Nanostructural differences in pectic polymers isolated from strawberry fruits with low expression levels of pectate lyase or polygalacturonase genes

Our research group has obtained transgenic strawberry plants expressing antisense sequences of either a pectate lyase (APEL lines) [1] or a polygalacturonase gene (APG lines) [2]. Both genes encode ripening-specific endo-pectinases with a common target, deesterified homogalacturonans, but each enzyme act by a different mechanism and pH range. Ripe fruits from both transgenic genotypes were significantly firmer than control, being APG fruits on average 25% firmer than APEL fruits. Cell wall analysis of both transgenic genotypes indicated that pectin fractions extracted with CDTA and sodium carbonate were significantly modified in transgenic fruits [2,3]. To gain insight in the role of these pectinases in pectin disassembly during ripening, CDTA and Na2CO3 pectins have been analyzed by atomic force microscopy (AFM). APEL and APG CDTA pectins had similar contour lengths but both were significantly longer than control. Similarly, APG carbonate chains were longer than control, showing APEL carbonate chains an intermediate length. Furthermore, transgenic pectins displayed a more complex branching pattern and a higher number of micellar aggregates, especially in the sodium carbonate fractions of APG samples. Acid hydrolysis of carbonate pectins reduced the number of micellar aggregates. AFM analyses confirm that the inhibition of both pectinases reduces pectin disassembly, and also suggest that each pectinase acts on specific pectin domains. Particularly, polygalacturonase silencing induces more significant pectin modifications, nicely correlated with the firmer phenotype of APG fruits, than the down-regulation of pectate lyase

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

Regeneración de plantas, vía embriogénesis somática, a partir de material adulto de olivo silvestre

La embriogénesis somática es una herramienta poderosa para la clonación de genotipos de interés. En algunas especies como el olivo, esta técnica se ve limitada por las dificultades que presenta la regeneración a partir de material adulto. En este trabajo, se ha inducido embriogénesis somática a partir de material adulto de Olea europaea var. sylvestris siguiendo el protocolo desarrollado por Mazri et al. (Scient. Hort. 159: 88-95, 2013) para el cultivar de olivo Dahbia. Se emplearon 4 genotipos con distinto nivel de resistencia al patógeno fúngico Verticillium dahliae: AC18, StopVert and Outvert (genotipos resistentes) y AC15 (genotipo susceptible) (Jiménez-Díaz, IAS-CSIC, Córdoba, comunicación personal). Inicialmente, los explantos se cultivaron en oscuridad en medio líquido de inducción conteniendo la formulación mineral MS con los macroelementos a la mitad, y un suplemento de 30 µM TDZ-0.5 µM NAA durante 4 días, a 80 rpm; posteriormente, fueron transferidos al mismo medio sin reguladores del crecimiento. A las 8 semanas, el callo que proliferó se cultivó en medio de expresión ECO suplementado con 0.25 µM IBA, 0.5 µM 2iP y 0.44 µM BA, durante varios subcultivos. Sólo los explantos de yema apical del genotipo Stopvert formaron callo embriogénico con una frecuencia del 5%. Tras la multiplicación de este callo, una parte del mismo se transfirió a medio de maduración ECO con membranas de acetato de celulosa para la maduración de embriones. Los embriones maduros mostraron un porcentaje de germinación del 35%, lo que ha permitido la recuperación de plantas. La estabilidad genética del material obtenido se analizó mediante marcadores microsatélites. Se compararon plantas regeneradas a partir de embriones somáticos con el callo embriogénico del que procedían, plantas iniciadas a partir de yemas laterales y mantenidas in vitro mediante proliferación de axilares y la planta donante.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Nanostructural changes in cell wall pectins during strawberry fruit ripening assessed by atomic force microscopy

Rapid loss of firmness occurs during strawberry (Fragaria × ananassa Duch) ripening, resulting in a short shelf life and high economic losses. The disassembly of cell walls is considered the main responsible for fruit softening, being pectins extensively modified during strawberry ripening (Paniagua et al. 2014). Atomic force microscopy allows the analysis of individual polymer chains at nanostructural level with a minimal sample preparation (Morris et al., 2001). The main objective of this research was to compare pectins of green and red ripe strawberry fruits at the nanostructural level to shed light on structural changes that could be related to softening. Cell walls from strawberry fruits were extracted and fractionated with different solvents to obtain fractions enriched in a specific component. The yield of cell wall material, as well as the amount of the different fractions, decreased in ripe fruits. CDTA and Na2CO3 fractions underwent the largest decrements, being these fractions enriched in pectins supposedly located in the middle lamella and primary cell wall, respectively. Uronic acid content also decreased significantly during ripening in both pectin fractions, but the amount of soluble pectins, those extracted with phenol:acetic acid:water (PAW) and water increased in ripe fruits. Monosaccharide composition in CDTA and Na2CO3 fractions was determined by gas chromatography. In both pectin fractions, the amount of Ara and Gal, the two most abundant carbohydrates, decreased in ripe fruits. The nanostructural characteristics of CDTA and Na2CO3 pectins were analyzed by AFM. Isolated pectic chains present in the CDTA fraction were significantly longer and more branched in samples from green fruits than those present in samples obtained from red fruit. In spite of slight differences in length distributions, Na2CO3 samples from unripe fruits displayed some longer chains at low frequency that were not detected in ripe fruits. Pectin aggregates were more frequently observed in green fruit samples from both fractions. These results support that pectic chain length and the nanostructural complexity of the pectins present in CDTA and Na2CO3 fractions diminish during strawberry fruit development, and these changes, jointly with the loss of neutral sugars, could contribute to the solubilization of pectins and fruit softening. Paniagua et al. (2014). Ann Bot, 114: 1375-1383 Morris et al. (2001). Food Sci Tech 34: 3-10 This research was supported by FEDER EU Funds and the Ministerio de Educación y Ciencia of Spain (grant reference AGL2011-24814)Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Enhancing shoot recovery from transgenic avocado somatic embryos

Use of biotechnological tools in avocado (Persea americana Mill.) is hampered by difficulties in obtaining mature somatic embryos with an acceptable germination capacity. Use of semi-permeable cellulose acetate membranes on top of maturation medium has improved the quality of obtained embryos and their germination rate; however, in the case of transgenic embryos the conversion rate is still rather low. In this investigation, a protocol for recovery of transgenic plants has been developed. Mature avocado somatic embryos, over cellulose acetate membranes, were obtained and induced to germinate following the protocol described in Palomo-Ríos (2012). Some transgenic embryos developed buds ≤ 2 mm, which failed to elongate. For shoot recovery, cotyledons were partially removed and the embryonic axis cultured over 4 weeks in MS medium supplemented with either 1 mg/l BA, 1 mg/l TDZ or 1 mg/l BA and 1 mg/l TDZ. Highest shoot recovery was obtained in medium supplemented with 1 mg/l BA and 1 mg/l TDZ, 53.6±6.7% versus 23.2±5,7% and 39.6±6.4% in media supplemented with 1 mg/l BA or 1 mg/l TDZ, respectively. Afterwards, sprouted embryos were cultured over 4 additional weeks in MS medium supplemented with 1 mg/l BA. In some cases, resulting shoots could be induced to proliferate in GD medium (Gamborg et al., 1968) supplemented with 0.3 mg/l BA, while in other cases, they had to be recovered through micrografting onto in vitro germinated seedlings as described in Pliego-Alfaro and Murashige (1987). MS medium supplemented with 1mg/l BA and solidified with 6 g/l Sigma A-1296 agar, was used for micrografts. Shoots derived from micrografts were either induced to proliferate in GD medium with 0.3 mg/l BA or further grafted and cultured in liquid MS medium supplemented with 0.1 mg/l BA, using perlite as substrate. After 8-12 weeks, micrografts could be transferred to ex vitro conditions. References: Gamborg O.L., Muller, R.A., Iojima, K., (1968). Nutrient requirements of suspension cultures of soybean roots cells. Exp. Cell. Res. 50:151-158. Palomo-Ríos, E., (2012). Thesis: Regeneración y Transformación Genética de Aguacate (Persea americana Mill.). Ph.D. Dissertation, University of Málaga, Málaga Pliego-Alfaro, F., Murashige, T., (1987). Possible rejuvenation of adult avocado by graftage onto juvenile root stocks in vitro. Hort Sci. 22:1321-1324.AGL2011-30354-C02-0

Unravelling the nanostructure of strawberry fruit pectins by atomic force microscopy

Atomic force microscopy (AFM) allows the analysis of individual polymers at nanostructural level with a minimal sample preparation. This technique has been used to analyse the pectin disassembly process during the ripening and postharvest storage of several fleshy fruits. In general, pectins analysed by AFM are usually visualized as isolated chains, unbranched or with a low number of branchs and, occasionally, as large aggregates. However, the exact nature of these structures is unknown. It has been suggested that pectin aggregates represent a mixture of rhamnonogalacturonan I and homogalacturonan, while isolated chains and their branches are mainly composed by polygalacturonic acid. In order to gain insight into the nature of these structures, sodium carbonate soluble pectins from ripe strawberry (Fragaria x ananassa, Duch.) fruits were subjected to enzymatic digestion with endo-Polygalacturonase M2 from Aspergillus aculeatus, and the samples visualized by AFM at different time intervals. Pectins isolated from control, non-transformed plants, and two transgenic genotypes with low level of expression of ripening-induced pectinase genes encoding a polygalacturonase (APG) or a pectate lyase (APEL) were also included in this study. Before digestion, isolated pectin chains from control were shorter than those from transgenic fruits, showing number-average (LN) contour length values of 73.2 nm vs. 95.9 nm and 91.4 nm in APG and APEL, respectively. The percentage of branched polymers was significantly higher in APG polyuronides than in the remaining genotypes, 33% in APG vs. 6% in control and APEL. As a result of the endo-PG treatment, a gradual decrease in the main backbone length of isolated chains was observed in the three samples. The minimum LN value was reached after 8 h of digestion, being similar in the three genotypes, 22 nm. By contrast, the branches were not visible after 1.5-2 h of digestion. LN values were plotted against digestion time and the data fitted to a first-order exponential decay curve, obtaining R2 values higher than 0.9. The half digestion time calculated with these equations were similar for control and APG pectins, 1.7 h, but significantly higher in APEL, 2.5 h, indicating that these polymer chains were more resistant to endo-PG digestion. Regarding the pectin aggregates, their volumes were estimated and used to calculate LN molecular weights. Before digestion, control and APEL samples showed complexes of similar molecular weights, 1722 kDa, and slightly higher than those observed in APG samples. After endo-PG digestion, size of complexes diminished significantly, reaching similar values in the three pectin samples, around 650 kDa. These results suggest that isolated polymer chains visualized by AFM are formed by a HG domain linked to a shorter polymer resistant to endo-PG digestion, maybe xylogalacturonan or RG-I. The silencing of the pectate lyase gene slightly modified the structure and/or chemical composition of polymer chains making these polyuronides more resistant to enzymatic degradation. Similarly, polygalacturonic acid is one of the main component of the aggregates.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

AFM study of strawberry pectin nanostructure and its relevance on fruit texture

Atomic force microscopy (AFM) has been used to characterize the nanostructure of cell wall pectins during strawberry fruit growth and ripening, as well as in transgenic fruits with pectinase genes downregulated. This technique allows the imaging of individual polymers at high magnification with minimal sample preparation. AFM studies during fruit development show that pectin size, ramification and aggregation is reduced in ripe fruits. Additionally, transgenic lines with different pectinase genes downregulated (polygalacturonase, pectate lyase and B-galactosidase) also show a more complex pectin nanostructure, including longer chains, higher branching degree and larger presence of aggregates. In all those cases the higher pectin complexity at nanoscale correlates with a reduced softening in strawberry fruits at macroscale level. Globally, our results support the key role of pectins in fruit structure and highlights the use of AFM as a powerful tool to gain insights about the bases of textural fruit quality not only in strawberry, but also in other commercial crops.AGL2017-86531-C2-1-R, Ministerio de Economía, Industria y Competitividad of Spain and FEDER EU funds. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Evaluación del efecto del filtrado crudo del hongo Rosellinia necatrix en el crecimiento de brotes y cultivos embriogénicos de olivo

Rosellinia necatrix es un patógeno muy extendido en plantaciones de aguacate del sur de la Península Ibérica, afectando también, ocasionalmente, a plantaciones de olivo. Nuestro grupo está abordando la obtención de variantes somaclonales de olivo y aguacate, que muestren mayor tolerancia a este patógeno. En este trabajo se presentan los resultados en olivo, tras la exposición de cultivos embriogénicos a dosis crecientes de filtrado crudo del hongo. Se utilizó callo embriogénico, obtenido a partir de radícula, cultivado en medio líquido ECO (Pérez-Barranco et al. 2009 PCTOC 97: 243-251) durante 3 semanas en agitación, y posteriormente filtrado (malla de 2 mm) para separar la fracción fina. Esta fracción fue cultivada durante otras 3 semanas en medio ECO líquido conteniendo un 20, 40, 60 u 80% de filtrado crudo de Rosellinia necatrix, tras su esterilización en frio. Finalmente, el callo embriogénico fue cultivado en placa en medio ECO sólido para su recuperación. El filtrado del hongo al 20% no afectó al crecimiento del callo embriogénico; sin embargo, a partir del 40% el crecimiento fue inhibido, salvo en algunas placas, donde se observó proliferación de callo tras 2 meses en medio de cultivo sin filtrado. Se han obtenido varias líneas que han sobrevivido tras la exposición al 40, 60 y 80% de filtrado del hongo. Estas líneas están siendo cultivadas de nuevo en presencia de la misma concentración de filtrado del hongo para evaluar su comportamiento. Posteriormente, se abordará la recuperación de plantas. Paralelamente, brotes de olivo regenerados a partir de la línea embriogénica P1 han sido cultivados en medio de multiplicación (Vidoy-Mercado et al. 2012, Acta Hort. 949: 27-30) suplementado con filtrado crudo del hongo (60%), durante varios subcultivos, para evaluar el efecto del filtrado en el crecimiento de los brotes.Universidad de Málaga, Campus de Exelencia Internac ional Andalucía Tech. Proyecto AGR-7992 (P11-Junta de Andalucía

Polygalacturonase gene FaPG1 downregulation is related to increased strawberry fruit resistance to fungal decay

Plant health is a major target in breading programs because crops are under constant biotic stress, and climate change is exacerbating pests and disease negative impacts in agriculture. Obtaining crop varieties armed with better defences is a potential strategy to reduce losses from biotic attacks. Plant cell walls perform crucial roles on many physiological processes, and under biotic stress, play crucial defensive roles as protecting barrier, as well as a source of integrity signalling molecules. Plant immunity has evolved a complex multi-layered system which first line of defence is initiated by conserved molecular patterns coming from pathogens, named pathogen-associated molecular patterns or PAMPs, or from their own corrupted cell walls due to pathogen invasion, named damaged-associated molecular patterns or DAMPs. Accumulating evidence from cell wall mutants has unveiled several components and mechanisms of plant innate immunity under biotic stresses, mostly in Arabidopsis, but still little is known from species with agronomic interest as strawberry. Our group has an established strawberry transgenic collection of cell wall mutants. Among them, RNAseq expression profiles of FaPG1 mutants has shown downregulation of other cell wall related genes than PG [1], but the mechanisms underneath required further investigation. FaPG genes code for enzymes with endo-PG activity related to oligogalacturonic acid (OGA) release, which would be associated to the changes in gene expression of other cell wall genes than FaPG. In this work, postharvest assays of FaPG1 fruits showed not only the increased fruit firmness typical of this mutant, but a better resistance to fungal infections by Botrytis cinerea, enhancing fruit shelf life in comparison with control fruits.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech