Fragaria para su uso en mejora

La fresa cultivada, Fragaria × ananassa Duch., es una especie octoploide procedente de un híbrido interespecífico entre las especies silvestres F. virginiana Duch. y F. chiloensis L. Los cruces intraespecíficos de F × ananassa se utilizan extensivamente para la obtención de nuevos cultivares con características agronómicas mejoradas. A pesar de esto, esta especie muestra una alta susceptibilidad a patógenos fúngicos y bacterianos y una baja tolerancia a estreses abióticos. Especies silvestres de menor ploidía, e.g. F. vesca (2x), pueden ser una fuente genética de gran valor para la mejora de esos caracteres en la fresa cultivada, pero las diferencias en ploidía representan una barrera reproductiva para los cruzamientos interespecíficos. La duplicación del número de cromosomas mediante tratamientos con colchicina de meristemos procedentes de estolones se ha utilizado para facilitar la hibridación interespecífica entre fresas diploides y comerciales. En este trabajo, se ha puesto a punto la metodología para la obtención de poliploides en fresa mediante la aplicación de colchicina durante la regeneración de brotes in vitro.Esta investigación ha sido financiada por los fondos FEDER EU y el Ministerio de Economía y Competitividad de España (AGL2017-86531-C2-1-R

Dimorphic Fungal Coinfection as a Cause of Chronic Diarrhea and Pancolitis

Histoplasma capsulatum and Paracoccidioides brasiliensis are dimorphic fungi that cause systemic mycosis mostly in tropical South America and some areas of North America. Gastrointestinal involvement is not uncommon among these fungal diseases, but coinfection has not previously been reported. We report a patient with chronic diarrhea and pancolitis caused by paracoccidioidomycosis and histoplasmosis

Exogenous attention to facial vs non-facial emotional visual stimuli

The capacity of the two types of non-symbolic emotional stimuli most widely used in research on affective processes, faces and (non-facial) emotional scenes, to capture exogenous attention, was compared. Negative, positive and neutral faces and affective scenes were presented as distracters to 34 participants while they carried out a demanding digit categorization task. Behavioral (reaction times and number of errors) and electrophysiological (event-related potentialsERPs) indices of exogenous attention were analyzed. Globally, facial expressions and emotional scenes showed similar capabilities to attract exogenous attention. Electrophysiologically, attentional capture was reflected in the P2a component of ERPs at the scalp level, and in left precentral areas at the source level. Negatively charged faces and scenes elicited maximal P2a/precentral gyrus activity. In the case of scenes, this negativity bias was also evident at the behavioral level. Additionally, a specific effect of facial distracters was observed in N170 at the scalp level, and in the fusiform gyrus and inferior parietal lobule at the source level. This effect revealed maximal attention to positive expressions. This facial positivity offset was also observed at the behavioral level. Taken together, the present results indicate that faces and non-facial scenes elicit partially different and, to some extent, complementary exogenous attention mechanismsThis work was supported by the grants PSI2008-03688, PSI2009-08607 and PSI2011-26314 from the Ministerio de Economía y Competitividad (MINECO) of Spain. MINECO also supports Jacobo Albert through a Juan de la Cierva contract (JCI-2010-07766

Unravelling the nanostructure of strawberry fruit pectins by endo-polygalacturonase digestion and atomic force microscopy.

https://v2.sherpa.ac.uk/id/publication/12825Este estudio analizó las pectinas de las fresas utilizando microscopía de fuerza atómica (AFM) para visualizar sus cadenas y agregados. Las pectinas se trataron con una enzima específica, lo que resultó en una reducción gradual de la longitud de las cadenas y la desaparición de las ramificaciones tras 2 horas de digestión. El tamaño de los complejos también disminuyó, pero un tratamiento para romper enlaces de rhamnogalacturonano II no afectó la longitud de las cadenas ni el tamaño de los complejos, aunque sí redujo la densidad de los agregados. Esto sugiere que las pectinas podrían estar compuestas por homogalacturonano y unidades más complejas, donde el rhamnogalacturonano II podría jugar un papel en la formación de los agregados.Ministerio de Economía y Competitividad of Spain (AGL2011-24814 y AGL2014-55784-C2-1-R) and Ramón y Cajal RYC-2011-0883