Supplementation of citrus pectin with whole-cell pectinase PG5 on Pichia pastoris promotes recovery of colitis and enhances intestinal barrier function in DSS-treated mice.

A whole-cell biocatalyst was developed by genetically engineering pectinase PG5 onto the cell surface of Pichia pastoris using Gcw12 as the anchoring protein. Whole-cell PG5 eliminated the need for enzyme extraction and purification, while also exhibiting enhanced thermal stability, pH stability, and resistance to proteases in vitro compared to free PG5. Magnetic resonance mass spectrometry analysis revealed that whole-cell PG5 efficiently degraded citrus pectin, resulting in the production of a mixture of pectin oligosaccharides. The primary components of the mixture were trigalacturonic acid, followed by digalacturonic acid and tetragalacturonic acid. Supplementation of citrus pectin with whole-cell PG5 resulted in a more pronounced protective effect compared to free PG5 in alleviating colitis symptoms and promoting the integrity of the colonic epithelial barrier in a mouse model of dextran sulfate sodium-induced colitis. Hence, this study demonstrates the potential of utilizing whole-cell pectinase as an effective biocatalyst to promote intestinal homeostasis in vivo.

Space exposure enhanced pectin-degrading enzymes expression and activity in Aspergillus costaricaensis.

Aspergillus is a well-studied fungal genus that is widely used in the processing of plant biomass in industries. This study investigated the effects of space exposure on the ability of Aspergillus costaricaensis, a filamentous fungus isolated from rotten orange peel, to degrade pectin. These fungal spores were carried into space by the Long March 5B carrier rocket and exposed to cosmic radiation for 79 h. After the flight, these spores were resuscitated, and then the growing strains were screened with pectin as the sole carbon source, and the pectinase activity was evaluated. A mutant with increased biomass accumulation ability and pectin-degrading activity compared to the ground control strain was obtained. Comparative transcriptome analysis revealed that several CAZymes genes were significantly upregulated in the mutant, especially those related to pectin degradation. Among the 44 pectinases identified from the annotated genome, 42 were up-regulated. The activities of these pectinases are able to synergistically break down the structure of pectin. In addition, the expression of some genes involved in metabolism, sugar transport, and stress response was altered. These results imply that space exposure might serve as a potential mutagenesis breeding technique, offering the opportunity to acquire biomass-degrading microbial strains with potential for industrial application.

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.

Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins.

Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-inhibiting proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics, and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development.

Metabolic novelty originating from horizontal gene transfer is essential for leaf beetle survival.

Horizontal gene transfer (HGT) provides an evolutionary shortcut for recipient organisms to gain novel functions. Although reports of HGT in higher eukaryotes are rapidly accumulating, in most cases the evolutionary trajectory, metabolic integration, and ecological relevance of acquired genes remain unclear. Plant cell wall degradation by HGT-derived enzymes is widespread in herbivorous insect lineages. Pectin is an abundant polysaccharide in the walls of growing parts of plants. We investigated the significance of horizontally acquired pectin-digesting polygalacturonases (PGs) of the leaf beetle Phaedon cochleariae. Using a CRISPR/Cas9-guided gene knockout approach, we generated a triple knockout and a quadruple PG-null mutant in order to investigate the enzymatic, biological, and ecological effects. We found that pectin-digestion 1) is exclusively linked to the horizontally acquired PGs from fungi, 2) became fixed in the host genome by gene duplication leading to functional redundancy, 3) compensates for nutrient-poor diet by making the nutritious cell contents more accessible, and 4) facilitates the beetles development and survival. Our analysis highlights the selective advantage PGs provide to herbivorous insects and demonstrate the impact of HGT on the evolutionary success of leaf-feeding beetles, major contributors to species diversity.

Synergistic action of thermophilic pectinases for pectin bioconversion into D-galacturonic acid.

Large amounts of pectin-rich biomass are generated worldwide yearly, which can be hydrolysed by pectinases to obtain bio-based chemical building blocks such as D-galacturonic acid (GalA). The aim of this work was to investigate thermophilic pectinases and explore their synergistic application in the bioconversion of pectic substrates into GalA. Two exo-polygalacturonases (exo-PGs) from Thermotoga maritima (TMA01) and Bacillus licheniformis (BLI04) and two pectin methylesterases (PMEs) from Bacillus licheniformis (BLI09) and Streptomyces ambofaciens (SAM10) were cloned and expressed in Escherichia coli BL21 (DE3), purified and fully characterised. These pectinases exhibited optimum activity at temperatures above 50 °C and good stability at high temperature (40-90 °C) for up to 24 h. Exo-PGs preferred non-methylated substrates, suggesting that previous pectin demethylation by PMEs was necessary to achieve an efficient pectin monomerisation into GalA. Synergistic activity between PMEs and exo-PGs was tested using pectin from apple, citrus and sugar beet. GalA was obtained from apple and citrus pectin in a concentration of up to 2.5 mM after 4 h reaction at 50 °C, through the combined action of BLI09 PME with either TMA01 or BLI04 exo-PGs. Overall, this work contributes to expand the knowledge of pectinases from thermophiles and provides further insights into their application in the initial valorisation of sustainable pectin-rich biomass feedstocks.

Botrytis cinerea mediated cell wall degradation accelerates spike stalk browning in Munage grape.

Munage grape (Vitis vinifera L. cv. Munage.) is a unique cultivar in southern Xinjiang, China. Spike stalk browning in this species has becomes more common in recent years, negatively impacting the shelf life, and causing severe economic losses during storage. This study investigated the changes in metabolisms of cell wall by Botrytis cinerea infection in association with spike stalk browning. Morphological and physiological observations showed that preharvest B. cinerea infection accelerates the spike stalk browning during storage in Munage grapes by promoting cell wall degradation. Accordingly, the cell structures in infected spike stalk showed severe collapse, while the cell structures in uninfected spike stalk remained relatively complete. Furthermore, the contents of CDTA-soluble pectin (CSP), Na2 CO3 -soluble pectin (NSP), cellulose, and hemicellulose were reduced, while the water-soluble pectin (WSP) content was increased during infection. In addition, the activities of polygalacturonase (PG), pectin methylesterase (PME), beta-galactosidase (ß-Gal), and cellulase (Cx) were highly promoted by B. cinerea. Correspondingly, the expression levels of VvPG were markedly upregulated after inoculation and played a major role in cell wall degradation. Additionally, the spike stalk inoculated by B. cinerea showed higher activities of PPO and POD, and content of total phenolics. These results contribute to elucidating the relationship between cell wall degradation induced by B. cinerea during spike stalk browning and provide a basis for future research on improving the ability of the host cell wall to resist degrading enzymes. PRACTICAL APPLICATIONS: Botrytis cinerea is the main fungal pathogen causing the gray mold of grapes. It usually enters the tissue early in crop development, has a long incubation period, and rapidly infects the tissue when the environment is favorable and the host physiology changes. Gray mold has been reported as one of the major postharvest diseases of grapes. However, there are relatively few reports on the pathways through which B. cinerea causes the browning of grape stalks. Controlling browning caused by B. cinerea may require clarification of the physiological and molecular mechanisms by which browning occurs. The elucidation of the role of B. cinerea in causing browning of grape stalks through the cell wall degradation pathway will help to provide scientific basis for further controlling browning, maintaining freshness of stalks, developing biological agents to prevent browning, improving grape quality, and extending storage period.

The gene TaPG encoding a polygalacturonase is critical for pollen development and male fertility in thermo-sensitive cytoplasmic male-sterility wheat.

Thermo-sensitive cytoplasmic male sterility is of great significance to heterosis and hybrid seed production in wheat. Consequently, it is worthwhile to research the genes associated with male sterility. Although polygalacturonases (PGs) have been studied to play a crucial role in male reproduction of many plants, their functions in the reproductive development of wheat remain unclear. Here, TaPG (TraesCS7A02G404900) encoding a polygalacturonase was isolated from the anthers of KTM3315A, a wheat thermo-sensitive cytoplasmic male sterile with Aegilops kotschyi cytoplasm. Expression pattern analyses showed that TaPG was strongly expressed in fertile anthers and its protein was localized in the cell wall. Further verification via barley stripe mosaic virus revealed that the silencing of TaPG exhibited abnormal anthers, premature degradation of tapetum, pollen abortion, and defective pollen wall formation, resulting in the declination of fertility. Conclusively, our research suggested that TaPG contributed to the pollen development and male fertility, which will provide a novel insight into the fertility conversion of thermo-sensitive cytoplasmic male sterility in wheat.

Degradation of de-esterified pctin/homogalacturonan by the polygalacturonase GhNSP is necessary for pollen exine formation and male fertility in cotton.

The pollen wall exine provides a protective layer for the male gametophyte and is largely composed of sporopollenin, which comprises fatty acid derivatives and phenolics. However, the biochemical nature of the external exine is poorly understood. Here, we show that the male sterile line 1355A of cotton mutated in NO SPINE POLLEN (GhNSP) leads to defective exine formation. The GhNSP locus was identified through map-based cloning and confirmed by genetic analysis (co-segregation test and allele prediction using the CRISPR/Cas9 system). In situ hybridization showed that GhNSP is highly expressed in tapetum. GhNSP encodes a polygalacturonase protein homologous to AtQRT3, which suggests a function for polygalacturonase in pollen exine formation. These results indicate that GhNSP is functionally different from AtQRT3, the latter has the function of microspore separation. Biochemical analysis showed that the percentage of de-esterified pectin was significantly increased in the 1355A anthers at developmental stage 8. Furthermore, immunofluorescence studies using antibodies to the de-esterified and esterified homogalacturonan (JIM5 and JIM7) showed that the Ghnsp mutant exhibits abundant of de-esterified homogalacturonan in the tapetum and exine, coupled with defective exine formation. The characterization of GhNSP provides new understanding of the role of polygalacturonase and de-esterified homogalacturonan in pollen exine formation.

A novel method of affinity purification and characterization of polygalacturonase of Aspergillus flavus by galacturonic acid engineered magnetic nanoparticle.

Pectinases hydrolyze pectin and make up 25% of global food processing enzyme sales. In this study, we aimed to purify exo-polygalacturonase (Exo-PG) by using galacturonic acid conjugated magnetic nanoparticles (MNPs) and examined its application in juice purification. The submerged fermentation was carried out in the presence of apple pectin (1%) to promote production of exo-PG from Aspergillus flavus. Maximum exo-PG activity was observed after 4 days (30 °C and pH 5.0). A single protein band (66 kDa) of purified exo-PG was observed in SDS-PAGE. Purification of exo-PG enzyme was âˆ¼ 10 fold with a yield of 29%. The enzyme retained 98% activity in the presence of 15 % glycerol at 4 °C. The purified exo-PG using MNPs yielded a 10-12% increase in juice production as compare to without treated fruit juice. To the best of our knowledge, this is the first report of affinity purification of exo-PG enzyme, using engineered magnetic nanoparticles.

Production and molecular characterization of tailored citrus pectin-derived compounds.

In this study, tailored-made citrus pectin-derived compounds were produced through controlled enzymatic and/or chemical modifications of commercial citrus pectin with different degrees of methylesterification (DM) and similar average molecular weight (MW). In the first treatment, degradation of the citrus pectin (CP) materials by endo-polygalacturonase (EPG) yielded pectins with average Mw's (between 2 and 60 kDa). Separation and identification of the oligosaccharide fraction present in these samples, revealed the presence of non-methylesterified galacturonic acid oligomers with degree of polymerization (DP) 1-5. In the second treatment, exploiting the combined effect of EPG and pectin lyase, compounds with MW between 2 and 21 kDa, containing methylesterified and non-methylesterified polygalacturonans (DP 1-6), were generated. Finally, CP was sequentially modified by chemical saponification and the action of EPG. A sample of DM 11% and MW 2.7 kDa, containing POS (DP 1-5), was produced. Diverse pectin-derived compounds were successfully generated for further studies exploring their functionality.

Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain.

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

Machine learning prediction of novel pectinolytic enzymes in Aspergillus niger through integrating heterogeneous (post-) genomics data.

Pectinolytic enzymes are a variety of enzymes involved in breaking down pectin, a complex and abundant plant cell-wall polysaccharide. In nature, pectinolytic enzymes play an essential role in allowing bacteria and fungi to depolymerize and utilize pectin. In addition, pectinases have been widely applied in various industries, such as the food, wine, textile, paper and pulp industries. Due to their important biological function and increasing industrial potential, discovery of novel pectinolytic enzymes has received global interest. However, traditional enzyme characterization relies heavily on biochemical experiments, which are time consuming, laborious and expensive. To accelerate identification of novel pectinolytic enzymes, an automatic approach is needed. We developed a machine learning (ML) approach for predicting pectinases in the industrial workhorse fungus, Aspergillus niger. The prediction integrated a diverse range of features, including evolutionary profile, gene expression, transcriptional regulation and biochemical characteristics. Results on both the training and the independent testing dataset showed that our method achieved over 90 % accuracy, and recalled over 60 % of pectinolytic genes. Application of the ML model on the A. niger genome led to the identification of 83 pectinases, covering both previously described pectinases and novel pectinases that do not belong to any known pectinolytic enzyme family. Our study demonstrated the tremendous potential of ML in discovery of new industrial enzymes through integrating heterogeneous (post-) genomimcs data.

Structural Alteration of Rice Pectin Affects Cell Wall Mechanical Strength and Pathogenicity of the Rice Blast Fungus Under Weak Light Conditions.

Pectin, a component of the plant cell wall, is involved in cell adhesion and environmental adaptations. We generated OsPG-FOX rice lines with little pectin due to overexpression of the gene encoding a pectin-degrading enzyme [polygalacturonase (PG)]. Overexpression of OsPG2 in rice under weak light conditions increased the activity of PG, which increased the degradation of pectin in the cell wall, thereby reducing adhesion. Under weak light conditions, the overexpression of OsPG decreased the pectin content and cell adhesion, resulting in abnormally large intercellular gaps and facilitating invasion by the rice blast fungus. OsPG2-FOX plants had weaker mechanical properties and greater sensitivity to biotic stresses than wild-type (WT) plants. However, the expression levels of disease resistance genes in non-infected leaves of OsPG2-FOX were more than twice as high as those of the WT and the intensity of disease symptoms was reduced, compared with the WT. Under normal light conditions, overexpression of OsPG2 decreased the pectin content, but did not affect cell adhesion and sensitivity to biotic stresses. Therefore, PG plays a role in regulating intercellular adhesion and the response to biotic stresses in rice.

New insights into the specificity and processivity of two novel pectinases from Verticillium dahliae.

Pectin, the major non-cellulosic component of primary cell wall can be degraded by polygalacturonases (PGs) and pectin methylesterases (PMEs) during pathogen attack on plants. We characterized two novel enzymes, VdPG2 and VdPME1, from the fungal plant pathogen Verticillium dahliae. VdPME1 was most active on citrus methylesterified pectin (55-70%) at pH 6 and a temperature of 40 °C, while VdPG2 was most active on polygalacturonic acid at pH 5 and a temperature of 50 °C. Using LC-MS/MS oligoprofiling, and various pectins, the mode of action of VdPME1 and VdPG2 were determined. VdPME1 was shown to be processive, in accordance with the electrostatic potential of the enzyme. VdPG2 was identified as endo-PG releasing both methylesterified and non-methylesterified oligogalacturonides (OGs). Additionally, when flax roots were used as substrate, acetylated OGs were detected. The comparisons of OGs released from Verticillium-susceptible and partially resistant flax cultivars identified new possible elicitor of plant defence responses.

Allergen Homologues, Pathogenesis-Related 1, Polygalacturonase, and Pectin Methyl Esterase from a Japanese Hop.

BACKGROUND: Japanese hop is an important cause of weed pollinosis in East Asia. Its pollen is abundant in autumn. This pollen is known to be the cause of many allergic diseases. However, molecular characteristics of its allergens have not been elucidated. OBJECTIVE: In this study, we produced recombinant proteins of allergen homologues from Japanese hop by the analysis of expressed sequence tags (EST), and evaluated its allergenicity. METHODS: cDNA library was constructed using as little as 50 ng of total RNA from Japanese hop pollen. Allergen homologues were identified by the initial screening of 963 EST clones. Recombinant proteins were overexpressed in the E. coli expression system and purified using Ni-nitrilotriacetic acid-agarose. Purified proteins were analyzed by ELISA. RESULTS AND DISCUSSION: Japanese hop pathogenesis-related 1 protein (PR-1) shares 37.0 to 44.4% of amino acid sequence identity with Art v 2, Cuc m 3, and Cyn d 24. Pectin methyl esterase (PME) shows 23.2 to 50.2% of identities to Act d 7, Ole e 11, and Sal k 1. Polygalacturonase (PGs) shows 16.7 to 19.3% of identities to Phl p 13, Cry j 2, Cha o 2, Jun a 2, Pla a 2, and Pla or 2. IgE antibodies from Japanese hop allergy patients' sera recognized PR-1 (3.4%), PME (13.8%), PGs (3.7%), and profilin (13.8%), respectively. CONCLUSION: Novel allergenic components were identified, even though low IgE reactivity was displayed reflecting the low degree of cross-reactivity with other pollen allergens. We believe that these molecules have worth further studies.

Identification of pollen and pistil polygalacturonases in Nicotiana tabacum and their function in interspecific stigma compatibility.

KEY MESSAGE: The pollen and pistil polygalacturonases in Nicotiana tabacum were identified and found to regulate pollen tube growth and interspecific compatibility. Polygalacturonase (PG) is one of the enzymes catalyzing the hydrolysis of pectin. This process plays important roles in the pollen and pistil. In this research, the pollen and pistil PGs in Nicotiana tabacum (NtPGs) were identified, and their expression, localization and the potential function in the pollen and interspecific stigma incompatibility were explored. The results showed that 118 NtPGs were retrieved from the genome of N. tabacum. The phylogenetic tree and RT-qPCR analysis led to the identification of 10 pollen PGs; among them, two, seven and one showed specifically higher expression levels in the early development of anthers, during pollen maturation and in mature anthers, respectively, indicating their function difference. Immunofluorescence analysis showed that PGs were located in the cytoplasm of (1) mature pollen and (2) in vitro grown pollen tubes, as well as in the wall of in vivo grown pollen tubes. Four NtPGs in clade A were identified as the pistil PGs, and the pistil PGs were not found in clade E. Significantly higher PGs expression was recorded after incompatible pollination in comparison with the compatible stigma, indicating a potential function of PGs in regulating stigma incompatibility. The influence of PGs on pollen tube growth was explored in vitro and partly in vivo, showing that high PGs activity inhibited pollen tube growth. The application of PGs on the otherwise compatible stigma resulted in pollen tube growth inhibition or failure of germination. These results further supported that increased PGs expression in incompatible stigma might be partially responsible for the interspecific stigma incompatibility in Nicotiana.

Comprehensive analysis of polygalacturonase gene family highlights candidate genes related to pollen development and male fertility in wheat (Triticum aestivum L.).

MAIN CONCLUSION: Four polygalacturonase gene family members were highlighted that contribute to elucidate the roles of polygalacturonase during the fertility conversion process in male-sterile wheat. Polygalacturonase (PG) belongs to a large family of hydrolases with important functions in cell separation during plant growth and development via the degradation of pectin. Specific expressed PGs in anthers may be significant for male sterility research and hybrid wheat breeding, but they have not been characterized in wheat (Triticum aestivum L.). In this study, we systematically studied the PG gene family using the latest published wheat reference genomic information. In total, 113 wheat PG genes were identified, which could be classified into six categories A-F according to their structure characteristics and phylogenetic comparisons with Arabidopsis and rice. Polyploidy and segmental duplications in wheat were proved to be mainly responsible for the expansion of the wheat PG gene family. RNA-seq showed that TaPGs have specific temporal and spatial expression characteristics, in which 12 TaPGs with spike-specific expression patterns were detected by qRT-PCR in different fertility anthers of KTM3315A, a thermo-sensitive cytoplasmic male-sterile wheat. Four of them specific upregulated (TaPG09, TaPG95, and TaPG93) or downregulated (TaPG87) at trinucleate stage of fertile anthers, and further aligning with the homologous in Arabidopsis revealed that they may undertake functions such as anther dehiscence, separation of pollen, pollen development, and pollen tube elongation, thereby inducing male fertility conversion in KTM3315A. These findings facilitate function investigations of the wheat PG gene family and provide new insights into the fertility conversion mechanism in male-sterile wheat.

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.

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.