Alginate binding enhances the structural stability and potentiates the lytic activity of bacteriophage endolysin's partially folded conformation.

Polysaccharide polymers are increasingly being used as chaperon-like macromolecules in assisting protein folding of unfolded protein molecules. They interact with unfolded or partially folded proteins in a charge and conformation specific manner that results in the formation of stable protein-polysaccharide complexes. In most of the cases, the complex formation of protein-polysaccharide is driven via non-covalent interactions that have found to endorse the activity of proteins. T4L (18.7 kDa) and T7L (17 kDa) endolysins belong to the hydrolase and amidase class of peptidoglycan degrading enzymes. Both T4L and T7L exist in partially folded forms and are devoid of lytic activity at low pH conditions. In the current study, we assessed the binding of alginate with T4L and T7L at pH 7 and 3 using variety of biophysical and biochemical techniques. Spectroscopic studies revealed differential structural modulations of partially folded T4L and T7L upon their interaction with alginate. Further, the complex formation of alginate with partially folded T4L/T7L was confirmed by ITC and STEM. Additionally, the formed complexes of alginate with both T4L/T7L PF endolysins were found to be chemically and enzymatically stable. Moreover, such complexes were also marked with differential enhancement in their lytic activities at acidic pH conditions. This implied the potency of alginate as an excellent choice of matrix to preserve the structural and functional integrity of partially folded forms of T4L and T7L at highly acidic conditions.

Discovery and characterization of a novel poly-mannuronate preferred alginate lyase: The first member of a new polysaccharide lyase family.

Alginate is one of the most important marine colloidal polysaccharides, and its oligosaccharides have been proven to possess diverse biological functions. Alginate lyases could specifically degrade alginate and therefore serve as desirable tools for the research and development of alginate. In this report, a novel catalytic domain, which demonstrated no significant sequence similarity with all previously defined functional domains, was verified to exhibit a random endo-acting lyase activity to alginate. The action pattern analysis revealed that the heterologously expressed protein, named Aly44A, preferred to degrade polyM. Its minimum substrates and the minimum products were identified as unsaturated alginate trisaccharides and disaccharides, respectively. Based on the sequence novelty of Aly44A and its homologs, a new polysaccharide lyase family (PL44) was proposed. The discovery of the novel enzyme and polysaccharide lyase family provided a new entrance for the gene-mining and acquiring of alginate lyases, and would facilitate to the utilization of alginate and its oligosaccharides.

In vitro colon fermentation behaviors of Ca2+ cross-linked guluronic acid block from sodium alginate.

The degradation of sodium alginate by human gut microbiota was found to be retarded via calcium cross-linking in our previous study. We hypothesized that the guluronic acid block (GB) on the alginate molecule might be the key structural region affecting alginate degradation by the gut microbiota when cross-linked with calcium. This study aims to prove this hypothesis by studying the structural features of the cross-linked GB on its in vitro fecal fermentation behaviors concerning the aspects of total carbohydrate contents, monosaccharide contents, short-chain fatty acids production, calcium state variations, and structural variations. Herein, GB isolated from sodium alginate was cross-linked under ranges of molar ratios of [Ca2+]/[-COOH] that further restricted the degradation by gut microbiota similar to the cross-linked alginates. First, total carbohydrate contents, short-chain fatty acids production, monosaccharides contents, and calcium state analyses confirmed that the degradation of GB by gut microbiota was restricted by calcium cross-linking. Furthermore, the tracking analysis of structural variations during in vitro fermentation revealed that the "granules" structure could further restrict degradation by the gut microbiota, leaving more cross-linked GB fragments surviving in comparison to the "networks" structure. In addition, Bacteroides xylanisolvens showed a significant positive correlation to the "cross-linking porosity (R = 0.825, p < 0.001), which supported our previous findings on fermentation behaviors of cross-linked alginate. Together, guluronic acid blocks are the key structural regions that retard the degradation of sodium alginate by the gut microbiota when cross-linked with calcium.

Physicochemical properties and fractal characterizations of the functionalized porous clinoptilolites for controlling alginate delivery in growing cauliflower and leaf mustard.

Using natural clinoptilolite (NCP) as a carrier and alginate (Alg)-calcium as an active species, the porous silicon calcium alginate nanocomposite (Alg-Ca-NCP) was successfully fabricated via adsorption-covalence-hydrogen bond. Its structural features and physicochemical properties were detailed investigated by various characterizations. The results indicated that Alg-Ca-NCP presented the disordered lamellar structures with approximately uniform particles in size of 300-500 nm. Specially, their surface fractal evolutions between the irregular roughness and dense structures were demonstrated via the SAXS patterns. The results elucidated that the abundant micropores of NCP were beneficial for unrestricted diffusing of Alg-Ca, which was conducive to facilitate a higher loading and sustainable releasing. The Ca content of leaf mustard treated with Alg-Ca-NCP-0.5 was 484.5 mg/100g on the 21st day, higher than that by water (CK) and CaCl2 solution treatments, respectively. Meanwhile, the prepared Alg-Ca-NCPs presented the obvious anti-aging effects on peroxidase drought stress of mustard leaves. These demonstrations provided a simple and effective method to synthesize Alg-Ca-NCPs as delivery nanocomposites, which is useful to improve the weak absorption and low utilization of calcium alginate by plants.

Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum.

Among the epimerases specific to alginate, some of them in Azotobacter genera convert ß-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C-5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers.

Batch-mode degradation of high-strength phenolic pollutants by Pseudomonas aeruginosa strain STV1713 immobilized on single and hybrid matrices.

Controlled environments are pivotal in all bioconversion processes, influencing the efficacy of biocatalysts. In this study, we designed a batch bioreactor system with a packed immobilization column and a decontamination chamber to enhance phenol and 2,4-dichlorophenol degradation using the hyper-tolerant bacterium Pseudomonas aeruginosa STV1713. When free cells were employed to degrade phenol and 2,4-DCP at a concentration of 1000 mg/L, the cells completely removed the pollutants within 28 h and 66 h, respectively. Simultaneous reductions in chemical oxygen demand and biological oxygen demand were observed (phenol: 30.21 mg/L/h and 16.92 mg/L/h, respectively; 2,4-dichlorophenol: 12.85 mg/L/h and 7.21 mg/L/h, respectively). After assessing the degradation capabilities, the bacterium was immobilized on various matrices (sodium alginate, alginate-chitosan-alginate and polyvinyl alcohol-alginate) to enhance pollutant removal. Hybrid immobilized cells exhibited greater tolerance and degradation capabilities than those immobilized in a single matrix. Among them, polyvinyl alcohol-alginate immobilized cells displayed the highest degradation capacities (up to 2000 mg/L for phenol and 2500 mg/L for 2,4-dichlorophenol). Morphological analysis of the immobilized cells revealed enhanced cell preservation in hybrid matrices. Furthermore, the elucidation of the metabolic pathway through the catechol dioxygenase enzyme assay indicated higher activity of the catechol 1,2-dioxygenase enzyme, suggesting that the bacterium employed an ortho-degradation mechanism for pollutant removal. Additionally, enzyme zymography confirmed the presence of catechol 1,2-dioxygenase, with the molecular weight of the enzyme determined as 245 kDa.

Yeast extracts from different manufacturers and supplementation of amino acids and micro elements reveal a remarkable impact on alginate production by A. vinelandii ATCC9046.

BACKGROUND: In research and production, reproducibility is a key factor, to meet high quality and safety standards and maintain productivity. For microbial fermentations, complex substrates and media components are often used. The complex media components can vary in composition, depending on the lot and manufacturing process. These variations can have an immense impact on the results of biological cultivations. The aim of this work was to investigate and characterize the influence of the complex media component yeast extract on cultivations of Azotobacter vinelandii under microaerobic conditions. Under these conditions, the organism produces the biopolymer alginate. The focus of the investigation was on the respiration activity, cell growth and alginate production. RESULTS: Yeast extracts from 6 different manufacturers and 2 different lots from one manufacturer were evaluated. Significant differences on respiratory activity, growth and production were observed. Concentration variations of three different yeast extracts showed that the performance of poorly performing yeast extracts can be improved by simply increasing their concentration. On the other hand, the results with well-performing yeast extracts seem to reach a saturation, when their concentration is increased. Cultivations with poorly performing yeast extract were supplemented with grouped amino acids, single amino acids and micro elements. Beneficial results were obtained with the supplementation of copper sulphate, cysteine or a combination of both. Furthermore, a correlation between the accumulated oxygen transfer and the final viscosity (as a key performance indicator), was established. CONCLUSION: The choice of yeast extract is crucial for A. vinelandii cultivations, to maintain reproducibility and comparability between cultivations. The proper use of specific yeast extracts allows the cultivation results to be specifically optimised. In addition, supplements can be applied to modify and improve the properties of the alginate. The results only scratch the surface of the underlying mechanisms, as they are not providing explanations on a molecular level. However, the findings show the potential of optimising media containing yeast extract for alginate production with A. vinelandii, as well as the potential of targeted supplementation of the media.

Structure of the AlgKX modification and secretion complex required for alginate production and biofilm attachment in Pseudomonas aeruginosa.

Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.

Differential Impact of Hexuronate Regulators ExuR and UxuR on the Escherichia coli Proteome.

ExuR and UxuR are paralogous proteins belonging to the GntR family of transcriptional regulators. Both are known to control hexuronic acid metabolism in a variety of Gammaproteobacteria but the relative impact of each of them is still unclear. Here, we apply 2D difference electrophoresis followed by mass-spectrometry to characterise the changes in the Escherichia coli proteome in response to a uxuR or exuR deletion. Our data clearly show that the effects are different: deletion of uxuR resulted in strongly enhanced expression of D-mannonate dehydratase UxuA and flagellar protein FliC, and in a reduced amount of outer membrane porin OmpF, while the absence of ExuR did not significantly alter the spectrum of detected proteins. Consequently, the physiological roles of proteins predicted as homologs seem to be far from identical. Effects of uxuR deletion were largely dependent on the cultivation conditions: during growth with glucose, UxuA and FliC were dramatically altered, while during growth with glucuronate, activation of both was not so prominent. During the growth with glucose, maximal activation was detected for FliC. This was further confirmed by expression analysis and physiological tests, thus suggesting the involvement of UxuR in the regulation of bacterial motility and biofilm formation.

[Production of mucic acid from pectin-derived D-galacturonic acid: a review].

Mucic acid is a hexaric acid that can be biosynthesized by oxidation of D-galacturonic acid, which is the main constituent of pectin. The structure and properties of mucic acid are similar to that of glucaric acid, and can be widely applied in the preparation of important platform compounds, polymers and macromolecular materials. Pectin is a cheap and abundant renewable biomass resource, thus developing a process enabling production of mucic acid from pectin would be of important economic value and environmental significance. This review summarized the structure and hydrolysis of pectin, the catabolism and regulation of D-galacturonic acid in microorganisms, and the strategy for mucic acid production based on engineering of corresponding pathways. The future application of mucic acid are prospected, and future directions for the preparation of mucic acid by biological method are also proposed.

Effect of Extraction Procedures with Ultrasound and Cellulolytic Enzymes on the Structural and Functional Properties of Citrus grandis Osbeck Seed Mucilage.

The structural and functional properties of Citrus grandis Osbeck (CGO) seed mucilage by different extraction practices, including conventional citrate buffer, ultrasonic-assisted (UAE), enzymatic-assisted extraction (EAE) with cellulase or Celluclast® 1.5 L and various ultrasonic-assisted enzymatic extraction (UAEE) procedures were investigated. It was found that CGO seed from agricultural and processing byproducts is an excellent new source of high methoxyl pectin with quite high intrinsic viscosity (about 108.64 dL/g) and molecular weight (about 1.9 × 106) as compared with other pectin sources. UAEE with Celluclast® 1.5 L enhanced the extraction yield most pronouncedly (about 2.3 times). Moreover, the monosaccharide composition of CGO seed mucilage is least affected by EAE with Celluclast® 1.5 L. In contrast, EAE with cellulase dramatically reduces the galacturonic acid (GalA) content to less than 60 molar%, and increases the glucose (Glc) content pronouncedly (to about 40 molar%), which may be considered as an adverse effect in terms of pectin purity. Though extraction procedures involved with ultrasound and cellulolytic enzymes generally show a decrease in GalA contents, weight average molar mass and intrinsic viscosity, EAE with Celluclast® 1.5 L is least affected, followed by UAE and UAEE with Celluclast® 1.5 L. These features can be leveraged in favor of diversified applications.

4-Deoxy-l-erythro-5-hexoseulose Uronate (DEH) and DEH Reductase: Key Molecule and Enzyme for the Metabolism and Utilization of Alginate.

4-Deoxy-l-erythro-5-hexoseulose uronate (DEH), DEH reductase, and alginate lyase have key roles in the metabolism of alginate, a promising carbon source in brown macroalgae for biorefinery. In contrast to the widely reviewed alginate lyase, DEH and DEH reductase have not been previously reviewed. Here, we summarize the current understanding of DEH and DEH reductase, with emphasis on (i) the non-enzymatic and enzymatic formation and structure of DEH and its reactivity to specific amino groups, (ii) the molecular identification, classification, function, and structure, as well as the structural determinants for coenzyme specificity of DEH reductase, and (iii) the significance of DEH for biorefinery. Improved understanding of this and related fields should lead to the practical utilization of alginate for biorefinery.

Tau and amyloid beta differentially affect the innate immune genes expression in Drosophila models of Alzheimer's disease and ß- D Mannuronic acid (M2000) modulates the dysregulation.

Alzheimer's disease (AD) is the most common cause of dementia and neuroinflammation is considered as one of the main culprits. The aim of this study was to evaluate the independent role of Aß42 and tau on the inflammatory pathway in the Drosophila models of AD and investigating the potential modulating effect of M2000 as a novel NSAIDs in those flies. The expression levels of relish, orthologs of NF-κB, antimicrobial peptide (AMP) including attacin A, diptericin B and a dual oxidase (Duox) as a ROS mediator, were evaluated in both M2000 treated and untreated groups followed by brain histology analysis to assess the extent of neurodegeneration. The potential inhibitory role of M2000 (ß-D Mannuronic acid) on the aggregation of tau protein was also investigated in vitro. According to the result, there was a significant induction of Duox, AMPs and its transcription factor expression in both aged and Drosophila models of AD which was in accordance with the increase in the number of vacuoles in the brain section of Drosophila models of AD. Interestingly M2000 treatment revealed a significant reduction in all neurodegeneration indexes; in vivo and anti-aggregating property; in vitro. Findings suggest that M2000 has potential to be an AD therapeutic agent.

Mechanistic Basis for Understanding the Dual Activities of the Bifunctional Azotobacter vinelandii Mannuronan C-5-Epimerase and Alginate Lyase AlgE7.

The structure and functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5-epimerases determine the monomer composition by catalyzing the epimerization of ß-d-mannuronic acid (M) residues into α-l-guluronic acid (G) residues. The molecular weight is affected by alginate lyases, which catalyze a ß-elimination mechanism that cleaves alginate chains. The reaction mechanisms for the epimerization and lyase reactions are similar, and some enzymes can perform both reactions. These dualistic enzymes share high sequence identity with mannuronan C-5-epimerases without lyase activity. The mechanism behind their activity and the amino acid residues responsible for it are still unknown. We investigate mechanistic determinants involved in the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by nuclear magnetic resonance (NMR) spectroscopy. Our results show that calcium promotes lyase activity, whereas NaCl reduces the lyase activity of AlgE7. By using defined polymannuronan (polyM) and polyalternating alginate (polyMG) substrates, the preferred cleavage sites of AlgE7 were found to be M|XM and G|XM, where X can be either M or G. From the study of AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study, we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 functions as the catalytic acid. IMPORTANCE Postharvest valorization and upgrading of algal constituents are promising strategies in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine, and material industries. By providing a better understanding of the catalytic mechanism and of how the two enzyme actions can be altered by changes in reaction conditions, this study opens further applications of bacterial epimerases and lyases in the enzymatic tailoring of alginate polymers.

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.

Autotoxin Rg1 Induces Degradation of Root Cell Walls and Aggravates Root Rot by Modifying the Rhizospheric Microbiome.

Management of crop root rot disease is one of the key factors in ensuring sustainable development in agricultural production. The accumulation of autotoxins and pathogens in soil has been reported as a primary driver of root rot diseases; however, less is known about the correlation of plants, their associated pathogens and microbiome mediated by autotoxins as well as the contributions autotoxins make to the occurrence of root rot disease. Here, we integrated metabolomic, transcriptomic, and rhizosphere microbiome analyses to identify the root cell wall degradants cellobiose and d-galacturonic acid as being induced by the autotoxic ginsenoside Rg1 of Panax notoginseng, and we found that exogenous cellobiose and d-galacturonic acid in addition to Rg1 could aggravate root rot disease by modifying the rhizosphere microbiome. Microorganisms that correlated positively with root rot disease were enriched and those that correlated negatively were suppressed by exogenous cellobiose, d-galacturonic acid, and Rg1. In particular, they promoted the growth and infection of the soilborne pathogen Ilyonectria destructans by upregulating pathogenicity-related genes. Cellobiose showed the highest ability to modify the microbiome and enhance pathogenicity, followed by Rg1 and then d-galacturonic acid. Collectively, autotoxins damaged root systems to release a series of cell wall degradants, some of which modified the rhizosphere microbiome so that the host plant became more susceptible to root rot disease. IMPORTANCE The accumulation of autotoxins and pathogens in soil has been reported as a primary driver of root rot disease and one of the key factors limiting sustainable development in agricultural production. However, less is known about the correlation of plants, their associated pathogens, and the microbiome mediated by autotoxins, as well as the contributions autotoxins make to the occurrence of root rot disease. In our study, we found that autotoxins can damage root systems, thus releasing a series of cell wall degradants, and both autotoxins and the cell wall degradants they induce could aggravate root rot disease by reassembling the rhizosphere microbiome, resulting in the enrichment of pathogens and microorganisms positively related to the disease but the suppression of beneficial microorganisms. Deciphering this mechanism among plants, their associated pathogens, and the microbiome mediated by autotoxins will advance our fundamental knowledge of and ability to degrade autotoxins or employ microbiome to alleviate root rot disease in agricultural systems.

Cation complexation by mucoid Pseudomonas aeruginosa extracellular polysaccharide.

Mucoid Pseudomonas aeruginosa is a prevalent cystic fibrosis (CF) lung colonizer, producing an extracellular matrix (ECM) composed predominantly of the extracellular polysaccharide (EPS) alginate. The ECM limits antimicrobial penetration and, consequently, CF sufferers are prone to chronic mucoid P. aeruginosa lung infections. Interactions between cations with elevated concentrations in the CF lung and the anionic EPS, enhance the structural rigidity of the biofilm and exacerbates virulence. In this work, two large mucoid P. aeruginosa EPS models, based on ß-D-mannuronate (M) and ß-D-mannuronate-α-L-guluronate systems (M-G), and encompassing thermodynamically stable acetylation configurations-a structural motif unique to mucoid P. aeruginosa-were created. Using highly accurate first principles calculations, stable coordination environments adopted by the cations have been identified and thermodynamic stability quantified. These models show the weak cross-linking capability of Na+ and Mg2+ ions relative to Ca2+ ions and indicate a preference for cation binding within M-G blocks due to the smaller torsional rearrangements needed to reveal stable binding sites. The geometry of the chelation site influences the stability of the resulting complexes more than electrostatic interactions, and the results show nuanced chemical insight into previous experimental observations.

Glycinebetaine: a versatile protectant to improve rice performance against aluminium stress by regulating aluminium uptake and translocation.

KEY MESSAGE: Glycinebetaine alleviates the detrimental effects of aluminium stress by regulating aluminium uptake and translocation, maintaining PSII activity, and activating the oxidative defence, thereby maintaining the growth and development of rice. Aluminium (Al) toxicity is one of the primary growth-limiting factors that limits plant growth and crop productivity in acidic soils. Rice (Oryza sativa L.) plants are susceptible to Al stress and do not naturally accumulate glycinebetaine (GB), one of the most effective protectants. Therefore, the objective of this study was to investigate whether exogenous GB can ameliorate the detrimental effects of Al stress on rice plants. Our results showed that the growth, development and biomass of rice were clearly inhibited under Al stress. However, exogenous GB application increased rice shoot growth and photosynthetic pigments contents, maintained photosystem II (PSII) activity, and activated the antioxidant defence system under Al stress. More importantly, GB may mediate the expression of Al uptake- and translocation-related genes, including OsALS1, OsNrat1, OsSTAR1 and OsSTAR2, and the galacturonic acid contents in rice roots under Al stress. Therefore, our findings highlight exogenous GB application is a valid approach to effectively combat Al toxicity by regulating physiological and biochemical processes in crops.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra.

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Elevating Ascorbate in Arabidopsis Stimulates the Production of Abscisic Acid, Phaseic Acid, and to a Lesser Extent Auxin (IAA) and Jasmonates, Resulting in Increased Expression of DHAR1 and Multiple Transcription Factors Associated with Abiotic Stress Tolerance.

Gene expression and phytohormone contents were measured in response to elevating ascorbate in the absence of other confounding stimuli such as high light and abiotic stresses. Young Arabidopsis plants were treated with 25 mM solutions of l-galactose pathway intermediates l-galactose (l-gal) or l-galactono-1,4-lactone (l-galL), as well as L-ascorbic acid (AsA), with 25 mM glucose used as control. Feeding increased rosette AsA 2- to 4-fold but there was little change in AsA biosynthetic gene transcripts. Of the ascorbate recycling genes, only Dehydroascorbate reductase 1 expression was increased. Some known regulatory genes displayed increased expression and included ANAC019, ANAC072, ATHB12, ZAT10 and ZAT12. Investigation of the ANAC019/ANAC072/ATHB12 gene regulatory network revealed a high proportion of ABA regulated genes. Measurement of a subset of jasmonate, ABA, auxin (IAA) and salicylic acid compounds revealed consistent increases in ABA (up to 4.2-fold) and phaseic acid (PA; up to 5-fold), and less consistently certain jasmonates, IAA, but no change in salicylic acid levels. Increased ABA is likely due to increased transcripts for the ABA biosynthetic gene NCED3. There were also smaller increases in transcripts for transcription factors ATHB7, ERD1, and ABF3. These results provide insights into how increasing AsA content can mediate increased abiotic stress tolerance.