Pectate Lyase Activity Research Articles

Portulaca oleracea (L.) essential oil inhibits Pestalotiopsis neglecta and controls black spot needle blight in Pinus sylvestris var. mongolica (Litv.)

Black spot needle blight in Pinus sylvestris var. mongolica is a common disease caused by Pestalotiopsis neglecta which leads to yellowing and withering of needles, eventually leading to the death of the affected trees. The study investigated the antifungal activity and mechanism of Portulaca oleracea essential oil (POEO) against P. neglecta and its efficacy in controlling black spot needle blight in Pinus sylvestris var. mongolica. In this study, we identified the main components of POEO as linalool and limonene. The antifungal assay proved that POEO could inhibit the growth of P. neglecta, 6 μL/mL of POEO inhibited P. neglecta by 100 % with an EC50 value of 1.16 μL/mL, and the results of scanning electron microscopy confirmed that POEO inhibited the growth of the P. neglecta mycelium. In addition, POEO treatment induced oxidative stress and exacerbated membrane lipid peroxidation of P. neglecta, which ultimately caused serious damage to P. neglecta cell membranes and cell contents leakage. POEO also reduced the activity and virulence of polygalacturonase (PG), endo-1,4-β-D-glucose hydratase (EG), and pectate lyase (PL) secreted by P. neglecta, thereby controlling black-spotted needle blight of Pinus sylvestris var. mongolica, which was able to control black spot needle blight in Pinus sylvestris var. mongolica. In summary, our results revealed the antifungal mechanism of POEO on P. neglecta and provided potential options for controlling black spot needle blight in Pinus sylvestris var. mongolica and other plant diseases.

Identification and Characterization of Pectate Lyase as a Novel Allergen in Artemisia sieversiana Pollen

Introduction: Artemisia species are widely spread in north hemisphere. Artemisia sieversiana pollen is one of the common pollen allergens in the north of China. At present, seven allergens were identified and had been listed officially from A. sieversiana pollen, but the remaining allergens are still insufficiently studied, which need to be found. Methods: Pectate lyase was purified from the extracts of A. sieversiana pollen by anion exchange, size exclusion, and HPLC-hydrophobic interaction chromatography. The gene of A. sieversiana pectate lyase (Art si pectate lyase) was cloned and expressed in Escherichia coli. The enzyme activity and circular dichroism (CD) spectrum of natural and recombinant proteins were analyzed. The allergenicity of Art si pectate lyase was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test. The allergen’s physicochemical properties, three-dimensional structure, sequence profiles with homologous allergens and phylogenetic tree were analyzed by in silico methods. Results: Natural Art si pectate lyase (nArt si pectate lyase) was purified from A. sieversiana pollen extracts by three chromatographic strategies. The cDNA sequence of Art si pectate lyase had a 1191-bp open reading frame encoding 396 amino acids. Both natural and recombinant pectate lyase (rArt si pectate lyase) exhibited similar CD spectrum, and nArt si pectate lyase had higher enzymatic activity. Moreover, the specific immunoglobulin E (IgE) binding rate against nArt si pectate lyase and rArt si pectate lyase was determined as 40% (6/15) in patients’ serum with Artemisia species pollen allergy by ELISA. The nArt si pectate lyase and rArt si pectate lyase could inhibit 76.11% and 47.26% of IgE binding activities to the pollen extracts, respectively. Art si pectate lyase was also confirmed to activate patients’ basophils. Its structure contains a predominant motif of classic parallel helical core, consisting of three parallel β-sheets, and two highly conserved features (vWiDH, RxPxxR) which may contribute to pectate lyase activity. Moreover, Art si pectate lyase shared the highest sequence identity of 73.0% with Art v 6 among currently recognized pectate lyase allergen, both were clustered into the same branch in the phylogenetic tree. Conclusion: In this study, pectate lyase was identified and comprehensively characterized as a novel allergen in A. sieversiana pollen. The findings enriched the allergen information for this pollen and promoted the development of component-resolved diagnosis and molecular therapy of A. sieversiana pollen allergy.

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.

Feasibility insights into the application of Paenibacillus pabuli E1 in animal feed to eliminate non-starch polysaccharides.

The goal of the research was to find alternative protein sources for animal farming that are efficient and cost-effective. The researchers focused on distillers dried grains with solubles (DDGS), a co-product of bioethanol production that is rich in protein but limited in its use as a feed ingredient due to its high non-starch polysaccharides (NSPs) content, particularly for monogastric animals. The analysis of the Paenibacillus pabuli E1 genome revealed the presence of 372 genes related to Carbohydrate-Active enzymes (CAZymes), with 98 of them associated with NSPs degrading enzymes that target cellulose, hemicellulose, and pectin. Additionally, although lignin is not an NSP, two lignin-degrading enzymes were also examined because the presence of lignin alongside NSPs can hinder the catalytic effect of enzymes on NSPs. To confirm the catalytic ability of the degrading enzymes, an in vitro enzyme activity assay was conducted. The results demonstrated that the endoglucanase activity reached 5.37 U/mL, while beta-glucosidase activity was 4.60 U/mL. The filter paper experiments did not detect any reducing sugars. The xylanase and beta-xylosidase activities were measured at 11.05 and 4.16 U/mL, respectively. Furthermore, the pectate lyase and pectin lyase activities were found to be 8.19 and 2.43 U/mL, respectively. The activities of laccase and MnP were determined as 1.87 and 4.30 U/mL, respectively. The researchers also investigated the effect of P. pabuli E1 on the degradation of NSPs through the solid-state fermentation of DDGS. After 240 h of fermentation, the results showed degradation rates of 11.86% for hemicellulose, 11.53% for cellulose, and 8.78% for lignin. Moreover, the crude protein (CP) content of DDGS increased from 26.59% to 30.59%. In conclusion, this study demonstrated that P. pabuli E1 possesses various potential NSPs degrading enzymes that can effectively eliminate NSPs in feed. This process improves the quality and availability of the feed, which is important for animal farming as it seeks alternative protein sources to replace traditional nutrients.

Comparison of cell wall changes of two different types of apple cultivars during fruit development and ripening

Fruit development and ripening is a complex procedure (Malus×domestica Borkh.) and can be caused by various factors such as cell structure, cell wall components, and cell wall hydrolytic enzymes. In our study, we focused on the variations in fruit firmness, cell wall morphology and components, the activity of cell wall hydrolytic enzymes and the expression patterns of associated genes during fruit development in two different types of apple cultivars, the hard-crisp cultivar and the loose-crisp cultivar. In this paper, the aim was to find out the causes of the texture variations between the different type cultivars. Cell wall materials (CWMs), hemicellulose and cellulose content were strongly associated with variations in fruit firmness during the fruit development. The content of water soluble pectin (WSP) and chelator soluble pectin (CSP) gradually increased, while the content of ionic soluble pectin (ISP) showed inconsistent trends in the four cultivars. The activities of polygalacturonase (PG), β-galactosidase (β-gal), cellulase (CEL), and pectate lyase (PL) gradually increased in four cultivars. And the activities of PG, β-gal, and CEL were higher in ‘Fuji’ and ‘Honeycrisp’ fruit with the fruit development, while the activity of PL of ‘Fuji’ and ‘Honeycrisp’ was lower than that of ‘ENVY’ and ‘Modi’. Both four cultivars of fruit cells progressively became bigger as the fruit expanded, with looser cell arrangements and larger cell gaps. According to the qRT-PCR, the relative expression levels of MdACO and Mdβ-gal were notably enhanced. Our study showed that there were large differences in the content of ISP and hemicellulose, the activity of PL and the relative expression of Mdβ-gal between two different types of apple cultivars, and these differences might be responsible for the variations in the texture of the four cultivars.

Improvement of optimum pH and specific activity of pectate lyase from Bacillus RN.1 using loop replacement.

Background: Alkaline pectate lyase plays an important role in papermaking, biological refining and wastewater treatment, but its industrial applications are largely limited owing to its low activity and poor alkali resistance. Methods: The alkaline pectate lyase BspPel from Bacillus RN.1 was heterologously expressed in Escherichia coli BL21 (DE3) and its activity and alkali resistance were improved by loop replacement. Simultaneously, the effect of R260 on enzyme alkaline tolerance was also explored. Results: Recombinant pectate lyase (BspPel-th) showed the highest activity at 60°C and pH 11.0, and showed significant stability over a wide pH range (3.0-11.0). The specific enzyme activity after purification was 139.4U/mg, which was 4.4 times higher than that of the wild-type enzyme. BspPel-th has good affinity for apple pectin, since the V max and K m were 29μmol/min. mL and 0.46mol/L, respectively. Molecular dynamics simulation results showed that the flexibility of the loop region of BspPel-th was improved. Conclusion: The modified BspPel-th has considerable potential for industrial applications with high pH processes.

Gene-specific silencing of SlPL16, a pectate lyase coding gene, extends the shelf life of tomato fruit

Pectate lyase plays an important role in fruit development and ripening. Although earlier studies showed SlPL, a pectate lyase encoding gene, functions dominantly in tomato fruit softening, the functions of many other pectate lyase encoding genes involved in tomato fruit development and softening remain unknown. Here, gene-specific silencing of SlPL16 (Solyc06g083580) by RNA interference (RNAi) resulted in reduced pectate lyase enzyme activity, pericarp thickness, and soluble sugar content, and increased both the firmness and shelf life of tomato fruit. Through microscopic observation, it was found that the SlPL16-RNAi pericarp had reduced cell size, in comparison to the wild-type (WT). Moreover, gene silencing of SlPL16 resulted in the down-regulation of genes involved in cell wall degradation and pectin synthesis at various fruit developmental stages. Together, these results indicate that SlPL16 provides an important supplement of pectate lyase activity in tomato fruit softening, along with a fundamental role in cell expansion during early fruit development. Additionally, this study provides a potential gene locus in genetic engineering to improve fruit storage tolerance.

Improvement of shelf-life quality of ivy gourd (Coccinia grandis L. Voigt) using an exogenous coating of mannitol and sorbitol

Mannitol and sorbitol are considered as suitable osmolytes. In light of this, the two reagents were applied (100 µM to 400 µM individually and combinations of 100 µM + 100 µM to 400 µM + 400 µM) by dipping the ivy gourd fruits in them. The physiological along with physical parameters were assessed with and without (considered to be controlled) dipping in these reagents. The results of the present study reveal that the rate of physiological and physical changes in ivy gourds during storage is influenced by the amount of exogenous mannitol-sorbitol used. In addition to significantly lowering weight, pH, and titrable acidity, the mannitol-sorbitol coating of ivy gourds increases phosphomolybdate activity, total soluble sugar, phenolic content, and pectate lyase activity. It has enzymes that fight free radicals, including peroxidase and catalase, whose activity decreases in stressful situations. In comparison to the other treatments and the untreated ivy gourds, the 300 µM mannitol treatment offered the highest amount of protection. Hence, ivy gourds' postharvest quality can be effectively extended by mannitol and sorbitol for up to 10 days without significantly affecting their phytochemical and physiological qualities.

Pectate Lyase Gene VvPL1 Plays a Role in Fruit Cracking of Table Grapes

Fruit cracking seriously affects the commercial value of table grapes. To explore whether cell wall disassembly influences grape berry cracking, first, the differences in the cell wall metabolism were compared between cracking-resistant "Shennongjinhuanghou" (SN) and cracking-susceptible "Xiangfei" (XF) varieties. Our results showed that cell wall disassembly events were extremely different between "SN" and "XF." The cracking-resistant "SN" had a higher pectinmethylesterase activity in the early stage and lower polygalacturonase, β-galactosidase, pectate lyase, and cellulase activities from veraison, cooperatively yielding higher ionically bound pectin, covalently bound pectin, hemicellulose, and lower water-soluble pectin, leading to a stronger skin break force and elasticity and conferring "SN" with higher cracking resistance. Furthermore, the function of the VvPL1 gene in fruit cracking was verified by heterologously transforming tomatoes. The transgenic experiment showed that overexpressed fruits had a higher activity of pectate lyase from the breaking stage and a lower level of covalently bound pectin, ionically bound pectin, cellulose, and hemicellulose and a higher level of water-soluble pectin at the red ripe stage, which resulted in a significantly reduced skin break force and flesh firmness and increased fruit cracking incidences. In conclusion, our results demonstrated that the cracking susceptibility of the grape berry is closely related to cell wall disassembly events and VvPL1 plays an important role in fruit cracking.

Pectinolytic arsenal of Colletotrichum lindemuthianum and other fungi with different lifestyles.

To identify and analyse genes that encode pectinases in the genome of the fungus Colletotrichum lindemuthianum, evaluate the expression of these genes, and compare putative pectinases found in C. lindemuthianum with pectinases produced by other fungi and oomycetes with different lifestyles. Genes encoding pectinases in the genome of C. lindemuthianum were identified and analysed. The expression of these genes was analysed. Pectinases from C. lindemuthianum were compared with pectinases from other fungi that have different lifestyles, and the pectinase activity in some of these fungi was quantified. Fifty-eight genes encoding pectinases were identified in C. lindemuthianum. At least six types of enzymes involved in pectin degradation were identified, with pectate lyases and polygalacturonases being the most abundant. Twenty-seven genes encoding pectinases were differentially expressed at some point in C. lindemuthianum during their interactions with their host. For each type of pectinase, there were at least three isoenzyme groups. The number of pectinases present in fungi with different lifestyles seemed to be related more to the lifestyle than to the taxonomic relationship between them. Only phytopathogenic fungi showed pectate lyase activity. The collective results demonstrate the pectinolytic arsenal of C. lindemuthianum, with many and diverse genes encoding pectinases more than that found in other phytopathogens, which suggests that at least part of these pectinases must be important for the pathogenicity of the fungus C. lindemuthianum. Knowledge of these pectinases could further the understanding of the importance of this broad pectinolytic arsenal in the common bean infection and could be exploited for biotechnological purposes.

Pectinases Secretion by Saccharomyces cerevisiae: Optimization in Solid-State Fermentation and Identification by a Shotgun Proteomics Approach.

A sequential design strategy was applied to optimize the secretion of pectinases by a Saccharomyces cerevisiae strain, from Brazilian sugarcane liquor vat, on passion fruit residue flour (PFRF), through solid-state fermentation (SSF). A factorial design was performed to determine the influence variables and two rotational central composite designs were executed. The validated experimental result was of 7.1 U mL−1 using 50% PFRF (w/w), pH 5, 30 °C for 24 h, under static SSF. Polygalacturonase, pectin methyl esterase, pectin–lyase and pectate–lyase activities were 3.5; 0.08; 3.1 and 0.8 U mL−1, respectively. Shotgun proteomics analysis of the crude extract enabled the identification of two pectin–lyases, one pectate–lyase and a glucosidase. The crude enzymatic extract maintained at least 80% of its original activity at pH values and temperatures ranging from 2 to 8 and 30 to 80 °C, respectively, over 60 min incubation. Results revealed that PFRF might be a cost-effective and eco-friendly substrate to produce pectinases. Statistical optimization led to fermentation conditions wherein pectin active proteins predominated. To the extent of our knowledge, this is the first study reporting the synthesis of pectate lyase by S. cerevisiae.

Study on the effect of icariin on the preservation of postharvest mango fruit

The effect of exogenous icariin (0.005% and 0.01%) on mango cultivar ‘Zill’ fruit during 25°C storage was investigated in this study. The results showed 0.005% icariin treatment could delay the decline of fruit firmness and yellowing process of flesh tissues, inhibit the conversion of soluble sugar, synthesis of carotenoids and degradation of vitamin C, and reduce the disease spots, and to maintain the appearance and nutrient quality. Moreover, the treatment could improve the disease resistance of fruit by inhibiting catalase (CAT) activity in the early stage of storage, but promoting peroxidase (POD) activity in the later stage. Furthermore, icariin treatment reduced the activities of polygalacturonase (PG) and pectate lyase (PL), thus attenuated the degradation of cell wall and decelerated the softening process. The above results indicate 0.005% icariin treatment could extend the shelf life of postharvest mango and present application value for fruits preservation. Practical applications Mango is a highly perishable fruit and undergoes a rapid ripening process after harvest. Icariin is the main active ingredient of Chinese herbal medicine Herba epimedin ‘Yinyanghuo’. The efficacy of icariin in mammals has been widely studied, while its' preservation functions in food were rarely reported. Our results suggested that icariin treatment could delay the ripening and senescence, and enhance disease resistance of postharvest mango, which is expected to be a promising approach for fruit preservation in further industrial application.

FvWRKY48 binds to the pectate lyase FvPLA promoter to control fruit softening in Fragaria vesca.

The regulatory mechanisms that link WRKY gene expression to fruit ripening are largely unknown. Using transgenic approaches, we showed that a WRKY gene from wild strawberry (Fragaria vesca), FvWRKY48, may be involved in fruit softening and ripening. We showed that FvWRKY48 is localized to the nucleus and that degradation of the pectin cell wall polymer homogalacturonan, which is present in the middle lamella and tricellular junction zones of the fruit, was greater in FvWRKY48-OE (overexpressing) fruits than in empty vector (EV)-transformed fruits and less substantial in FvWRKY48-RNAi (RNA interference) fruits. Transcriptomic analysis indicated that the expression of pectate lyase A (FvPLA) was significantly downregulated in the FvWRKY48-RNAi receptacle. We determined that FvWRKY48 bound to the FvPLA promoter via a W-box element through yeast one-hybrid, electrophoretic mobility shift, and chromatin immunoprecipitation quantitative polymerase chain reaction experiments, and β-glucosidase activity assays suggested that this binding promotes pectate lyase activity. In addition, softening and pectin degradation were more intense in FvPLA-OE fruit than in EV fruit, and the middle lamella and tricellular junction zones were denser in FvPLA-RNAi fruit than in EV fruit. We speculated that FvWRKY48 maybe increase the expression of FvPLA, resulting in pectin degradation and fruit softening.

A highly alkaline pectate lyase from the Himalayan hot spring metagenome and its bioscouring applications

From the vast enzymatic weaponry encrypted in the metagenomic resource of the Himalayan hot spring, a novel gene encoding pectate lyase (eplM) was identified. Mapping against the nucleotide non-redundant NCBI database could not reveal any match for this gene sequence. At the protein level, it was about 56.86% identical to an uncharacterized sequence from Rheinheimera tangshanensis. The novel gene, eplM, was cloned and expressed in a heterologous host, Escherichia coli. The purified protein, EPLM, was biochemically characterized for pectate lyase activity towards pectin. It exhibited the maximum activity in the alkaline pH range, with optimum activity at 11.0 pH (1524.10 U/mg). The enzyme was functional in the temperature range of 30–50 °C, with the optimum activity at 40 °C. EPLM was found to be a calcium-dependent enzyme that can efficiently act on the methylated pectin obtained from a diverse source of plant biomass. The catalytic utility of EPLM was tested by demonstrating bioscouring of banana pseudostem biomass. The micrographic changes in surface structures of the fibers and release of unsaturated galacturonides endorsed EPLM as a promising biocatalyst for pectin-removal processing applications.

Characterization of a novel phage depolymerase specific to Escherichia coli O157:H7 and biofilm control on abiotic surfaces.

The increasing prevalence of foodborne diseases caused by Escherichia coli O157:H7 as well as its ability to form biofilms poses major threats to public health worldwide. With increasing concerns about the limitations of current disinfectant treatments, phage-derived depolymerases may be used as promising biocontrol agents. Therefore, in this study, the characterization, purification, and application of a novel phage depolymerase, Dpo10, specifically targeting the lipopolysaccharides of E. coli O157, was performed. Dpo10, with a molecular mass of 98 kDa, was predicted to possess pectate lyase activity via genome analysis and considered to act as a receptor-binding protein of the phage. We confirmed that the purified Dpo10 showed O-polysaccharide degrading activity only for the E. coli O157 strains by observing its opaque halo. Dpo10 maintained stable enzymatic activities across a wide range of temperature conditions under 55°C and mild basic pH. Notably, Dpo10 did not inhibit bacterial growth but significantly increased the complement-mediated serum lysis of E. coli O157 by degrading its O-polysaccharides. Moreover, Dpo10 inhibited the biofilm formation against E. coli O157 on abiotic polystyrene by 8-fold and stainless steel by 2.56 log CFU/coupon. This inhibition was visually confirmed via fieldemission scanning electron microscopy. Therefore, the novel depolymerase from E. coli siphophage exhibits specific binding and lytic activities on the lipopolysaccharide of E. coli O157 and may be used as a promising anti-biofilm agent against the E. coli O157:H7 strain.

Characterization and Sensitivity Test of the Allergenic Pollen Proteins from Litchi Chimensis Plant

Pollen of Litchi chinensis (Litchi) is a major aeroallergen of Bangladesh. Pollen of this fruits plant was collected from full bloomed flower growing in different places of Rajshahi in Bangladesh. Pollen protein was extracted and partial purified by means of long-term PBS extraction, salting out, dialysis, gel filtrations and DEAE-Cellulose chromatography and the protein was designated as LFPP (Litchi flowers pollen protein). Gel filtration of the purified pollen protein gives two main peaks. The major peak gives four bands on SDS-PAGE. The enzyme (pectate lyase) proteins after gel filtration again re-purified by Ion exchange chromatography, a single band in the protein profile of LFPP, (M.W. 28kDa) was the major allergenic component of Litchi chinensis (Litchi) flower pollen. The homogeneity and the molecular weight of the protein were estimated by SDS-PAGE, and Gel filtration was 28kDa. The allergenic protein was identified by skin prick tests and showed the pectate lyase (Pel) activity. Skin-prick tests also revealed highest degree of sensitivity to the Nawabgang sample giving positive response in 80% of the patients. Skin reactivity ranged between 1+ and 3+.

Inhibitory effects of high molecular weight chitosan coating on ‘Hom Thong’ banana fruit softening

This study was undertaken to examine the effects of different molecular weights of 1% (w/v) chitosan coating on physicochemical characteristics and softening of ‘Hom Thong’ banana. The results indicated that high and medium molecular weights of chitosan coating (HMW-CTS; 540 kDa and MMW-CTS; 265 kDa) maintained banana fruit qualities during 25 °C storage compared to a low molecular weight of chitosan coating (LMW-CTS; 65 kDa) and control treatments. HMW-CTS expressed the highest ability to reduce respiration rate and ethylene production of the fruit and preserved fruit firmness by inhibiting cell wall degrading enzymes including polygalacturonase and pectate lyase activities which play an important role in pectin degradation. Additionally, HMW-CTS influenced a decrease in disease severity and cell membrane injury in relation to low malondialdehyde content and high antioxidant activities of coated fruit. The overall results suggest that HMW-CTS is a promising postharvest chitosan coating in maintaining fruit firmness and extending the shelf life of ‘Hom Thong’ banana fruit after harvest.

Dynamic changes of pectin epitopes and daylily tepals during flower opening and senescence of Hemerocallis citrina

Ephemeral flower deaths occur within 24 h of flowering in the genus Hemerocallis. The senescence of plant organs is owing to the loss of cell wall cohesion. Pectin is the most structurally complex plant cell wall polysaccharide and has an important role in cell-to-cell adhesion. In this study, we used histological stains, immunolocalization and pectin characterization analyses to examine the dynamic changes in pectin in Hemerocallis citrina ‘Datonghuanghua’ tepals at eight stages (S1–S8). The pectin-specific antibodies JIM5 and JIM7, which hybridize to differently esterified regions of the homogalacturonan (HG) backbone, showed that demethylesterification of HG-type pectins was evident after the outer tepals had opened. Before flower opening, these two antibodies labelled tissue continuously across the cell wall, and the cells were round and tightly packed. The labeling of highly esterified epitopes recognized by JIM7 became deformed when the outer tepals opened. However, 12 h after blooming, low-and high-esterified epitope signals were disrupted near the vascular cylinder, and air space then appeared until these signals disappeared. labeling with the antibody LM5, which is specific for the (1 → 4)-β galactan side chains of rhamnogalacturonan-I, was disrupted near the vascular cylinder at the same stage. Moreover, the contents of neutral sugars in pectin analyses showed that galactose decreased significantly at 12 h after blooming. Three enzymes were involved in pectin remodeling at different developmental phases. The activities of pectin methylesterase continued to increase after the outer tepals opened and reached a significant peak at 12 h after blooming. The activities of polygalacturonase and pectate lyase also increased, but the difference was not significant. Our findings revealed that demethylesterification of HG-type pectins was evident after the outer tepals opened, and this remodeling greatly facilitated their degradation by pectic enzymes. The loss of cell wall material and cohesion that accompanied the activities of pectic enzymes was associated with senescence. Therefore, remodeling of the pectin in cell walls of the tepals is necessary for the occurrence of senescence.

RNA Chaperones Hfq and ProQ Play a Key Role in the Virulence of the Plant Pathogenic Bacterium Dickeya dadantii

Dickeya dadantii is an important pathogenic bacterium that infects a number of crops including potato and chicory. While extensive works have been carried out on the control of the transcription of its genes encoding the main virulence functions, little information is available on the post-transcriptional regulation of these functions. We investigated the involvement of the RNA chaperones Hfq and ProQ in the production of the main D. dadantii virulence functions. Phenotypic assays on the hfq and proQ mutants showed that inactivation of hfq resulted in a growth defect, a modified capacity for biofilm formation and strongly reduced motility, and in the production of degradative extracellular enzymes (proteases, cellulase, and pectate lyases). Accordingly, the hfq mutant failed to cause soft rot on chicory leaves. The proQ mutant had reduced resistance to osmotic stress, reduced extracellular pectate lyase activity compared to the wild-type strain, and reduced virulence on chicory leaves. Most of the phenotypes of the hfq and proQ mutants were related to the low amounts of mRNA of the corresponding virulence factors. Complementation of the double mutant hfq-proQ by each individual protein and cross-complementation of each chaperone suggested that they might exert their effects via partially overlapping but different sets of targets. Overall, it clearly appeared that the two Hfq and ProQ RNA chaperones are important regulators of pathogenicity in D. dadantii. This underscores that virulence genes are regulated post-transcriptionally by non-coding RNAs.

Understanding the effects of process parameters in the bioscouring of cotton and their interactions on pectate lyase activity by factorial design analysis

Pectinases can be used to remove impurities and hydrophobic material from cotton fibers by the degradation of pectic substances. The biochemical characterization and the evaluation of process parameters that influence the enzyme’s activity are mandatory to optimize the industrial application. In the present study, a factorial experimental design was conducted to evaluate the activity of a commercial pectate lyase at different reaction conditions of temperature, pH, and nonionic surfactant concentration, aiming its application in bioscouring of textiles. The main effects of all three factors were found to be statistically significant. The second-order interactions between temperature and pH, and pH and surfactant concentration, were also important. Maximum activity on polygalacturonic acid was achieved at 60 °C, pH 9, and without surfactant. These results provided support for the subsequent bioscouring assays, resulting in a high level of cleanliness and good wettability of knitted cotton fabric at the lowest enzyme concentration (1 g/l), and could make textile processing more sustainable.