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

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

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

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

Cell wall disassembly is delayed by rhamnogalacturonate lyase gene silencing: potential role in fruit firmness

Strawberry fruits greatly reduce their quality due to softening during ripening with economically important losses. Texture changes of fleshy fruits during ripening are mainly due to middle lamellae dissolution, cell-to-cell adhesion losses and wall weakening of parenchyma cells by the coordinated action of several cell wall enzymes. Pectin degradation has been proven a key factor in strawberry softening by functional analysis of several pectinase genes (polygalacturonase, pectate lyase and -galactosidase). The complexity and highly dynamic nature of pectins remains a challenge to fully elucidate structure-function relationships of pectins. In this work, we present the functional analysis of two independent strawberry transgenic lines with more than 95% silencing of a rhamnogalacturonate lyase gene (FaRGLyase1). Firmness of ripe fruit was significantly higher in both transgenic lines than in the control. Cell walls from these fruits were extracted and analyzed by glycan microarray profiling. This high‐throughput technique allows a wide screening of cell-wall glycan occurrence based on the detection of specific cell wall oligosaccharide epitopes by monoclonal antibodies and reveals profiles which can be used as potential fingerprints specific for a singular organ and/or developmental stage. Our microarray results showed that the silencing of FaRGLyase1 reduced degradation of several rhamnogalacturonan-I related epitopes, as expected. Additionally, comparison of transgenic cell walls from ripe fruits with those extracted from control fruits at different developmental stages (green, white and red) by hierarchical clustering, demonstrated a higher similarity of transgenic fruit cell walls with the control cell walls from fruits at the white stage. Glycan microarray profiles revealed less degraded fruit cell walls as result of FaRGLyase1 down-regulation which could contribute to the increased firmness of transgenic fruitsUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

High-throughput mapping of cell wall glycans to unveil cell wall disassembly, a key process determining strawberry fruit softening

The short shelf life of strawberry fruit is a major limitation that produces important economic losses related to postharvest spoiling. Fruit texture of fleshy fruits is a complex trait but mainly rely on mechanical properties of parenchyma cell walls. Several studies support the relevance of cell wall modifying enzymes on cell wall deconstruction, decreasing cell wall strength and cell to cell adhesion, and ultimately producing the softening of the fruit at macroscopic level. Previous studies on our group showed that transgenic silencing of ripening-specific genes encoding some of these enzymes reduced softening and increased postharvest shelf life in strawberry (Fragaria × ananassa, cv. ‘Chandler’) fruits. In this research, to further investigate the cell wall remodelling process associated to strawberry softening a high-throughput analysis of cell wall composition based on monoclonal antibodies against different polysaccharide epitopes has been performed. To this purpose, cell walls were isolated from non-transgenic fruits at different developmental stages as well as from ripe fruits of selected transgenic lines with genes involved in metabolism of pectins (pectate lyase, polygalacturonase, β-galactosidase, pectin acetil esterase), hemicellulose/cellulose (endo-β-glucanase) or lignin (cinnamyl alcohol dehydrogenase) down-regulated. These transgenic lines showed a large variability in fruit firmness at ripening. Cell walls were fractionated and subjected to a carbohydrate microarray. The results obtained unveiled a common pattern of cell wall composition on those transgenic lines with firmer phenotypes, specially defined by the higher content of pectins on those cell wall fractions more imbricated in the matrix, which can be interpreted as a less degraded cell wall structure.This research was supported by FEDER EU Funds and the Ministerio de Economía y Competitividad of Spain (grant reference AGL2017-86531-C2-1-R). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Downregulation of NAC transcription factors modifies cell wall composition and increases strawberry fruit firmness

The strawberry is a soft fruit with a very short post-harvest shelf life. The changes in texture during fruit ripening are mainly due to the dissolution of the middle lamellae, reducing cell-to-cell adhesion, and the weakening of parenchymal cell walls as result of the action of cell wall modifying enzymes. At present, no master regulator of this process has been discovered yet. NAC transcription factors have been involved in numerous physiological processes, including fruit ripening. In strawberry, the NAC family comprises more than 110 genes, and at least 6 of them are expressed during fruit development. In this research, we performed a functional analysis of two ripening-related NAC genes, FaNAC2 and FaNAC3, in Fragaria x ananassa Duch. cv. Chandler. Several RNAi transgenic lines showing low FaNAC2 or FaNAC3 mRNA levels in fruit were obtained through Agrobacterium-mediated transformation. These lines produced fruits significantly firmer than control at the ripe stage, being the increase in firmness higher in FaNAC2 silenced plants. Cell walls were extracted from ripe transgenic fruits and characterized by ELISA and Epitope Detection Chromatography (EDC), using monoclonal antibodies against different polysaccharide epitopes. FaNAC2 transgenic lines showed more extensive changes than FaNAC3; these modifications involved increased amounts of demethylated pectins (LM19) in water and CDTA fractions and an alteration of the lateral branches of RG-I, decreasing the amount of arabinan epitopes and increasing galactan epitopes detected by LM6 and LM5, respectively. The amount of arabinogalactan proteins recognized by the JIM13 antibody was also affected, decreasing in the Na2CO3 fraction and increasing in the 4M KOH and cellulase fraction of the transgenic lines.The results obtained indicate that NAC genes could be involved in the regulation of cell wall disassembly associated to strawberry fruit softening.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech