Experimental and Molecular Dynamics Simulation Insights into Adsorption of Co(II), Cr(III), and Cu(II) on Chitosan and Chitosan/Tripolyphosphate Nanoparticles.

Chitosan (CS)/tripolyphosphate (TPP) nanoparticles were synthesized using the ionic gelation method based on the mass ratio and volume ratio between CS and TPP and then subsequently characterized using XRD, FT-IR, and SEM. The interaction between the metal ions Co(II), Cr(III), and Cu(II) on CS and 2CS/TPP was simulated using molecular dynamics (MD), and the findings were compared with the experimental data. CS/TPP nanoparticles were more favorable than using pure chitosan at a % removal efficiency of 91.47, 89.11, and 78.11 for Cu(II), Cr(III), and Co(II), respectively. The binding energy between 2CS/TPP and the metals was more favorable than that for CS at -214.95, -106.87, and -58.11 kcal/mol for Cr(III), Co(II), and Cu(II), respectively. The CS/TPP nanoparticles greatly affect metal adsorption and are therefore considered materials for wastewater treatment.

Integrative pathway and network analysis provide insights on flooding-tolerance genes in soybean.

Soybean is highly sensitive to flooding and extreme rainfall. The phenotypic variation of flooding tolerance is a complex quantitative trait controlled by many genes and their interaction with environmental factors. We previously constructed a gene-pool relevant to soybean flooding-tolerant responses from integrated multiple omics and non-omics databases, and selected 144 prioritized flooding tolerance genes (FTgenes). In this study, we proposed a comprehensive framework at the systems level, using competitive (hypergeometric test) and self-contained (sum-statistic, sum-square-statistic) pathway-based approaches to identify biologically enriched pathways through evaluating the joint effects of the FTgenes within annotated pathways. These FTgenes were significantly enriched in 36 pathways in the Gene Ontology database. These pathways were related to plant hormones, defense-related, primary metabolic process, and system development pathways, which plays key roles in soybean flooding-induced responses. We further identified nine key FTgenes from important subnetworks extracted from several gene networks of enriched pathways. The nine key FTgenes were significantly expressed in soybean root under flooding stress in a qRT-PCR analysis. We demonstrated that this systems biology framework is promising to uncover important key genes underlying the molecular mechanisms of flooding-tolerant responses in soybean. This result supplied a good foundation for gene function analysis in further work.

An advanced systems biology framework of feature engineering for cold tolerance genes discovery from integrated omics and non-omics data in soybean.

Soybean is sensitive to low temperatures during the crop growing season. An urgent demand for breeding cold-tolerant cultivars to alleviate the production loss is apparent to cope with this scenario. Cold-tolerant trait is a complex and quantitative trait controlled by multiple genes, environmental factors, and their interaction. In this study, we proposed an advanced systems biology framework of feature engineering for the discovery of cold tolerance genes (CTgenes) from integrated omics and non-omics (OnO) data in soybean. An integrative pipeline was introduced for feature selection and feature extraction from different layers in the integrated OnO data using data ensemble methods and the non-parameter random forest prioritization to minimize uncertainties and false positives for accuracy improvement of results. In total, 44, 143, and 45 CTgenes were identified in short-, mid-, and long-term cold treatment, respectively, from the corresponding gene-pool. These CTgenes outperformed the remaining genes, the random genes, and the other candidate genes identified by other approaches in an independent RNA-seq database. Furthermore, we applied pathway enrichment and crosstalk network analyses to uncover relevant physiological pathways with the discovery of underlying cold tolerance in hormone- and defense-related modules. Our CTgenes were validated by using 55 SNP genotype data of 56 soybean samples in cold tolerance experiments. This suggests that the CTgenes identified from our proposed systematic framework can effectively distinguish cold-resistant and cold-sensitive lines. It is an important advancement in the soybean cold-stress response. The proposed pipelines provide an alternative solution to biomarker discovery, module discovery, and sample classification underlying a particular trait in plants in a robust and efficient way.

Structural analysis of rice Os4BGlu18 monolignol ß-glucosidase.

Monolignol glucosides are storage forms of monolignols, which are polymerized to lignin to strengthen plant cell walls. The conversion of monolignol glucosides to monolignols is catalyzed by monolignol ß-glucosidases. Rice Os4BGlu18 ß-glucosidase catalyzes hydrolysis of the monolignol glucosides, coniferin, syringin, and p-coumaryl alcohol glucoside more efficiently than other natural substrates. To understand more clearly the basis for substrate specificity of a monolignol ß-glucosidase, the structure of Os4BGlu18 was determined by X-ray crystallography. Crystals of Os4BGlu18 and its complex with δ-gluconolactone diffracted to 1.7 and 2.1 Å resolution, respectively. Two protein molecules were found in the asymmetric unit of the P212121 space group of their isomorphous crystals. The Os4BGlu18 structure exhibited the typical (ß/α)8 TIM barrel of glycoside hydrolase family 1 (GH1), but the four variable loops and two disulfide bonds appeared significantly different from other known structures of GH1 ß-glucosidases. Molecular docking studies of the Os4BGlu18 structure with monolignol substrate ligands placed the glycone in a similar position to the δ-gluconolactone in the complex structure and revealed the interactions between protein and ligands. Molecular docking, multiple sequence alignment, and homology modeling identified amino acid residues at the aglycone-binding site involved in substrate specificity for monolignol ß-glucosides. Thus, the structural basis of substrate recognition and hydrolysis by monolignol ß-glucosidases was elucidated.

Demonstration of monolignol ß-glucosidase activity of rice Os4BGlu14, Os4BGlu16 and Os4BGlu18 in Arabidopsis thaliana bglu45 mutant.

The glycoside hydrolase family 1 members Os4BGlu14, Os4BGlu16, and Os4BGlu18 were proposed to be rice monolignol ß-glucosidases. In vitro studies demonstrated that the Os4BGlu16 and Os4BGlu18 hydrolyze the monolignol glucosides coniferin and syringin with high efficiency compared to other substrates. The replacement of the conserved catalytic acid/base glutamate residue by a nonionizable glutamine residue in Os4BGlu14 suggested that it may be inactive as a ß-glucosidase. Here, we investigated the activities of Os4BGlu14, Os4BGlu16, and Os4BGlu18 in planta by recombinant expression of their genes in the Arabidopsis bglu45-2 (monolignol ß-glucosidase) mutant and analysis of monolignol glucosides by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MSMS). The bglu45-2 line exhibits elevated monolignol glucoside levels, but lower amounts of coniferin, syringin, and p-coumaryl alcohol glucoside were seen in Arabidopsis bglu45-2 rescued lines complemented by the Os4BGlu14, Os4BGlu16, and Os4BGlu18 genes. These data suggest that the bglu45-2 mutant has a broader effect on monolignols than previously reported and that the Os4BGlu14, Os4BGlu16 and Os4BGlu18 proteins act as monolignol ß-glucosidases to complement the defect. An OsBGlu16-GFP fusion protein localized to the cell wall. This apoplastic localization and the effect of these enzymes on monolignol glucoside levels suggest monolignol glucosides from the vacuole may meet the monolignol ß-glucosidases, despite their different localization.

ß-Glucosidases: Multitasking, moonlighting or simply misunderstood?

ß-Glucosidases have a wide range of functions in plants, including roles in recycling of cell-wall oligosaccharides, defense, phytohormone signaling, secondary metabolism, and scent release, among others. It is not always clear which one is responsible for a specific function, as plants contain a large set of ß-glucosidases. However, progress has been made in recent years in elucidating these functions. To help understand what is known and what remains ambiguous, we review the general approaches to investigating plant ß-glucosidase functions. We consider information that has been gained regarding glycoside hydrolase family 1 enzyme functions utilizing these approaches in the past decade. In several cases, one enzyme has been assigned different biological functions by different research groups. We suggest that, at least in some cases, the ambiguity of an enzyme's function may come from having multiple functions that may help coordinate the response to injury or other stresses.

Expression and enzymatic properties of rice (Oryza sativa L.) monolignol ß-glucosidases.

Monolignol glucosides and their ß-glucosidases are found in monocots, but their biological roles are unclear. Phylogenetic analysis of rice (Oryza sativa L.) glycoside hydrolase family GH1 ß-glucosidases indicated that Os4BGlu14, Os4BGlu16, and Os4BGlu18 are closely related to known monolignol ß-glucosidases. An optimized Os4BGlu16 cDNA and cloned Os4BGlu18 cDNA were used to express fusion proteins with His6 tags in Pichia pastoris and Escherichia coli, respectively. The secreted Os4BGlu16 fusion protein was purified from media by immobilized metal affinity chromatography (IMAC), while Os4BGlu18 was extracted from E. coli cells and purified by anion exchange chromatography, hydrophobic interaction chromatography and IMAC. Os4BGlu16 and Os4BGlu18 hydrolyzed the monolignol glucosides coniferin (kcat/KM, 21.6mM(-1)s(-1) for Os4BGlu16 and for Os4BGlu18) and syringin (kcat/KM, 22.8mM(-1)s(-1) for Os4BGlu16 and 24.0mM(-1)s(-1) for Os4BGlu18) with much higher catalytic efficiencies than other substrates. In quantitative RT-PCR, highest Os4BGlu14 mRNA levels were detected in endosperm, embryo, lemma, panicle and pollen. Os4BGlu16 was detected highest in leaf from 4 to 10 weeks, endosperm and lemma, while Os4BGlu18 mRNA was most abundant in vegetative stage from 1 week to 4 weeks, pollen and lemma. These data suggest a role for Os4BGlu16 and Os4BGlu18 monolignol ß-glucosidases in both vegetative and reproductive rice tissues.

Recombinant expression and characterization of the cytoplasmic rice ß-glucosidase Os1BGlu4.

The Os1BGlu4 ß-glucosidase is the only glycoside hydrolase family 1 member in rice that is predicted to be localized in the cytoplasm. To characterize the biochemical function of rice Os1BGlu4, the Os1bglu4 cDNA was cloned and used to express a thioredoxin fusion protein in Escherichia coli. After removal of the tag, the purified recombinant Os1BGlu4 (rOs1BGlu4) exhibited an optimum pH of 6.5, which is consistent with Os1BGlu4's cytoplasmic localization. Fluorescence microscopy of maize protoplasts and tobacco leaf cells expressing green fluorescent protein-tagged Os1BGlu4 confirmed the cytoplasmic localization. Purified rOs1BGlu4 can hydrolyze p-nitrophenyl (pNP)-ß-D-glucoside (pNPGlc) efficiently (kcat/Km  =  17.9 mM(-1) · s(-1)), and hydrolyzes pNP-ß-D-fucopyranoside with about 50% the efficiency of the pNPGlc. Among natural substrates tested, rOs1BGlu4 efficiently hydrolyzed ß-(1,3)-linked oligosaccharides of degree of polymerization (DP) 2-3, and ß-(1,4)-linked oligosaccharide of DP 3-4, and hydrolysis of salicin, esculin and p-coumaryl alcohol was also detected. Analysis of the hydrolysis of pNP-ß-cellobioside showed that the initial hydrolysis was between the two glucose molecules, and suggested rOs1BGlu4 transglucosylates this substrate. At 10 mM pNPGlc concentration, rOs1BGlu4 can transfer the glucosyl group of pNPGlc to ethanol and pNPGlc. This transglycosylation activity suggests the potential use of Os1BGlu4 for pNP-oligosaccharide and alkyl glycosides synthesis.