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

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

Polμ deficiency increases resistance to oxidative damage and delays liver aging

Polμ is an error-prone PolX polymerase that contributes to classical NHEJ DNA repair. Mice lacking Polμ (Polμ(-/-)) show altered hematopoiesis homeostasis and DSB repair and a more pronounced nucleolytic resection of some V(D)J junctions. We previously showed that Polμ(-/-) mice have increased learning capacity at old ages, suggesting delayed brain aging. Here we investigated the effect of Polμ(-/-) deficiency on liver aging. We found that old Polμ(-/-) mice (>20 month) have greater liver regenerative capacity compared with wt animals. Old Polμ(-/-) liver showed reduced genomic instability and increased apoptosis resistance. However, Polμ(-/-) mice did not show an extended life span and other organs (e.g., heart) aged normally. Our results suggest that Polμ deficiency activates transcriptional networks that reduce constitutive apoptosis, leading to enhanced liver repair at old age.This work was supported by grants to AB from the Ministry of Science and Innovation (SAF 2008-02099; PLE2009-0147 and PSE-010000-2009-3), Comunidad Auto´noma de Madrid (S2010/BMD-2420), and the European Commission (FP7-HEALTH-2009/CARE-MI. CC is supported by The Spanish Ministry of Economy and Competiveness and the Pro-CNIC Foundation.S

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis