Fruit softening: evidence for rhamnogalacturonan lyase action in vivo in ripe fruit cell walls.

BACKGROUND AND AIMS: The softening of ripening fruit involves partial depolymerization of cell-wall pectin by three types of reaction: enzymic hydrolysis, enzymic elimination (lyase-catalysed) and non-enzymic oxidative scission. Two known lyase activities are pectate lyase and rhamnogalacturonan lyase (RGL), potentially causing mid-chain cleavage of homogalacturonan and rhamnogalacturonan-I (RG-I) domains of pectin respectively. However, the important biological question of whether RGL exhibits action in vivo had not been tested. METHODS: We developed a method for specifically and sensitively detecting in-vivo RGL products, based on Driselase digestion of cell walls and detection of a characteristic unsaturated 'fingerprint' product (tetrasaccharide) of RGL action. KEY RESULTS: In model experiments, potato RG-I that had been partially cleaved in vitro by commercial RGL was digested by Driselase, releasing an unsaturated tetrasaccharide ('ΔUA-Rha-GalA-Rha'), taken as diagnostic of RGL action. This highly acidic fingerprint compound was separated from monosaccharides (galacturonate, galactose, rhamnose, etc.) by electrophoresis at pH 2, then separated from ΔUA-GalA (the fingerprint of pectate lyase action) by thin-layer chromatography. The 'ΔUA-Rha-GalA-Rha' was confirmed as 4-deoxy-ß-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnosyl-(1→4)-d-galacturonosyl-(1→2)-l-rhamnose by mass spectrometry and acid hydrolysis. Driselase digestion of cell walls from diverse ripe fruits [date, sea buckthorn, cranberry, yew (arils), mango, plum, blackberry, apple, pear and strawberry] yielded the same fingerprint compound, demonstrating that RGL had been acting in vivo in these fruits prior to harvest. The 'fingerprint' : (galacturonate + rhamnose) ratio in digests from ripe dates was approximately 1 : 72 (mol/mol), indicating that ~1.4 % of the backbone Rha→GalA bonds in endogenous RG-I had been cleaved by in-vivo RGL action. CONCLUSIONS: The results provide the first demonstration that RGL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.

Layer by layer application of chitosan and carboxymethyl cellulose coatings delays ripening of mango fruit by suppressing cell wall polysaccharides disassembly.

Mango is a climacteric fruit that ripens quickly after harvest due to its climacteric nature. Edible coatings have been reported to delay the ripening of various harvested fruit. The efficacy of the applied edible coatings could be improved by using in combination as a layer-by-layer (LBL) approach. So, the influence of LBL application of chitosan (CH) and carboxymethyl cellulose (CMC) was studied on mangoes during postharvest storage at 15 °C for 20 days. Mangoes were coated with monolayers of CH (1 % w/v) and CMC (1 % w/v) as well as LBL application of CH and CMC and were compared with control. The treatment of mangoes with CH and CMC-based LBL treatment resulted in lower decay percentage and weight loss along with higher total chlorophyll pigments and suppressed total carotenoid accumulation. The LBL application of CH and CMC showed lower activity of chlorophyll degrading such as chlorophyllase (CPS), pheophytinase (Phe), Mg-dechalatase (MGD) and chlorophyll degrading peroxidase (Chl-POD) enzymes as well as reduced changes in b*, a* and L* along with a suppressed increase in ethylene (EP) and CO2 production (CPR) rates having higher antioxidant such as catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD) and superoxide dismutase (SOD) enzymes activity. In addition, mangoes coated with LBL treatment of CH and CMC exhibited lower water-soluble pectin (WSP) and higher protopectin (PP) having higher concentrations of chelate soluble (CSP) and sodium carbonate-soluble pectin (SCP). Similarly, LBL-coated mangoes showed significantly higher hemicellulose (HCLS) and cellulose (CLS) contents in contrast with control. It was observed that mangoes coated with CH and CMC-based LBL coating exhibited higher flesh firmness and showed suppressed cellulase (CS), pectin methylesterase (PME), polygalacturonase (PG) and ß-galactosidase (ß-Gal) enzymes activity. The concentrations of total soluble solids and ripening index were markedly lower and titratable acidity was higher in the LBL-based coating treatment in comparison with control. In conclusion, LBL treatment based on CH and CMC coatings could be used for the delay of ripening and softening of harvested mangoes.

Stool-softening effect and action mechanism of free anthraquinones extracted from Rheum palmatum L. on water deficit-induced constipation in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese herbal medicine, rhubarb is said to remove accumulation with purgation, clearing heat, and discharging fire. Modern pharmacology has shown that rhubarb extract has a purgative effect when given to experimental animals in an appropriate dose. However, the active components and their mechanism of action are still not clearly defined. AIM OF THE STUDY: The current research aimed to evaluate the synergistic stool-softening effects and explore the action mechanism of rhubarb free anthraquinones (RhA) and their monomers on constipation in rats. MATERIALS AND METHODS: A rat model of water deficit-induced constipation was established to induce constipation, and these rats were treated with RhA and its monomers. ELISA, histopathology, immunohistochemistry, qPCR and Western blotting based on network pharmacology and molecular docking were conducted to explore the possible mechanism of action of RhA and its monomers. RESULTS: RhA, aloe-emodin, rhein, and chrysophanol showed stool-softening activity, and the combination of aloe-emodin and rhein had the strongest softening effect on faecal pellets. Aloe-emodin, rhein, and chrysophanol significantly increased the serum levels of vasoactive intestinal peptide (VIP), motilin (MTL), and substance P (SP), upregulated the expression of VIP, cyclase-associated protein 1 (CAP1), protein kinase A (PKA), cystic fibrosis transmembrane conductance regulator (CFTR), aquaporin 3 (AQP3), aquaporin 4 (AQP4), and aquaporin 8 (AQP8), decreased the expression of epithelial sodium channel (ENaC) and Na+/H+ exchanger 3 (NHE3), and reduced the colonic tissue concentration of Na+-K+-ATPase in the constipated rats. Osmolality of colonic fluid in model rats treated by RhA, aloe-emodin, rhein, and chrysophanol was increased. CONCLUSION: Aloe-emodin, rhein, and chrysophanol were the stool-softening components of the RhA extract, and there were certain drug-interactions between the components. RhA upregulated VIP expression, activated the cyclic adenosine monophosphate protein kinase A (cAMP/PKA) pathway, and further stimulated CFTR expression while inhibiting NHE3 and ENaC expression, resulting in a hypertonic state in the colonic lumen. Water transport could then be driven by an osmotic gradient, which in turn led to the upregulation of AQP3, AQP4, and AQP8 expression. In addition, RhA likely improved gastrointestinal motility by increasing serum VIP, SP, and MTL concentrations, thus promoting faecal excretion.

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.

Transcriptome Profiles Reveal the Promoting Effects of Exogenous Melatonin on Fruit Softening of Chinese Plum.

In this study, we investigated the effect of exogenous melatonin (MT) on cell wall metabolism leading to Chinese plum (Prunus salicina Lindl.) fruit softening. Exogenous MT treatment increased the endogenous MT content in plum fruits before fruit ripening. However, in mature plum fruits, exogenous MT treatment decreased the fruit hardness, pulp hardness, fruit elasticity, contents of ion-bound pectin, covalently-bound pectin, hemicellulose, and cellulose, and activities of xyloglucan endotransglycosylase/hydrolase and endo-ß-1,4-glucanase, and increased the water-soluble pectin content, and activities of pectin methyl esterase, pectin lyase, polygalacturonase, ß-galactopyranosidase, and α-L-arabinofuranosidase. Transcriptome analysis revealed that the differentially expressed genes (DEGs) associated with cell wall metabolism in the exogenous MT-treated plum fruits were mainly enriched in the pentose and glucuronate interconversions, phenylpropanoid biosynthesis, cyanoamino acid metabolism, and galactose metabolism pathways. Analysis of these DEGs revealed that exogenous MT treatment affected the expression of genes regulating the cell wall metabolism. Overall, exogenous MT treatment promotes the fruit softening of Chinese plum.

Low temperature inhibits pectin degradation by PpCBFs to prolong peach storage time.

Low-temperature storage is a widely used method for peach fruit storage. However, the impact of PpCBFs on pectin degradation during low-temperature storage is unclear. As such, in this study, we stored the melting-flesh peach cultivar "Fuli" at low temperature (LT, 6°C) and room temperature (RT, 25°C) to determine the effect of different temperatures on its physiological and biochemical changes. Low-temperature storage can inhibit the softening of "Fuli" peaches by maintaining the stability of the cell wall. It was found that the contents of water-soluble pectin and ionic-soluble pectin in peach fruit stored at RT were higher than those stored at LT. The enzyme activities of polygalacturonase (PG), pectate lyase (PL), and pectin methylesterase (PME) were all inhibited by LT. The expressions of PpPME3, PpPL2, and PpPG were closely related to fruit firmness, but PpCBF2 and PpCBF3 showed higher expression levels at LT than RT. The promoters of PpPL2 and PpPG contain the DER motif, which suggested that PpCBF2 and PpCBF3 might negatively regulate their expression by directly binding to their promoters. These results indicated that LT may maintain firmness by activating PpCBFs to repress pectin-degradation-related enzyme genes during storage.

Inhibition of cell wall pectin metabolism by plasma activated water (PAW) to maintain firmness and quality of postharvest blueberry.

Blueberry is a class of berries with high nutritional and economic value but has short shelf life due to its rapid softening at room temperature. This study investigated the effects of plasma-activated water (PAW) treatment on the softening quality and cell wall pectin metabolism of blueberries stored for 10 d at 25 °C after being immersed in PAW for 10 min. PAW was generated by plasma with different times (1 and 2 min), fixed frequency (10 kHz) and fixed voltage (50 kV). The analysis showed that the firmness of PAW-treated fruit significantly increased (P < 0.05) by 36.4% after 10 d storage. PAW treatment controlled the solubilization of pectin from water-insoluble to water-soluble. The activities of cell wall pectin-degrading enzymes like polygalacturonase (PG), ß-galactosidase (ß-Gal) and pectin methylesterase (PME) in PAW-treated blueberries decreased by 15.7%, 18.3%, and 27.9%, respectively, on day 10. After PAW treatment, blueberries also maintained better postharvest quality (firmness, colour, soluble solid content and anthocyanin content) and intact epidermal waxy and cell wall structure. These results suggested that PAW showed great potential for postharvest fresh-keeping of blueberry.

VvPL15 Is the Core Member of the Pectate Lyase Gene Family Involved in Grape Berries Ripening and Softening.

The process of ripening and softening in grape begins at veraison and is closely related to the depolymerization of pectin components. A variety of enzymes are involved in pectin metabolism and one class of enzyme, pectin lyases (PLs), have been reported to play an important role in softening in many fruits; however, little information is available on the VvPL gene family in grape. In this study, 16 VvPL genes were identified in the grape genome using bioinformatics methods. Among them, VvPL5, VvPL9, and VvPL15 had the highest expression levels during grape ripening, which suggests that these genes are involved in grape ripening and softening. Furthermore, overexpression of VvPL15 affects the contents of water-soluble pectin (WSP) and acid-soluble pectin (ASP) in the leaves of Arabidopsis and significantly changes the growth of Arabidopsis plants. The relationship between VvPL15 and pectin content was further determined by antisense expression of VvPL15. In addition, we also studied the effect of VvPL15 on fruit in transgenic tomato plants, which showed that VvPL15 accelerated fruit ripening and softening. Our results indicate that VvPL15 plays an important role in grape berry softening during ripening by depolymerizing pectin.

Transcriptome analysis reveals key metabolic pathways and gene expression involving in cell wall polysaccharides-disassembling and postharvest fruit softening in custard apple (Annona squamosa L.).

This study aimed to investigate the effect of custard apple cell wall polysaccharides-disassembling on postharvest fruit softening and to explore its key metabolic pathways and gene expression. Custard apple fruit was stored at 15 ± 0.5 °C for 12 days, it was found that the decreased significantly in fruit firmness, contents of Na2CO3-soluble pectin, hemicellulose and cellulose, and the increased significantly in water-soluble pectin and CDTA-soluble pectin. The activities of cell wall-degrading relevant enzymes in fruit were improved significantly during storage, including cellulase, polygalacturonase, pectin methyl esterase, neutral xylanase, ß-galactosidase, and ß-D-glucosidase. The RNA sequencing results revealed 41,545 nonredundant unigenes and 7571 differentially expressed genes (DEGs) in custard apple fruit samples. Functional annotation and DEGs data revealed cell wall degradation potentially involved in starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, galactose metabolism, pentose and glucuronate interconversions. Specifically, two EG and six ß-Glc genes controlled the cellulose decomposition, and one ß-xyl and one GATU genes involved in the degradation of hemicellulose, and two PME, one Pel, and four PG genes were the major regulators of pectin disassembling. These results provide a molecular foundation for explaining fruit softening and extending shelf life of custard apple.

Molecular dynamics simulation of the interaction of a raspberry polygalacturonase (RiPG) with a PG inhibiting protein (RiPGIP) isolated from ripening raspberry (Rubus idaeus cv. Heritage) fruit as a model to understand proteins interaction during fruit softening.

Polygalacturonase (PG) is an important hydrolytic enzyme involved in pectin disassembly and the subsequent textural changes during fruit ripening. Although the interaction of fungal PGs with other proteins has been documented, the interaction of plant PGs with other plant proteins has not yet been studied. In this study, the molecular mechanisms involved in raspberry fruit ripening, particularly the polygalacturonase (RiPG) interaction with polygalacturonase inhibiting protein (RiPGIP) and substrate, were investigated with a structural approach. The 3D model of RiPG2 and RiPGIP3 was built using a comparative modeling strategy and validated using molecular dynamics (MD) simulations. The RiPG2 model structure comprises 11 complete coils of right-handed parallel ß-helix architecture, with an average of 27 amino acid residues per turn. The structural model of the RiPGIP3 displays a typical structure of LRR protein, with the right-handed superhelical fold with an extended parallel ß-sheet. The conformational interaction between the RiPG2 protein and RiPGIP3 showed that RiPGIP3 could bind to the enzyme and thereby leave the active site cleft accessible to the substrate. All this evidence indicates that RiPG2 enzyme could interact with RiPGIP3 protein. It can be a helpful model for evaluating protein-protein interaction as a potential regulator mechanism of hydrolase activity during pectin disassembly in fruit ripening.

Bean softening during hydrothermal processing is greatly limited by pectin solubilization rather than protein denaturation or starch gelatinization.

The rate liming step of bean softening during cooking was evaluated. Red kidney beans (fresh/non-aged and aged) were cooked at different temperatures (70-95 °C) and their texture evolution established. Softening of beans (loss of hard texture) with cooking and increasing cooking temperature was evident at ≥ 80 °C more so for non-aged than aged beans, evidencing hard-to-cook development during storage. Beans at each cooking time and temperature were subsequently classified into narrow texture ranges and bean cotyledons in the most frequent texture class evaluated for the extent of starch gelatinization, protein denaturation and pectin solubilization. During cooking, starch gelatinization was shown to precede pectin solubilization and protein denaturation, with these reactions progressing faster and to a greater extent with increasing cooking temperature. At 95 °C for instance (practical bean processing temperature), complete starch gelatinization and protein denaturation is attained earlier (∼10 and 60 min cooking, respectively and at comparable time moments for both non-aged and aged beans) than plateau bean texture (∼120 and 270 min for non-aged and aged beans)/plateau pectin solubilization. The extent of pectin solubilization in the cotyledons was consequently most correlated (negatively, r = 0.95) with and plays the most significant role (P < 0.0001) in directing the relative texture of beans during cooking. Ageing was shown to significantly retard bean softening. Protein denaturation plays a less significant role (P = 0.007) while the contribution of starch gelatinization is insignificant (P = 0.181). Thermo-solubilization of pectin in bean cotyledons is therefore the rate limiting step of bean softening towards attaining a palatable texture during cooking.

Involvement of branched RG-I pectin with hemicellulose in cell-cell adhesion of tomato during fruit softening.

Cell adhesion of four cultivars of tomato fruit, "Micro Tom (MT)", "Heinz 1706 (H1706)", "Money Maker (MM)", "Ailsa Craig (AC)" were evaluated and cell walls were analyzed in order to assess the possible contribution of pectic and hemicellulosic polysaccharides to the softening and altered cell adhesion at two different stages of ripeness. Cell wall material (CWM) and solubilised fractions of green and red ripe fruit were analyzed by chemical, enzymatic techniques. In comparison with the four cultivars of tomato fruits, H1706 and MM are harder than MT and AC at both green and red ripe stage. The ripening-associated solubilisation of rhamnogalacturonan-riched pectic polysaccharides was reduced in H1706 and MM, and the content of side -chain sugars from RG-I reduced by more than 50% in MT and AC. In addition to recognized pectic modifying enzymes, RGase had a good effect on cell separation of H1706 and MM fruit at red ripe stage. The higher RG-I content and branching degree have been associated with increased cell adhesion and reduced cell wall porosity, thus maintained fruit firmness.

1-Methylcyclopropene treatment delays the softening of Actinidia arguta fruit by reducing cell wall degradation and modulating carbohydrate metabolism.

The rapid softening of hardy kiwifruit (Actinidia arguta) fruit significantly reduces its marketing potential. Therefore, the effect of 1-methylcyclopropene (1-MCP) on the softening of A. arguta fruit was investigated. Results indicated that A. arguta fruit treated with 1-MCP maintained a higher level of firmness, titratable acidity, ascorbic acid, total phenolics, and flavonoids content, relative to non-treated fruit. Fruit treated with 1-MCP and placed in long-term cold storage had higher sensory scores, as determined by a taste panel and supported by electronic nose and tongue data. Notably, 1-MCP delayed the degradation of cell wall components, including pectin, cellulose, and hemicellulose, by reducing the activity of cell-wall-modifying enzymes. In addition, 1-MCP reduced the activity of carbohydrate metabolism-related enzymes, resulting in fruit with higher levels of starch and sucrose and lower levels of glucose, fructose and sorbitol. Collectively, these results indicate that 1-MCP can be used to delay the softening of A. arguta fruit and extend its storage and shelf life.

Hydrogen-rich water delays fruit softening and prolongs shelf life of postharvest okras.

The effect of hydrogen-rich water (HRW) treatment on softening, cell wall components and cell wall metabolic genes in okras after harvest was studied. The results showed that HRW treatment could maintain fruit firmness and delay softening, thereby prolonging shelf life in okras during storage. The treated okras displayed significantly lower levels water- and chelate-soluble pectins while higher contents of Na2CO3-soluble pectin, hemicellulose and cellulose. The cell wall biosynthesis was maintained by HRW treatment via up-regulating genes involved in biosynthesis of pectin, hemicellulose and cellulose at the beginning of storage. On the contrary, the treatment could inhibit the cell wall disassembly due to the down-regulation of numerous cell wall degradative genes including AePME, AeGAL and AeCX at the end of storage. Taken together, our results suggested that HRW treatment delayed softening and extended shelf life in postharvest okras through modifying cell wall biosynthesis and disassembly at different times of storage.

Tragacanth gum coating suppresses the disassembly of cell wall polysaccharides and delays softening of harvested mango (Mangifera indica L.) fruit.

Mango is a climacteric fruit and is prone to high perishability. The rapid softening and ripening (due to degradation and disassembly of cell wall polysaccharides) are the major limitations in extending the storability of the harvested mango fruits. Various types of gum-based edible coatings have been reported for the shelf life extension of mango fruits. Tragacanth gum (TCG) also has appropriate coating properties. Its use as an edible coating has been reported on certain fruits. However, the effect of TCG coating in the regulation of harvested mango fruits ripening and softening has not been reported yet. So, the objective of this work was to investigate the effect of TCG (control, 0.5 %, 1 % and 1.5 %) coating on postharvest softening and ripening of harvested mango fruits. TCG coating affected the ripening and softening of mango in a dose-dependent manner. Results exhibited that mango fruits coated with 1.5 % TCG showed substantially lower disease incidence and weight loss. The 1.5 % TCG-coated mangoes showed substantially lower ethylene biosynthesis and respiration rate peaks as well as superoxide anion and hydrogen peroxide contents compared with the control. In the same way, 1.5 % TCG-coated mango fruits had markedly higher total chlorophyll content and lower L*, b* and a* along with substantially lower total carotenoids in peel tissues. Mango fruits coated with 1.5 % TCG exhibited markedly lower water-soluble pectin and higher chelate-soluble pectin, Na2CO3-soluble pectin, protopectin, cellulose and hemicellulose in flesh tissues compared with control. The activity of polygalacturonase (PG), cellulase (CX), pectin methylesterase (PME), ß-galactosidase (ß-Gal) and ß-glucosidase (ß-Glu) were significantly lower in flesh of 1.5 % TCG treated fruits along with substantially higher firmness in contrast with control. In addition, 1.5 % TCG coating treatment showed significantly higher activity of antioxidative enzymes and delayed the increase in soluble solids content (SSC) and ripening index (RI) along with considerably higher titratable acidity (TA) compared with the untreated control. So, pre-storage TCG based edible coating (1.5 %) could be applied to delay ripening and softening in mango fruit industry under postharvest ambient conditions.

Aloe vera gel coating delays softening and maintains quality of stored persimmon (Diospyros kaki Thunb.) Fruits.

The effect of Aloe vera (AV) gel coating was studied on antioxidant enzymes activities, oxidative stress, softening and associated quality attributes of persimmon fruits. The fruits were coated with 0 and 50% AV-gel coating and stored for 20 days at 20 ± 1 ºC. AV-gel coated fruits exhibited considerably less weight loss, hydrogen peroxide level, electrolyte leakage and malondialdehyde content. AV-gel coated fruits had significantly higher ascorbate peroxidase, peroxidase, superoxide dismutase and catalase activities. In addition, AV-gel coating suppressed pectin methylesterase, polygalacturonase and cellulase activities and showed higher ascorbic acid, DPPH scavenging antioxidants and phenolics, and lower sugars and carotenoids. To the best of our knowledge, these results are the first evidence that AV-gel coating modulates the activities of cell wall degrading enzymes to delay ripening in climacteric fruits. So, AV-gel coating prohibited the onset of senescence by activating enzymatic antioxidant system, accumulating bioactive compounds and suppressing cell wall degradation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05412-5.

Cell-wall microdomain remodeling controls crucial developmental processes.

Plant cell walls display cellular and subcellular specificities. At the subcellular level, wall regional territories with specific compositions are necessary for macroscopic developmental processes. These regional specificities were named differently throughout the years, and are unified here under the term 'cell-wall microdomains' that define the local composition and organization of wall polymers underlying territories of wall loosening and/or softening or stiffening. We review the occurrence and developmental role of wall microdomains in different cell types. We primarily focus on the contribution of two categories of wall-remodeling molecular actors: fine-tuning of homogalacturonan (HG; pectin) demethylesterification patterns and two classes of oxidoreductases [class III peroxidases (CIII PRXs) and laccases (LACs)], but we also highlight two different molecular scaffolds recently identified for positioning specific CIII PRXs.

Putrescine Treatment Delayed the Softening of Postharvest Blueberry Fruit by Inhibiting the Expression of Cell Wall Metabolism Key Gene VcPG1.

The postharvest shelf life of blueberries is very short at room temperature owing to softening, which reduces their edible value. Putrescine (Put) plays an important role in maintaining the firmness and prolonging the storage time of fruits. Therefore, we investigated the relationship between Put and the cell wall metabolism and their roles in the postharvest softening of blueberry. Harvested blueberry fruit was immersed in 1 mM Put aqueous solution for 10 min. After treatment, the blueberries were stored at 20 ± 0.5 °C and 80% relative humidity for 10 days. The results show that Put delayed the softening of the blueberries. Compared to the control, the blueberry fruit treated with Put showed higher levels of firmness and protopectin. Moreover, the activity and expression levels of the cell wall metabolism enzymes were markedly inhibited by the Put treatment, including polygalacturonase (PG), ß-galactosylase (ß-Gal), and ß-glucosidase (ß-Glu). The Put treatment promoted the expression of the Put synthesis gene VcODC and inhibited the expression of the Put metabolism gene VcSPDS. Further tests showed that the fruit firmness decreased significantly after the overexpression of VcPG1, which verified that VcPG1 is a key gene for fruit softening. The key transcription factors of fruit softening were preliminarily predicted and the expressions were analyzed, laying a foundation for the subsequent study of transcriptional regulation. These results indicate that Put delays the softening of postharvest blueberry by restraining the cell wall metabolism and maintaining the fruit firmness.

Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls.

BACKGROUND AND AIMS: The programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by ß-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested. METHODS: We developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action. KEY RESULTS: In model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide ('ΔUA-GalA'), taken as diagnostic of PL action. ΔUA-GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA-GalA from higher homologues. The ΔUA-GalA was confirmed as 4-deoxy-ß-l-threo-hex-4-enopyranuronosyl-(1→4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA-GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA-GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA-GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol-1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action. CONCLUSIONS: The results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.

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.