Pectin Disassembly Research Articles

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 β-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 β-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.

Pectin as Carbon Source for Monilinia laxa Exoproteome and Expression Profiles of Related Genes.

Pectin, as part of the fruit cell wall, can be degraded by brown rot fungi by coordinating the production, secretion, and action of extracellular enzymes. In this study, pectin utilization by the necrotroph Monilinia laxa 8L was studied by in vitro and in silico approaches. A total of 403 genes encoding carbohydrate-active enzymes (CAZymes) were identified, including 38 coding a predicted pectin-degrading activity. Analyzing the differences between M. laxa 8L exoproteomes in media containing glucose and pectin as sole carbon sources, we identified 107 pectin-specific proteins, among them, 64.48% harbor a classical secretory activity, including 42 CAZymes and six pectin-degrading proteins. Analyzing the gene-expression patterns of some pectinase families revealed their possible sequential action in pectin disassembly. We found, in vitro, an early pectin-dependent induction of MlRGAE1, MlPG1, and three members of the rhamnosidase family (MlαRHA2, MlαRHA3, and MlαRHA6) and late response of MlPG2 and MlPNL3. M. laxa 8L has the ability to use both pectin and byproducts as carbon sources, based on a functional pectinolytic machinery encoded in its genome, subjected to pectin-dependent regulation and appropriate secretion mechanisms of these pectinolytic enzymes. Differences in the secretion and transcription profile of M. laxa 8L provided insights into the different mechanisms that contribute to brown rot development.

Genome-Wide Identification and Analysis of Polygalacturonase Genes in Solanum lycopersicum

Polygalacturonase (PG), a large hydrolase family in plants, is involved in pectin disassembly of the cell wall in plants. The present study aims to characterize PG genes and investigate their expression patterns in Solanum lycopersicum. We identified 54 PG genes in the tomato genome and compared their amino acid sequences with their Arabidopsis counterpart. Subsequently, we renamed these PG genes according to their Arabidopsis homologs. Phylogenetic and evolutionary analysis revealed that these tomato PG genes could be classified into seven clades, and within each clade the exon/intron structures were conserved. Expression profiles analysis through quantitive real-time polymerase chain reaction (qRT-PCR) revealed that most SlPGs had specific or high expression patterns in at least one organ, and particularly five PG genes (SlPG14, SlPG15, SlPG49, SlPG70, and SlPG71) associated with fruit development. Promoter analysis showed that more than three cis-elements associated with plant hormone response, environmental stress response or specific organ/tissue development exhibited in each SlPG promoter regions. In conclusion, our results may provide new insights for the further study of PG gene function during plant development.

Effects of heat treatment on enzyme activity and expression of key genes controlling cell wall remodeling in strawberry fruit

Modification of cell wall polymers composition and structure is one of the main factors contributing to textural changes during strawberry (Fragaria x ananassa, Duch.) fruit ripening and storage. The present study aimed to provide new data to understand the molecular basis underlying the postharvest preservation of strawberry cell wall structure by heat treatment. Ripe fruit (cv. Aroma) were heat-treated in air oven (3 h at 45 °C) and then stored 8 days at 4 °C + 2 days at 20 °C, while maintaining a set of non-treated fruit as controls. The effect of heat stress on the expression pattern of key genes controlling strawberry cell wall metabolism, as well as some enzymatic activities was investigated. The expression of genes proved to be relevant for pectin disassembly and fruit softening process (FaPG1, FaPLB, FaPLC, FaAra1, FaβGal4) were down-regulated by heat treatment, while the expression of genes being involved in the reinforcement of cell wall as pectin-methylesterase (FaPME1) and xyloglucan endo-transglycosilase (FaXTH1) was up-regulated. Total cell wall amount as well as cellulose, hemicellulose, neutral sugars and ionically and covalently bounded pectins were higher in heat-stressed fruit compared to controls, which might be related to higher firmness values. Interestingly, heat stress was able to arrest the in vitro cell wall swelling process during postharvest fruit ripening, suggesting a preservation of cell wall structure, which was in agreement with a lower growth rate of Botrytis cinerea on plates containing cell walls from heat-stressed fruit when compared to controls.

Dissecting the complex molecular evolution and expression of polygalacturonase gene family in Brassica rapa ssp. chinensis

Polygalacturonases (PGs) participate in pectin disassembly of cell wall and belong to one of the largest hydrolase families in plants. In this study, we identified 99 PG genes in Brassica rapa. Comprehensive analysis of phylogeny, gene structures, physico-chemical properties and coding sequence evolution demonstrated that plant PGs should be classified into seven divergent clades and each clade's members had specific sequence and structure characteristics, and/or were under specific selection pressures. Genomic distribution and retention rate analysis implied duplication events and biased retention contributed to PG family's expansion. Promoter divergence analysis using "shared motif method" revealed a significant correlation between regulatory and coding sequence evolution of PGs, and proved Clades A and E were of ancient origin. Quantitative real-time PCR analysis showed that expression patterns of PGs displayed group specificities in B. rapa. Particularly, nearly half of PG family members, especially those of Clades C, D and F, closely relates to reproductive development. Most duplicates showed similar expression profiles, suggesting dosage constraints accounted for preservation after duplication. Promoter-GUS assay further indicated PGs' extensive roles and possible redundancy during reproductive development. This work can provide a scientific classification of plant PGs, dissect the internal relationships between their evolution and expressions, and promote functional researches.

Difference in activity and gene expression of pectin-degrading enzymes during softening process in two cultivars of Chinese pear fruit

Cell wall pectin-related physiological and molecular properties changes in two different cultivars of Chinese pear fruit, ‘Jingbaili’ and ‘Yali’, were investigated in this study. ‘Jingbaili’ pear fruit, which softened quickly, showed higher pectin fracture solubilization, while ‘Yali’ pear fruit, which maintained a firm texture, presented little change in the content of pectin fractions during storage. The activities of PG, β-GAL and α-ARF that involved in fruit softening were significantly higher in ‘Jingbaili’ pear fruit compared to ‘Yali’ pear fruit. Furthermore, the mRNA accumulation of the genes, such as PG1, PG2, GAL4, ARF1 and ARF2 that encoded above-mentioned cell wall hydrolase dramatically increased in ‘Jingbaili’ pear fruit during fruit softening. Thus, we concluded that ‘Jingbaili’ pear fruit may be more prone to degradation in cell wall pectin compared to ‘Yali’ pear fruit, suggesting that their different softening patterns determine the different modes of cell wall pectin disassembly between these two different types of Chinese pear 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 84nm in the control to 95.5 and 101nm, in antisense FaplC and antisense FaPG1 samples, respectively. Similarly, SSP polyuronides were longer in transgenic fruits (61, 67.5 and 71nm, 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.

Pectin Nanostructure Visualization by Atomic Force Microscopy

Pectins, complex polysaccharides rich in galacturonic acid, are a major component of primary plant cell walls. These macromolecules regulate cell wall porosity and intercellular adhesion, being important in the control of cell expansion and differentiation through their effect on the rheological properties of the cell wall. In fruits, pectin disassembly during ripening is one the main event leading to textural changes and softening. Changes in pectic polymer size, composition and structure have been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides but studies of detailed structure of isolated polymer chains are scarce (Paniagua et al., 2014). Atomic force microscopy (AFM) is a versatile and powerful technique able to analyze force measurements, as well as to visualize roughness of biological samples at nanoscale (Morris et al., 2010). Using this technique, recent research has found a close relationship between pectin nanostructural complexity and texture and postharvest behavior in several fruits (Liu and Cheng, 2011; Cybulska et al., 2014; Posé et al., 2015). Here, we describe an AFM procedure to topographically visualize pectic polymers from fruit cell wall extracts that has successfully been used in the study of strawberry ripening (Posé et al., 2012; Posé et al., 2015). Thus, from AFM images the 3D structural analysis of isolated chains (length, height, and branch pattern) can be resolved at high magnification and with minimal sample preparation. A full description of AFM fundamentals and the different sampling modes are described in Morris et al. (2010).

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

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. 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.

Benzyl-aminopurine (BAP) treatments delay cell wall degradation and softening, improving quality maintenance of refrigerated summer squash

Round summer squash are harvested before reaching full maturity and even though they are highly perishable, fruit postharvest handling is mostly based on storage at non-chilling temperatures. Finding complementary treatments minimizing deterioration and reducing postharvest losses would be extremely useful. In this work we evaluated the effect of postharvest cytokinin (CK) treatments on refrigerated round soft rind squash. Fruit were harvested at commercial maturity and sprayed with 1mmolL−1 benzylaminopurine (BAP) or water (control) prior to storage at 5°C for 0, 13 or 25 days. Quality was assessed upon removal from cold storage as well as after a 2 day shelf-life period at 20°C. CK-treated fruit showed slower deterioration and dehydration and remained firmer than the control. BAP sprays did not affect color, respiration or sugar-acid balance. The treatments prevented phenolic compound accumulation, and decreased pectin solubilization. By the end of the storage period BAP-treated squash had higher levels (45%) of tightly-bound polyuronides than untreated controls, indicating a substantial delay in cell wall dismantling. CK sprays also reduced neutral sugar solubilization from pectin-rich fractions, but no changes were found in the cross-linking glycans or cellulose. To our knowledge, this is the first work showing that CK can regulate pectin disassembly in developing fruit. Postharvest BAP sprays preventing texture deterioration may be a simple treatment to complement refrigeration of round, soft rind, summer squash.

DIFFERING PATTERNS OF PECTIN DISASSEMBLY AND PERICARP SOFTENING IN RIPENING AND CHILL-INJURED TOMATO FRUIT WITH DOWNREGULATED POLYGALACTURONASE ACTIVITY

ABSTRACT Pectin disassembly was examined in transgenic tomato fruit with reduced polygalacturonase (PG). Mature-green tomato fruit were stored at 5 and 12C for 21 days and subsequently transferred to 18C for an additional 5 days. Pericarp softened during storage at 12C and the softening rate increased after fruit were transferred to 18C. Firmness remained unchanged during storage at 5C but decreased after transfer of chilled fruit to 18C. Water-soluble pectins increased five- and ninefold during ripening of PG-antisense and wild-type fruit, respectively, while trans-1,2-cyclohexanediaminetetraacetic acid-soluble pectins increased only slightly. Solubilization of pectins was very restricted in fruit stored at 5C and upon subsequent transfer to 18C. Pectins exhibited a PG-mediated reduction in molecular mass during ripening at 12C, but no depolymerization was observed in chill-injured fruit. Chill-injured tomato fruit softened even though pectin solubilization and molecular mass distribution remained unchanged, suggesting that the softening mechanism differs in ripening and chill-injured tomato fruit. PRACTICAL APPLICATIONS Chilling injury poses serious problems in the supply chain of sensitive commodities. Abnormal textural changes are one of the chilling-injury symptoms described in sensitive fruit, including tomato, but the mechanism for the accelerated softening rate and development of mealiness is not yet fully understood. Although ripening-related softening and pectin metabolism have been extensively characterized, the pectic metabolism in chill-injured fruit remains elusive. Transgenic plants with altered enzyme expression are excellent tools to test hypothesis regarding the physiological role of the enzymes. Because polygalacturonase (PG) expression is inhibited during low-temperature storage, it is possible that, at least in part, the differences in textural changes between chill-injured and normally ripening fruit are related to this enzyme. The data presented here indicate that PG plays an even smaller role in the softening of chilled fruit than in that of ripening fruit. Tomato can provide a model for the understanding of the relationships between cell wall metabolism and texture that applies to other chilling-sensitive fruit.

Regulation of tomato fruit polygalacturonase mRNA accumulation by ethylene: A Re-examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 L/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 L/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 L/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 L/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 L/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.

Effect of Antisense Suppression of Endopolygalacturonase Activity on Polyuronide Molecular Weight in Ripening Tomato Fruit and in Fruit Homogenates.

Fruit of tomato (Lycopersicon esculentum Mill.) in which endopolygalacturonase (PG) activity had been suppressed to <1% of wild-type levels were slightly firmer than nontransgenic controls later in ripening. Enzymically inactive cell walls were prepared from these ripening fruit using Tris-buffered phenol. When extracted with chelator followed by Na2CO3, the amounts of pectin solubilized from cell walls of nontransgenic control or from transgenic antisense PG fruit were similar. Size-exclusion chromatography analysis showed that, relative to controls, in antisense PG fruit polyuronide depolymerization was delayed in the chelator-soluble fraction throughout ripening and reduced in the Na2CO3-soluble fraction at the overripe stage. Reduced pectin depolymerization rather than altered extractability thus may have contributed to enhanced fruit firmness. Substantially larger effects of suppressed PG activity were detected in tomato fruit homogenates processed to paste. In control paste the majority of the polyuronide was readily soluble in water and was very highly depolymerized. In antisense PG paste the proportion of polyuronide solubilized by water was reduced, and polyuronides retained a high degree of polymerization. The suppression of fruit PG activity thus has a small effect on polyuronide depolymerization in the fruit but a much larger effect in paste derived from these fruit. This indicates that in the cell wall PG-mediated degradation of polyuronide is normally restricted but that in tissue homogenates or in isolated cell walls this restriction is removed and extensive pectin disassembly results unless PG is inactivated.