Strawberry fruit softening and pectin disassembly: I. Nanostructural characterization of fruit pectins by atomic force microscopy II. Role of the β-Galactosidase gene FaβGal4

La fresa se caracteriza por sufrir un rápido reblandecimiento durante el periodo de maduración, llegando a adquirir una textura semilíquida al final del desarrollo. Esta disminución de firmeza limita el periodo postcosecha considerablemente y provoca pérdidas de entre el 5 y el 25% de la producción. El reblandecimiento del fruto durante la maduración es consecuencia principalmente de cambios en las características y/o composición de las paredes celulares, siendo las pectinas uno de los polímeros de pared que más se modifican. El objetivo principal de esta tesis ha sido profundizar en el estudio del papel de las pectinas y las pectinasas en el proceso de maduración de la fresa, con el fin de avanzar en el conocimiento del proceso de reblandecimiento del fruto. Para ello, se han realizado estudios a nivel nanoestructural mediante microscopía de fuerza atómica (AFM) de las fracciones ricas en pectinas procedentes de frutos en diferente estadio de maduración. Además, se han llevado a cabo diferentes experimentos para determinar la estructura de los polímeros y complejos supramoleculares que se observan en las imágenes de AFM, utilizando para ello pectinas de frutos control y muestras procedentes de líneas transgénicas con genes que codifican pectinasas silenciados. Por último, se ha analizado el papel de un gen que codifica una β-galactosidasa, FaβGal4, mediante la generación de plantas transgénicas con dicho gen silenciado. Los resultados obtenidos sugieren la existencia de una importante solubilización de las pectinas de la pared celular durante la maduración del fruto de fresa. La principal causa de este proceso es la eliminación de las cadenas laterales de las pectinas, tanto de las unidas iónicamente a la pared como de las unidas covalentemente. Además, también se produce una despolimerización de las pectinas unidas iónicamente y una evidente pérdida de azúcares neutros, sobre todo en la fracción de pectinas que están unidas covalentemente a la pared. Por otro lado, experimentos de digestión de pectinas de fresa con endo-PG de hongos indican que tanto las ramificaciones como las cadenas lineales de pectinas visibles mediante AFM están compuestas principalmente por ácido galacturónico. Aún así, parte de estas cadenas podría contener otros carbohidratos dificultando la digestión total con la endo-PG. Por otro lado, también se puede concluir que los agregados que se observan en las imágenes de AFM contienen ácido galacturónico, siendo estas estructuras susceptibles a la digestión por endo-PG, y que el RGII puede estar implicado en su formación, ya que el número de estas estructuras macromoleculares disminuye significativamente como resultado de una hidrólisis ácida suave que rompe los dímeros de RGII. Por último, el gen FaβGal4 juega un papel importante durante la maduración del fruto de la fresa. Las líneas transgénicas con este gen silenciado mostraron un incremento de la firmeza del 30% en frutos maduros con respecto al control sin transformar. A nivel de la pared celular, los frutos transgénicos mostraron una mayor cantidad de Gal en todas sus fracciones, así como una reducción en la solubilización y despolimerización de las pectinas. Con estos resultados, postulamos que la disminución de la pérdida de los residuos de Gal durante el proceso de maduración en los frutos transgénicos disminuiría la movilidad de otras enzimas modificadoras de la pared, obstruyendo el acceso a sus sustratos, siendo ésta la causa de la mayor firmeza de los frutos de las líneas silenciadas

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

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

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

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

A core collection of modified strawberry germplasm as a resource tool for funghi infection and fruit texture studies

Strawberry has been extensively used as a model organism for functional genomics of genes related with fungi infection (Amil-Ruiz et al, 2011) and fruit ripening, specially fruit texture (Posé et al, 2011). These studies implied the genetic transformation and subsequent characterization of a wide range of traits, resulting in a considerable number of lines being created and more or less extensively studied. A preliminary data survey was performed previous publications, laboratory notebooks and project reports. Main categories and the relevant features (like cultivar, transgene sequence, germplasm availability, plant phenotype, experiment results, etc) were identified, establishing the appropriate relationships. The project followed the Chado schema used by the GMOD initiative http://www.gmod.org. The germplasm collection currently hosts two strawberry cultivar (Camarosa and Chandler), nine transgenic lines with resistance genes against fungi infections and over a dozen for fruit texture related genes. Collected information includes in vitro and in planta leaf symptomatology and fungus spore germination, as well as gene expression for each transgene, plant production, fruit color, shape and firmness. Data on cell wall fractionation, pectin and soluble sugars quantification, FT-IR and size exclusion chromatography, AFM pectin structure characterization and Immunohistological analysis. The intrinsic value of these studies makes necessary to preserve both the germplasm and the data generated, and to make it available to the community for further study and reuse. Community driven formats will facilitate the use of the data in new. studies. The present project aims to increase the value of the transgenic strawberry plants in the collection, using standard data formats and open source tools to facilitate access to the research and breeding communities as well as to facilitate the distribution of the germplasm.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. AGL2011-2481

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

Interactions between callose and cellulose revealed through the analysis of biopolymer mixtures.

The properties of (1,3)-β-glucans (i.e., callose) remain largely unknown despite their importance in plant development and defence. Here we use mixtures of (1,3)-β-glucan and cellulose, in ionic liquid solution and hydrogels, as proxies to understand the physico-mechanical properties of callose. We show that after callose addition the stiffness of cellulose hydrogels is reduced at a greater extent than predicted from the ideal mixing rule (i.e., the weighted average of the individual components' properties). In contrast, yield behaviour after the elastic limit is more ductile in cellulose-callose hydrogels compared with sudden failure in 100% cellulose hydrogels. The viscoelastic behaviour and the diffusion of the ions in mixed ionic liquid solutions strongly indicate interactions between the polymers. Fourier-transform infrared analysis suggests that these interactions impact cellulose organisation in hydrogels and cell walls. We conclude that polymer interactions alter the properties of callose-cellulose mixtures beyond what it is expected by ideal mixing

A nanostructural view of the cell wall disassembly process during fruit ripening and postharvest storage by atomic force microscopy

Background: The mechanical properties of parenchyma cell walls and the strength and extension of adhesion areas between adjacent cells, jointly with cell turgor, are main determinants of firmness of fleshy fruits. These traits are modified during ripening leading to fruit softening. Cell wall modifications involve the depolymerisation of matrix glycans and pectins, the solubilisation of pectins and the loss of neutral sugars from pectin side chains. These changes weaken the cell walls and increase cell separation, which in combination with a reduction in cell turgor, bring about textural changes. Atomic force microscopy (AFM) has been used to characterize the nanostructure of cell wall polysaccharides during the ripening and postharvest storage of several fruits. This technique allows the imaging of individual polymers at high magnification with minimal sample preparation. Scope and approach: This paper reviews the main features of the cell wall disassembly process associated to fruit softening from a nanostructural point of view, as has been provided by AFM studies. Key findings and conclusions: AFM studies show that pectin size, ramification and complexity is reduced during fruit ripening and storage, and in most cases these changes correlate with softening. Postharvest treatments that improve fruit quality have been proven to preserve pectin structure, suggesting a clear link between softening and pectin metabolism. Nanostructural characterization of cellulose and hemicellulose during ripening has been poorly explored by AFM and the scarce results available are not conclusive. Globally, AFM could be a powerful tool to gain insights about the bases of textural fruit quality in fresh and stored fruits