Characterization of Crude and Processed Pulp Cell Walls of Three Selected Mamey Accessions (Mammea americana L.).

Mamey (Mammea americana L.) is a tropical fleshy fruit native from the West Indies and northern South America. It is very appreciated for its flavor and color but has been little described. The present study investigates the composition and histochemistry of the pulp cell walls of three mamey accessions readily available in Martinique. The impact of pulp processing into puree on cell wall composition is evaluated. The histology and rheology of mamey puree are assessed considering these characterizations. Mamey pulp cell wall composition is dominated by highly methyl-esterified pectins (DM: 66.2-76.7%) of high molecular weight, and show few hemicelluloses, mainly xyloglucans. Processing reduced methyl-esterified uronic acid contents and gave purees with significantly different viscosities. Mamey puree was composed of polydisperse particles (20-2343 µm), which size distributions were different depending on the accession: Ti Jacques was dominated by smaller particles (50% had approximated diameters lower than 160 µm), Sonson's by larger particles (50% had approximated diameters higher than 900 µm), and Galion's had an intermediate profile. This new knowledge on mamey pulp is valuable for future works on mamey processing into new food products, even more so for those including cell wall polysaccharide-degrading enzymes.

A multimodal and multiscale investigation of factors affecting the juice yield of cider apples.

Apple cider juice yield at harvest and after 15 and 30 days of storage durations was studied by analyzing the mechanical properties of fresh and plasmolyzed flesh, water distribution, cell wall polysaccharide composition and organization of the apples; in this study, the apple varieties used were Avrolles, Douce coetligne, Douce moen, Judor, Petit jaune. Juice yield mainly depended on the apple variety and the storage duration. Cellulose organization and cell wall pectin hydration were affected by ripening and are related to fruit firmness. Flesh viscoelastic mechanical properties were not general indications of juice yields. However, these properties helped distinguish the varieties according to flesh damage caused by ice crystals upon freezing. Cell encapsulation of the juice in the flesh contributed to lower yields. The apple variety and harvesting mode are recommended as a means to better control juice yield variations.

Cuticle architecture and mechanical properties: a functional relationship delineated through correlated multimodal imaging.

Cuticles are multifunctional hydrophobic biocomposites that protect the aerial organs of plants. During plant development, plant cuticles must accommodate different mechanical constraints combining extensibility and stiffness, and the corresponding relationships with their architecture are unknown. Recent data showed a fine-tuning of cuticle architecture during fruit development, with several chemical clusters which raise the question of how they impact the mechanical properties of cuticles. We investigated the in-depth nanomechanical properties of tomato (Solanum lycopersicum) fruit cuticle from early development to ripening, in relation to chemical and structural heterogeneities by developing a correlative multimodal imaging approach. Unprecedented sharps heterogeneities were evidenced including an in-depth mechanical gradient and a 'soft' central furrow that were maintained throughout the plant development despite the overall increase in elastic modulus. In addition, we demonstrated that these local mechanical areas are correlated to chemical and structural gradients. This study shed light on fine-tuning of mechanical properties of cuticles through the modulation of their architecture, providing new insight for our understanding of structure-function relationships of plant cuticles and for the design of bioinspired material.

Adsorption of apple xyloglucan on cellulose nanofiber depends on molecular weight, concentration and building blocks.

Apple xyloglucan (XyG) structural features (molecular weight and building motifs) were studied in relation with the hemicellulose interaction with cellulose nanofiber (CNF) through adsorption isotherm, high performance size exclusion chromatography and high-performance anion exchange chromatography analyses. Up to 75.6 µg of apple XyG adsorbed/mg of CNF. XyG adsorption depended on both Mw and XyG/CNF ratio. The adsorption capacity increased with Mw irrespectively of XyG/CNF ratio. At XyG/CNF ratio > 0.05, the adsorption of high Mw populations (2.0 × 105, 7.9 × 104 g mol-1) gradually ceased, while it kept increasing for low Mw populations (2.6 × 104, 1.2 × 104 g mol-1). The unbranched glucan segments and the galactosylated XLLG motif showed the highest binding affinity to CNF. Most of the XLLG motif was found in "loops and tails", whereas most of the XyG unbranched glucan segments interact directly with CNF.

New insight into the mode of action of a GH74 xyloglucanase on tamarind seed xyloglucan: Action pattern and cleavage site.

Structural elucidation of plant cell wall xyloglucan through the analysis of enzymatically produced fragments requires detailed knowledge of enzymes hydrolytic mechanism. In this note, the mode of action and cleavage site of commercial recombinant xyloglucanases (GH74, Paenibacillus sp.) was studied on native and fluorescent-tagged tamarind xyloglucan. In complement to information provided by the manufacturer, GH74 hydrolysis was shown dual endo/exo- and exo-processive with low affinity towards labelled reducing-ends. GH74 accommodated X/G in its subsite -1 and X/L in its subsite +1. Moreover, hydrolysis kinetic indicated a GH74 activity inhibition by excess products. These results will help for application of this enzyme in xyloglucans structural analysis or for processing cell walls.

The cutin polymer matrix undergoes a fine architectural tuning from early tomato fruit development to ripening.

The cuticle is a complex polymer matrix that protects all aerial organs of plants, fulfills multiple roles in plant-environment interactions, and is critical for plant development. These functions are associated with the structural features of cuticles, and the architectural modeling of cuticles during plant development is crucial for understanding their physical properties and biological functions. In this work, the in-depth architecture of the cutin polymer matrix during fruit development was investigated. Using cherry tomato fruit (Solanum lycopersicum) as a model from the beginning of the cell expansion phase to the red ripe stage, we designed an experimental scheme combining sample pretreatment, Raman mapping, multivariate data analyses, and biochemical analyses. These approaches revealed clear chemical areas with different contributions of cutin, polysaccharides, and phenolics within the cutin polymer matrix. Besides, we demonstrated that these areas are finely tuned during fruit development, including compositional and macromolecular rearrangements. The specific spatiotemporal accumulation of phenolic compounds (p-coumaric acid and flavonoids) suggests that they fulfill distinct functions during fruit development. In addition, we highlighted an unexpected dynamic remodeling of the cutin-embedded polysaccharides pectin, cellulose, and hemicellulose. Such structural tuning enables consistent adaption of the cutin-polysaccharide continuum and the functional performance of the fruit cuticle at the different developmental stages. This study provides insights into the plant cuticle architecture and in particular into the organization of the epidermal cell wall-cuticle.

Biorefinery of apple pomace: New insights into xyloglucan building blocks.

Within the apple pomace biorefinery cascade processing framework aiming at adding value to an agroindustrial waste, after pectin recovery, this study focused on hemicellulose. The structure of the major apple hemicellulose, xyloglucan (XyG), was assessed as a prerequisite to potential developments in industrial applications. DMSO-LiCl and 4 M KOH soluble hemicelluloses from pectin-extracted apple pomace were purified by anion exchange chromatography. XyG structure was assessed by coupling xyloglucanase and endo-ß-1,4-glucanase digestions to HPAEC and MALDI-TOF MS analyses. 71.9% of pomaces hemicellulose were recovered with starch. DMSO-LiCl and 4 M KOH soluble XyG exhibited Mw of 19 and 140 kDa, respectively. Besides the XXXG, XLXG, XXLG, XXFG, XLFG and XLLG structures, novel oligosaccharides with degree of polymerization of 6-10 were observed after xyloglucanase digestion. Cellobiose and cellotriose were revealed randomly distributed in XyG backbone and were more present in DMSO-LiCl soluble XyG. Residual pomace remains a potential source of other materials.

The SlSHN2 transcription factor contributes to cuticle formation and epidermal patterning in tomato fruit.

Tomato (Solanum lycopersicum) is an established model for studying plant cuticle because of its thick cuticle covering and embedding the epidermal cells of the fruit. In this study, we screened an EMS mutant collection of the miniature tomato cultivar Micro-Tom for fruit cracking mutants and found a mutant displaying a glossy fruit phenotype. By using an established mapping-by-sequencing strategy, we identified the causal mutation in the SlSHN2 transcription factor that is specifically expressed in outer epidermis of growing fruit. The point mutation in the shn2 mutant introduces a K to N amino acid change in the highly conserved 'mm' domain of SHN proteins. The cuticle from shn2 fruit showed a ~ fivefold reduction in cutin while abundance and composition of waxes were barely affected. In addition to alterations in cuticle thickness and properties, epidermal patterning and polysaccharide composition of the cuticle were changed. RNAseq analysis further highlighted the altered expression of hundreds of genes in the fruit exocarp of shn2, including genes associated with cuticle and cell wall formation, hormone signaling and response, and transcriptional regulation. In conclusion, we showed that a point mutation in the transcriptional regulator SlSHN2 causes major changes in fruit cuticle formation and its coordination with epidermal patterning.

Unraveling Cuticle Formation, Structure, and Properties by Using Tomato Genetic Diversity.

The tomato (Solanum lycopersicum) fruit has a thick, astomatous cuticle that has become a model for the study of cuticle formation, structure, and properties in plants. Tomato is also a major horticultural crop and a long-standing model for research in genetics, fruit development, and disease resistance. As a result, a wealth of genetic resources and genomic tools have been established, including collections of natural and artificially induced genetic diversity, introgression lines of genome fragments from wild relatives, high-quality genome sequences, phenotype and gene expression databases, and efficient methods for genetic transformation and editing of target genes. This mini-review reports the considerable progresses made in recent years in our understanding of cuticle by using and generating genetic diversity for cuticle-associated traits in tomato. These include the synthesis of the main cuticle components (cutin and waxes), their role in the structure and properties of the cuticle, their interaction with other cell wall polymers as well as the regulation of cuticle formation. It also addresses the opportunities offered by the untapped germplasm diversity available in tomato and the current strategies available to exploit them.

The Complex Architecture of Plant Cuticles and Its Relation to Multiple Biological Functions.

Terrestrialization of vascular plants, i.e., Angiosperm, is associated with the development of cuticular barriers that prevent biotic and abiotic stresses and support plant growth and development. To fulfill these multiple functions, cuticles have developed a unique supramolecular and dynamic assembly of molecules and macromolecules. Plant cuticles are not only an assembly of lipid compounds, i.e., waxes and cutin polyester, as generally presented in the literature, but also of polysaccharides and phenolic compounds, each fulfilling a role dependent on the presence of the others. This mini-review is focused on recent developments and hypotheses on cuticle architecture-function relationships through the prism of non-lipid components, i.e., cuticle-embedded polysaccharides and polyester-bound phenolics.

Comparison of cell wall chemical evolution during the development of fruits of two contrasting quality from two members of the Rosaceae family: Apple and sweet cherry.

Cell wall composition was studied during the development of apple cultivars (14-161/182 days after full bloom, DAA) maintaining firm fruit (Ariane) or evolving to mealy texture (Rome Beauty) when ripe and in sweet cherry cultivars (21/26-70/75 DAA) to assess their skin-cracking susceptibility (tolerant Regina and susceptible Garnet). Pectin sugar composition and hemicellulose fine structure assessed by enzymatic degradation coupled to MALDI-TOF MS analysis were shown to vary markedly between apples and cherries during fruit development. Apple showed decreasing rhamnogalacturonan I (RGI) and increasing homogalacturonan (HG) pectic domain proportions from young to mature fruit. Hemicellulose-cellulose (HC) sugars peaked at the beginning of fruit expansion corresponding to the maximum cell wall content of glucose and mannose. In contrast, HG peaked very early in the cell wall of young developing cherries and remained constant until ripening whereas RGI content continuously increased. HC content decreased very early and remained low in cell walls. Only the low content of mannose and to a lesser extent fucose increased and then slowly decreased from the beginning of the fruit expansion phase. Hemicellulose structural profiling showed strong varietal differences between cherry cultivars. Both apples and cherries demonstrated a peak of glucomannan oligomers produced by ß-glucanase hydrolysis of the cell wall at the onset of cell expansion. The different glucomannan contents and related oligomers released from cell walls are discussed with regard to the contribution of glucomannan to cell wall mechanical properties. These hemicellulose features may prove to be early markers of apple mealiness and cherry skin-cracking susceptibility.

Sequential natural deep eutectic solvent pretreatments of apple pomace: A novel way to promote water extraction of pectin and to tailor its main structural domains.

To establish a "green" biorefinery extraction of apple pomace pectin, a sequential pretreatment with three natural deep eutectic solvents (NADES, choline chloride (CC): glycerol (G); CC: lactic acid (LA); potassium carbonate (K): G) was used prior to hot water extraction. A synergistic effect of CC:G and CC:LA pretreatments was observed and led to the highest recovery of pectin. The sequential NADES/water extraction process also provided a mean to tailor pectin main structure. It was explained as resulting from ion exchange and individual NADES components effects. The 13C solid state NMR T1ρH and THH parameters indicated a reorganization of cellulose in the residues following extraction of pectin, notably after alkaline K:G pretreatment/water extraction. Hence, sequential NADES pretreatments/water extraction represents a "green" alternative to mild mineral acid to extract pectin and to tailor its main structures, while the residual pomace can be further sources of valuable compounds and polymers.

Cellulose, pectin and water in cell walls determine apple flesh viscoelastic mechanical properties.

The viscoelastic mechanical properties are important quality traits for fleshy fruit uses. The contribution of cell wall polysaccharides chemistry and organization on their variability was studied in six varieties of apple. Correlation between damping and storage modulus of plasmolyzed tissue distinguished better apple varieties on their viscoelasticity than fresh samples. Galactose, arabinose and uronic acids correlated positively with the storage modulus of fresh apple samples (E'f). These corresponded to 4-linked galactan but no specific arabinose linkage. Galacturonic acid branched on O-3 and terminal rhamnose correlated negatively with E'f. These correlations formed two groups of fruit except for branched methyl-esterified galacturonic. Solid-state 13C NMR spectroscopy analyses showed that E'f correlated negatively with cellulose C4 T1ρH relaxation and positively with pectin methyl esters THH proton diffusion. The results point to the key roles of pectin structure and hydration and cellulose microfibrils distribution on apple mechanical properties.

Data set on hydroxycinnamic acid ester analysis from the cell walls of apples and grapes.

Data on the esters of hydroxycinnamic acids (HCAs) from the cell walls of wine grapes (Cabernet franc) and cider apples (Douce Moen and Guillevic) were acquired. Caffeic acid, p-coumaric acid (pCA) and ferulic acid (FA) monomers were identified by HPLC-UV/MS. Means to limit the oxidative degradation during cell wall preparation were assessed by the yield of HCA recovered after alkaline extraction. Following the optimum cell wall preparation, the pCA content varied between 2.3 and 32.5 mg kg-1 dry cell wall and that of FA varied between 0.3 and 17.2 mg kg-1 dry cell wall. Higher HCA quantities were found in the peels compared to the flesh and in apples compared to grapes. The Douce Moen apple was richer in HCAs than the Guillevic apple. pCA was localized in the cell wall as observed by TEM after labeling with the INRA-COU1 antibody that recognizes pCA linked to O-5 of arabinose. The anti-FerAra antibody targeting FA on O-5 of arabinose failed to locate FA esters in the apple and grape cell walls.

Assembly of tomato fruit cuticles: a cross-talk between the cutin polyester and cell wall polysaccharides.

The cuticle is an essential and ubiquitous biological polymer composite covering aerial plant organs, whose structural component is the cutin polyester entangled with cell wall polysaccharides. The nature of the cutin-embedded polysaccharides (CEPs) and their association with cutin polyester are still unresolved Using tomato fruit as a model, chemical and enzymatic pretreatments combined with biochemical and biophysical methods were developed to compare the fine structure of CEPs with that of the noncutinized polysaccharides (NCPs). In addition, we used tomato fruits from cutin-deficient transgenic lines cus1 (cutin synthase 1) to study the impact of cutin polymerization on the fine structure of CEPs. Cutin-embedded polysaccharides exhibit specific structural features including a high degree of esterification (i.e. methylation and acetylation), a low ramification of rhamnogalacturonan (RGI), and a high crystallinity of cellulose. In addition to decreasing cutin deposition and polymerization, cus1 silencing induced a specific modification of CEPs, especially on pectin content, while NCPs were not affected. This new evidence of the structural specificities of CEPs and of the cross-talk between cutin polymerization and polysaccharides provides new hypotheses concerning the formation of these complex lipopolysaccharide edifices.

Meaning of xylan acetylation on xylan-cellulose interactions: A quartz crystal microbalance with dissipation (QCM-D) and molecular dynamic study.

In plant cell walls, xylan chains present various substituents including acetate groups. The influence of the acetyl substitution on the organization of xylan-cellulose complexes remains poorly understood. This work combines in vitro and in silico approaches to decipher the functional role of acetyl groups on the xylan/cellulose interaction. Acetylated xylans were extracted from apple pomace with dimethyl sulfoxide-lithium chloride (DMSO-LiCl) and deacetylated using a mild alkali treatment. The adsorption behavior of acetylated and deacetylated xylan fractions was investigated using quartz crystal microbalance with dissipation (QCM-D) and molecular dynamics. Acetylated xylans form a dense and poorly hydrated and rigid layer on cellulose with xylan chains that have two residues per helical turn conformation, whereas the deacetylated fraction forms a swollen and more viscous layer in which only the xylan chains in direct contact with the cellulose surface have two residues per helical turn conformation. The other chains have three residues per turn conformation.

Phenolic distribution in apple epidermal and outer cortex tissue by multispectral deep-UV autofluorescence cryo-imaging.

Phenolic compounds in fruit are involved in responses to biotic and abiotic stresses and are responsible for organoleptic properties. To establish the distribution of these secondary metabolites at the tissue and sub-cellular scales, mapping of fluorescence in apple epidermis and outer cortex tissue in cryogenic condition was performed after deep-UV excitation at 275 nm. Douce Moën and Guillevic cider apple varieties were sampled and frozen after harvest, after 30 days at 4 °C and after 20 days at room temperature. Image analysis of fluorescence emission images acquired between 300 and 650 nm allowed the assignment of fluorescence signals to phenolic compound families based on reference molecules. Emission attributed to monomeric and/or condensed flavanol was localized in whole tissue with major fluorescence in the cuticle region. Hydroxycinnamic acids were found predominantly in the outer cortex and appeared in the cell wall. Fluorescent pigments were mostly found in the epidermis. The distribution of flavanols in the sub-cuticle and phenolic acids in the outer cortex distinguished apple varieties. Storage conditions had no impact on phenolic distribution. The proposed fluorescent imaging and analysis approach enables studies on phenolic distribution in relation to fruit development, biotic/abiotic stress resistance and quality.

Functional exploration of Pseudoalteromonas atlantica as a source of hemicellulose-active enzymes: Evidence for a GH8 xylanase with unusual mode of action.

To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a ß-xylosidase, a α-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end.

Cryo-laser scanning confocal microscopy of diffusible plant compounds.

BACKGROUND: The in vivo observation of diffusible components, such as ions and small phenolic compounds, remains a challenge in turgid plant organs. The analytical techniques used to localize such components in water-rich tissue with a large field of view are lacking. It remains an issue to limit compound diffusion during sample preparation and observation processes. RESULTS: An experimental setup involving the infusion staining of plant tissue and the cryo-fixation and cryo-sectioning of tissue samples followed by fluorescence cryo-observation by laser scanning confocal microscopy (LSCM) was developed. This setup was successfully applied to investigate the structure of the apple fruit cortex and table grape berry and was shown to be relevant for localizing calcium, potassium and flavonoid compounds. CONCLUSION: The cryo-approach was well adapted and opens new opportunities for imaging other diffusible components in hydrated tissues.

Water and cell wall contributions to apple mechanical properties.

Relations between the apple cortex viscoelastic properties, water dynamics, histological, and chemical characteristics were investigated. Water mobility in four apple genotypes was studied by low-field NMR relaxometry prior and after plasmolysis of the cortex tissue. A discrete and a continuous method for decomposing the multi-exponential T2 curves were implemented and compared. The results show that both methods of relaxation curve decomposition had close ability to discriminate genotypes before and after plasmolysis. Although the sensitivity of T2 relaxometry allowed distinguishing microstructures among genotypes even after cellular fluids were mixed and diffused in plasmolyzed tissues, no relaxation component correlated with apple viscoelasticiy. Galactose and arabinose cell wall content were correlated with the storage modulus (E') prior and after plasmolysis though the correlation signs were opposite and pointed to a potential key role of pectin RGI side chains in regulating apple texture in turgid tissue.