Cellulose-Callose Hydrogels: Computational Exploration of Their Nanostructure and Mechanical Properties.

Polysaccharides play a crucial role in virtually all living systems. They also represent the biocompatible and fully sustainable component of a variety of nanoparticles, which are of increasing interest in biomedicine, food processing, cosmetics, and structural reinforcement of polymeric materials. The computational modeling of complex polysaccharide phases will assist in understanding the properties and behavior of all these systems. In this paper, structural, bonding, and mechanical properties of 10 wt % cellulose-callose hydrogels (ß-glucans coexisting in plant cell walls) were investigated by atomistic simulations. Systems of this kind have recently been introduced in experiments revealing unexpected interactions between the polysaccharides. Starting from initial configurations inspired by X-ray diffraction data, atomistic models made of ∼1.6 × 106 atoms provide a qualitatively consistent view of these hydrogels, displaying stability, homogeneity, connectivity, and elastic properties beyond those of a liquid suspension. The simulation shows that the relatively homogeneous distribution of saccharide nanofibers and chains in water is not due to the solubility of cellulose and callose, but to the formation of a number of cross-links among the various sample components. The broad distribution of strength and elasticity among the links implies a degree of anharmonicity and irreversible deformation already evident at low external load. Besides the qualitative agreement with experimental observations, the simulation results display also quantitative disagreements in the estimation of elastic coefficients, such as the Young's modulus, that require further investigation. Complementary simulations of dense cellulose-callose mixtures (no hydrogels) highlight the role of callose in smoothing the contact surface of different nanofibers forming larger bundles. Cellulose-callose structures in these systems displayed an enhanced water uptake and delayed dye release when compared to cellulose alone, highlighting potential new applications as drug delivery scaffolds. The simulation trajectories provide a tuning and testing ground for the development of coarse-grained models that are required for the large scale investigation of mechanical properties of cellulose and callose mixtures in a watery environment.

Elucidating the role of polygalacturonase genes in strawberry fruit softening.

To disentangle the role of polygalacturonase (PG) genes in strawberry softening, the two PG genes most expressed in ripe receptacles, FaPG1 and FaPG2, were down-regulated. Transgenic ripe fruits were firmer than those of the wild type when PG genes were silenced individually. Simultaneous silencing of both PG genes by transgene stacking did not result in an additional increase in firmness. Cell walls from ripe fruits were characterized by a carbohydrate microarray. Higher signals of homogalacturonan and rhamnogalacturonan I pectin epitopes in polysaccharide fractions tightly bound to the cell wall were observed in the transgenic genotypes, suggesting a lower pectin solubilization. At the transcriptomic level, the suppression of FaPG1 or FaPG2 alone induced few transcriptomic changes in the ripe receptacle, but the amount of differentially expressed genes increased notably when both genes were silenced. Many genes encoding cell wall-modifying enzymes were down-regulated. The expression of a putative high affinity potassium transporter was induced in all transgenic genotypes, indicating that cell wall weakening and loss of cell turgor could be linked. These results suggest that, besides the disassembly of pectins tightly linked to the cell wall, PGs could play other roles in strawberry softening, such as the release of oligogalacturonides exerting a positive feedback in softening.

Dirigent proteins in plants: modulating cell wall metabolism during abiotic and biotic stress exposure.

Dirigent (DIR) proteins were found to mediate regio- and stereoselectivity of bimolecular phenoxy radical coupling during lignan biosynthesis. Here we summarize the current knowledge of the importance of DIR proteins in lignan and lignin biosynthesis and highlight their possible importance in plant development. We focus on the still rather enigmatic Arabidopsis DIR gene family, discussing the few members with known functional importance. We comment on recent discoveries describing the detailed structure of two DIR proteins with implications in the mechanism of DIR-mediated catalysis. Further, we summarize the ample evidence for stress-induced dirigent gene expression, suggesting the role of DIRs in adaptive responses. In the second part of our work, we present a preliminary bioinformatics-based characterization of the AtDIR family. The phylogenetic analysis of AtDIRs complemented by comparison with DIR proteins of mostly known function from other species allowed us to suggest possible roles for several members of this family and identify interesting AtDIR targets for further study. Finally, based on the available metadata and our in silico analysis of AtDIR promoters, we hypothesize about the existence of specific transcriptional controls for individual AtDIR genes and implicate them in various stress responses, hormonal regulations, and developmental processes.

Antisense down-regulation of the strawberry ß-galactosidase gene FaßGal4 increases cell wall galactose levels and reduces fruit softening.

Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by ß-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative ß-galactosidase gene, FaßGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaßGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaßGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaßGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaßGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaßGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness.

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy.

BACKGROUND: One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening. SCOPE: In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.

Suppressing the rhamnogalacturonan lyase gene FaRGLyase1 preserves RGI pectin degradation and enhances strawberry fruit firmness.

Plant rhamnogalacturonan lyases (RGLyases) cleave the backbone of rhamnogalacturonan I (RGI), the "hairy" pectin and polymer of the disaccharide rhamnose (Rha)-galacturonic acid (GalA) with arabinan, galactan or arabinogalactan side chains. It has been suggested that RGLyases could participate in remodeling cell walls during fruit softening, but clear evidence has not been reported. To investigate the role of RGLyases in strawberry softening, a genome-wide analysis of RGLyase genes in the genus Fragaria was performed. Seventeen genes encoding RGLyases with functional domains were identified in Fragaria × ananassa. FaRGLyase1 was the most expressed in the ripe receptacle of cv. Chandler. Transgenic strawberry plants expressing an RNAi sequence of FaRGLyase1 were obtained. Three transgenic lines yielded ripe fruits firmer than controls without other fruit quality parameters being significantly affected. The highest increase in firmness achieved was close to 32%. Cell walls were isolated from ripe fruits of two selected lines. The amount of water-soluble and chelated pectins was higher in transgenic lines than in the control. A carbohydrate microarray study showed a higher abundance of RGI epitopes in pectin fractions and in the cellulose-enriched fraction obtained from transgenic lines. Sixty-seven genes were differentially expressed in transgenic ripe fruits when compared with controls. These genes were involved in various physiological processes, including cell wall remodeling, ion homeostasis, lipid metabolism, protein degradation, stress response, and defense. The transcriptomic changes observed in FaRGLyase1 plants suggest that senescence was delayed in transgenic fruits.

Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits.

Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell-cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity maintenance that results in firmer ripe fruit.

CRISPR/Cas9 editing of the polygalacturonase FaPG1 gene improves strawberry fruit firmness.

Firmness is one of the most important fruit quality traits in strawberries. The postharvest shelf life of this soft fruit is highly limited by the loss of firmness, where cell wall disassembly plays an important role. Previous studies demonstrated that the polygalacturonase FaPG1 has a key role in remodelling pectins during strawberry softening. In this study, FaPG1 knockout strawberry plants have been generated using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Ten independent lines, cv. "Chandler", were obtained, and all of them were successfully edited as determined by PCR amplification and T7 endonuclease assay. The targeted mutagenesis insertion and deletion rates were analyzed using targeted deep sequencing. The percentage of edited sequences varied from 47% up to almost 100%, being higher than 95% for seven of the selected lines. Phenotypic analyses showed that 7 out of the eight lines analyzed produced fruits significantly firmer than the control, ranging from 33 to 70% increase in firmness. There was a positive relationship between the degree of FaPG1 editing and the rise in fruit firmness. Minor changes were observed in other fruit quality traits, such as colour, soluble solids, titratable acidity or anthocyanin content. Edited fruits showed a reduced softening rate during postharvest, displayed a reduced transpirational water loss, and were less damaged by Botrytis cinerea inoculation. The analysis of four potential off-target sites revealed no mutation events. In conclusion, editing the FaPG1 gene using the CRISPR/Cas9 system is an efficient method for improving strawberry fruit firmness and shelf life.

Plasmodesmata and their role in the regulation of phloem unloading during fruit development.

Fruit consumption is fundamental to a balanced diet. The contemporary challenge of maintaining a steady food supply to meet the demands of a growing population is driving the development of strategies to improve the production and nutritional quality of fruit. Plasmodesmata, the structures that mediate symplasmic transport between plant cells, play an important role in phloem unloading and distribution of sugars and signalling molecules into developing organs. Targeted modifications to the structures and functioning of plasmodesmata have the potential to improve fruit development; however, knowledge on the mechanisms underpinning plasmodesmata regulation in this context is scarce. In this review, we have compiled current knowledge on plasmodesmata and their structural characterisation during the development of fruit organs. We discuss key questions on phloem unloading, including the pathway shift from symplasmic to apoplastic that takes place during the onset of ripening as potential targets for improving fruit quality.

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

The properties of (1,3)-ß-glucans (i.e., callose) remain largely unknown despite their importance in plant development and defence. Here we use mixtures of (1,3)-ß-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.

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

Pectins analysed by AFM are visualized as individual chains, branched or unbranched, and aggregates. To investigate the nature of these structures, sodium carbonate soluble pectins from strawberry fruits were digested with endo-polygalacturonase M2 from Aspergillus aculeatus and visualized by AFM. A gradual decrease in the length of chains was observed as result of the treatment, reaching a minimum LN value of 22nm. The branches were not visible after 2h of enzymatic incubation. The size of complexes also diminished significantly with the enzymatic digestion. A treatment to hydrolyse rhamnogalacturonan II borate diester bonds neither affected chains length or branching nor complex size but reduced the density of aggregates. These results suggest that chains are formed by a mixture of homogalacturonan and more complex molecules composed by a homogalacturonan unit linked to an endo-PG resistant unit. Homogalacturonan is a structural component of the complexes and rhamnogalacturonan II could be involved in their formation.

Structural changes in cell wall pectins during strawberry fruit development.

Strawberry (Fragaria × anannasa Duch.) is one of the most important soft fruit. Rapid loss of firmness occurs during the ripening process, resulting in a short shelf life and high economic losses. To get insight into the role of pectin matrix in the softening process, cell walls from strawberry fruit at two developmental stages, unripe-green and ripe-red, were extracted and sequentially fractionated with different solvents to obtain fractions enriched in a specific component. The yield of cell wall material as well as the per fresh weight contents of the different fractions decreased in ripe fruit. The largest reduction was observed in the pectic fractions extracted with a chelating agent (trans-1,2- diaminocyclohexane-N,N,N'N'-tetraacetic acid, CDTA fraction) and those covalently bound to the wall (extracted with Na2CO3). Uronic acid content of these two fractions also decreased significantly during ripening, but the amount of soluble pectins extracted with phenol:acetic acid:water (PAW) and water increased in ripe fruit. Fourier transform infrared spectroscopy of the different fractions showed that the degree of esterification decreased in CDTA pectins but increased in soluble fractions at ripen stage. The chromatographic analysis of pectin fractions by gel filtration revealed that CDTA, water and, mainly PAW polyuronides were depolymerised in ripe fruit. By contrast, the size of Na2CO3 pectins was not modified. The nanostructural characteristics of CDTA and Na2CO3 pectins were analysed by atomic force microscopy (AFM). Isolated pectic chains present in the CDTA fractions were significantly longer and more branched in samples from green fruit than those from red fruit. No differences in contour length were observed in Na2CO3 strands between samples of both stages. However, the percentage of branched chains decreased from 19.7% in unripe samples to 3.4% in ripe fruit. The number of pectin aggregates was higher in green fruit samples of both fractions. These results show that the nanostructural complexity of pectins present in CDTA and Na2CO3 fractions diminishes during fruit development, and this correlates with the solubilisation of pectins and the softening of the fruit.

The nanostructural characterization of strawberry pectins in pectate lyase or polygalacturonase silenced fruits elucidates their role in softening.

To ascertain the role of pectin disassembly in fruit softening, chelated- (CSP) and sodium carbonate-soluble (SSP) pectins from plants with a pectate lyase, FaplC, or a polygalacturonase, FaPG1, downregulated by antisense transformation were characterized at the nanostructural level. Fruits from transgenic plants were firmer than the control, although FaPG1 suppression had a greater effect on firmness. Size exclusion chromatography showed that the average molecular masses of both transgenic pectins were higher than that of the control. Atomic force microscopy analysis of pectins confirmed the higher degree of polymerization as result of pectinase silencing. The mean length values for CSP chains increased from 84 nm in the control to 95.5 and 101 nm, in antisense FaplC and antisense FaPG1 samples, respectively. Similarly, SSP polyuronides were longer in transgenic fruits (61, 67.5 and 71 nm, in the control, antisense FaplC and antisense FaPG1 samples, respectively). Transgenic pectins showed a more complex structure, with a higher percentage of branched chains than the control, especially in the case of FaPG1 silenced fruits. Supramolecular pectin aggregates, supposedly formed by homogalacturonan and rhamnogalacturonan I, were more frequently observed in antisense FaPG1 samples. The larger modifications in the nanostructure of pectins in FaPG1 silenced fruits when compared with antisense pectate lyase plants correlate with the higher impact of polygalacturonase silencing on reducing strawberry fruit softening.