Comparative expression analysis of sucrose phosphate synthase gene family in a low and high sucrose Pakistani sugarcane cultivars.

Sugarcane is the world's largest cultivated crop by biomass and is the main source of sugar and biofuel. Sucrose phosphate synthase (SPS) enzymes are directly involved in the synthesis of sucrose. Here, we analyzed and compared one of the important gene families involved in sucrose metabolism in a high and low sucrose sugarcane cultivar. A comprehensive in silico analysis of the SoSPS family displayed their phylogenetic relationship, gene and protein structure, miRNA targets, protein interaction network (PPI), gene ontology and collinearity. This was followed by a spatial expression analysis in two different sugarcane varieties. The phylogenetic reconstruction distributed AtSPS, ZmSPS, OsSPS, SoSPS and SbSPS into three main groups (A, B, C). The regulatory region of SoSPS genes carries ABRE, ARE, G-box, and MYC as the most dominant cis-regulatory elements. The PPI analysis predicted a total of 14 unique proteins interacting with SPS. The predominant expression of SPS in chloroplast clearly indicates that they are the most active in the organelle which is the hub of photosynthesis. Similarly, gene ontology attributed SPS to sucrose phosphate synthase and glucosyl transferase molecular functions, as well as sucrose biosynthetic and disaccharide biological processes. Overall, the expression of SPS in CPF252 (high sucrose variety) was higher in leaf and culm as compared to that of CPF 251 (low sucrose variety). In brief, this study adds to the present literature about sugarcane, sucrose metabolism and role of SPS in sucrose metabolism thereby opening up further avenues of research in crop improvement.

Cloning and in silico characterization of an abiotic stress-inducible U-box domain-containing protein gene GsPUB8 from Glycine soja.

The ubiquitination pathway is involved in the posttranslational modification of cellular proteins. However, the role of E3 ubiquitin ligase family proteins under abiotic stress conditions remains unclear, particularly in soybean. The core objective of the current study was to isolate and functionally characterize the GsPUB8 protein gene from wild soybean (Glycine soja) by using a homologous cloning method to investigate its abiotic stress responses. The GsPUB8 is a 40,562 Da molecular weight protein with 373 amino acid residues. The sequence alignment revealed the presence of U-box domain while the phylogenetic analysis showed an abundance of PUB8 proteins in both monocot and dicot plants. Analysis of gene structure predicted the absence of introns along with the presence of one exon. Furthermore, the activity of the GsPUB8 protein was anticipated in the plasma membrane and its expression was persuaded with NaCl, ABA, PEG6000, and NaHCO3 treatments with considerably higher manifestation in roots than leaves although, expressed in both vegetative and reproductive parts of G. soja. GsPUB8 protein showed 54% and 32% sequence identity to U-box domain containing 8 and 12 proteins from Arabidopsis thaliana and Oryza sativa subsp. japonica, respectively. GsPUB8 exhibited relatively higher expression under saline and drought stress particularly in roots. Whereas, the 3D model of GsPUB8 protein was generated using the SWISS-MODEL. This study can be used to manipulate the GsPUB8 protein or GsPUB8 gene for transformation purposes and its functional characterization under abiotic stress conditions.

Comprehensive Structural and Functional Characterization of a Seed γ-thionin as a Potent Bioactive Molecule Against Fungal Pathogens and Insect Pests.

BACKGROUND: Fungi and insect pests ruin stored crop grain, which results in millions of dollars of damage, presenting an ongoing challenge for farmers in addition to diminishing the safety of stored food. A wide-range defensive system against pathogens is needed to reduce or even eliminate the dependence of the crop yield upon the use of pesticides. Plant defensins (γ-thionins) are antimicrobial peptides (AMPs) that are a component of the host defense system. They are known to interact with cell membranes to exhibit antifungal and insecticidal activity. They exhibit a broad range of activities against fungi and insects and are effective at low concentrations. Thionins act on membranes, greatly reducing the development of pathogen resistance. OBJECTIVE: The aim of this study is to investigate a bioactive molecule that acts against fungal pathogens and stored grain insect pests. METHODS: γ-thionin protein was extracted from Brassica oleracea L. var. capitata f. alba (white cabbage) seed powder in phosphate buffer (100 mM, pH 7.0) and was identified by MALDI-TOF/TOF. The crude extract was subjected to 70% ammonium sulfate saturation followed by gel filtration chromatography. The disc diffusion assay along with a microtiter bioassay was used to determine the antifungal activity of the protein against phytopathogenic fungi. The insecticidal efficacy was evaluated by feeding insect pests with food contaminated with the purified protein. Additionally, an in silico molecular structure prediction study of the protein was performed using Auto Dock Vina for molecular docking of the protein with either fungal membrane moieties or α-amylase from Tenebrio molitor L. MD simulations of protein-ligand complexes were conducted using Schrodinger's Desmond module. RESULTS: γ-Thionin (BoT) was purified from white cabbage seeds and showed 100% homology with thionin (Brassica oleracea L. var. viridis) and 80% homology with defensin-like protein 1 (Raphanus sativus L.), respectively. BoT significantly inhibited the mycelial growth of Aspergillus niger van Tieghem and Aspergillus flavus Link at a concentration of 2 µM. Similarly, 0.12 µM BoT treatment resulted in significant mortality of Tribolium castaneum Herbst and Sitophilus oryzae L. Molecular docking and MD simulation of BoT confirmed the strong binding affinity with fungal membrane moieties (phosphatidylinositol 4,5-bisphosphate and phosphatidic acid), which causes disruption of the cell membrane and leakage of the cellular contents, leading to cell death. BoT blocked the active site of α-amylase, and as a result of the inactivation of this gut enzyme, the digestive systems of insects were disturbed, resulting in their deaths. CONCLUSION: This study revealed that γ-thionin is a good antifungal and insecticidal agent that could be used as an alternate to fungicides and insecticides.

In-silico Studies Calculated a New Chitin Oligomer Binding Site Inside Vicilin: A Potent Antifungal and Insecticidal Agent.

Vicilins are major seed storage proteins and show differential binding affinities toward sugar moieties of fungal cell wall and insect gut epithelium. Hence, purpose of study is the thorough in-silico characterization of interactions between vicilin and chitin oligomer followed by fungal and insecticidal bioassays. This work covers the molecular simulation studies explaining the interactions between Pisum sativum vicilin (PsV) and chitin oligomer followed by protein bioassay against different pathogens. LC-MS/MS of purified PsV (∼50 kDa) generated residual data along high pea vicilin homology (UniProtKB ID; P13918). Predicted model (PsV) indicated the characteristic homotrimer joined through head-to-tail association and each monomer is containing a bicupin domain. PsV site map analysis showed a new site (Site 4) into which molecular docking confirmed the strong binding of chitin oligomer (GlcNAc)4. Molecular dynamics simulation data (50 ns) indicated that chitin-binding site was comprised of 8 residues (DKEDRNEN). However, aspartate and glutamate significantly contributed in the stability of ligand binding. Computational findings were further verified via significant growth inhibition of Aspergillus flavus, A. niger, and Fusarium oxysporum against PsV. Additionally, the substantial adult population of Brevicoryne brassicae was reduced and different life stages of Tribolium castaneum also showed significant mortality.

In Silico Analysis and Functional Characterization of Antimicrobial and Insecticidal Vicilin from Moth Bean (Vigna aconitifolia (Jacq.) Marechal) Seeds.

Vicilin has nutraceutical potential and different noteworthy medicative health-promoting biotic diversions, and it is remarkable against pathogenic microorganisms and insects. In this study, Vigna aconitifolia vicilin (VacV) has been identified and characterized from the seed of Vigna aconitifolia (Jacq.) Marechal (Moth beans). LC-MS/MS analysis of VacV provided seven random fragmented sequences comprising 238 residues, showing significant homology with already reported Vigna radiata vicilin (VraV). VacV was purified using ammonium sulfate precipitation (60%) followed by size exclusion chromatography on Hi-Load 16/60 Superdex 200 pg column and anion-exchange chromatography (Hi trap Q FF column). Purified VacV showed a major ~50 kDa band and multiple lower bands on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under both reduced and non-reduced conditions. After all, a three-dimensional molecular structure of VacV was predicted, which showed ß-sheeted molecular conformation similar to crystallographic structure of VraV. All Vicilins from V. aconitifolia and other plants were divided into six sub-groups by phylogenetic analysis, and VacV shared a high degree of similarity with vicilins of Vigna radiata, Pisum sativum, Lupinus albus, Cicer arietinum and Glycine max. Additionally, VacV (20 µg) has significant growth inhibition against different pathogenic bacteria along strong antifungal activity (50 µg). Likewise, VacV (3.0 mg) produced significant growth reduction in Rice Weevil Sitophilus oryzae larvae after 9 days compared with control. Furthermore, by using MMT assay, the cytotoxicity effect of VacV on the growth of HepG2 liver cancerous cells was tested. VacV showed cytotoxicity against the HepG-2 line and the acquired value was 180 µg after 48 h. Finally, we performed molecular docking against caspase-3 protein (PDB ID: 3DEI) for VacV bioactive receptor interface residues. Hence, our results reveal that VacV, has nutraceutical potential and moth beans can be used as a rich resource of functional foods.

Eruca sativa seed napin structural insights and thorough functional characterization.

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC-MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an Rg of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm3, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ2-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.

Investigations on Acinetophage, QAB 3.4, Targeting Extensively Drug-Resistant Acinetobacter baumannii Isolates.

PURPOSE: Drug resistance against antimicrobials is on the rise at alarmingly high rates. Acinetobacter baumannii is one of the six ESKAPE pathogens which are a significant "one health" issue. Clinical isolates of A. baumannii exhibit MDR phenotype mostly and infrequently the XDR and PDR phenotype. As a result, these infections have one of the highest mortality rates in hospitals. Alternative therapies are urgently needed. METHODS: Various phages were enriched against XDR clinical strain of A. baumannii. A potent phage, QAB 3.4, was further tested against 100 clinical strains. Because of its broad lytic activity, it was further tested for stability, resistance development and as an infection control agent. RESULTS: Phage QAB 3.4 showed broad lytic activity against 100 MDR and XDR clinical isolates representing a wide diversity of infection sites. Assays conducted to document the phage's stability, and ability of clinical isolates to develop resistance against it, showed promising outcomes for its potential use in clinical applications. Phage QAB 3.4 was able to eradicate A. baumannii from pre-inoculated solid surfaces. It provides a proof of concept that phages can be used as environmentally friendly infection control agents. CONCLUSION: We propose the phage QAB 3.4 is a promising candidate for further pre-clinical and clinical studies to test its biosafety and efficacy.

Model prediction of a Kunitz-type trypsin inhibitor protein from seeds of Acacia nilotica L. with strong antimicrobial and insecticidal activity.

A Kunitz-type trypsin inhibitor protein has been purified and characterized from seeds of Acacia nilotica L. LC-MS/MS analysis of Acacia nilotica trypsin inhibitor (AnTI) provided the N-terminal fragment of 11 amino acids which yielded 100% identity with already reported Kunitz-type trypsin inhibitor protein of Acacia confusa (AcTI) in UniProtKB database search. SDS-PAGE showed a single band of ~21 kDa under nonreduced condition and appearance of a daughter band (17 kDa) in the presence of ß-mercaptoethanol indicating the presence of interchain disulfide linkage typical for Kunitz-type trypsin inhibitors. AnTI was purified from seed extract by using a combination of anion exchange and gel filtration chromatography. Since AnTI showed maximum homology with AcTI, a molecular structure of AcTI was predicted which showed highly ß-sheeted molecular conformation similar to crystallographic structure of Enterolobium contortisiliquum trypsin inhibitor (EcTI). AnTI (20 µg) produces significant population inhibition against different human pathogenic bacteria along strong antifungal activity (50 µg). Entomotoxin potential of AnTI was evaluated against two stored grain insect pests Tribolium castaneum (Herbst) (Tenebrionidae: Coleoptera) and Sitophilus oryzae (Linnaeus) (Curculionidae: Coleoptera). Statistically significant mortality of T. castaneum adults was observed at 1.5 mg after 15 days in comparison to control. Additionally, number of total eggs, larvae, pupae, adults, and their male/female ratio were also severely reduced in comparison to control. Similarly, two generation progeny of S. oryzae was studied after mixing AnTI with rice kernels. Mean percent mortality of adult population was significantly higher after 9 days of exposure in comparison to control group. AnTI significantly reduced the F1 generation while little mortality was observed for F2 generation. Exploration of such potent molecules is the prerequisite of our time regarding the anticipation of postantibiotic era and the development of insect resistance against chemical pesticides.

Mechanisms and Adaptation Strategies to Improve Heat Tolerance in Rice. A Review.

The incidence of short episodes of high temperature in the most productive rice growing region is a severe threat for sustainable rice production. Screening for heat tolerance and breeding to increase the heat tolerance of rice is major objective in the situation of recent climate change. Replacing sensitive genotypes with heat tolerant cultivars, modification in sowing time, and use of growth regulators are some of the adaptive strategies for the mitigation of yield reduction by climate change. Different strategies could be adopted to enhance the thermos-tolerance of rice by (1) the modification of agronomic practices i.e., adjusting sowing time or selecting early morning flowering cultivars; (2) induction of acclimation by using growth regulators and fertilizers; (3) selecting the genetically heat resistant cultivars by breeding; and, (4) developing genetic modification. Understanding the differences among the genotypes could be exploited for the identification of traits that are responsible for thermo-tolerance for breeding purpose. The selection of cultivars that flowers in early morning before the increase of temperature, and having larger anthers with long basal pore, higher basal dehiscence, and pollen viability could induce higher thermo-tolerance. Furthermore, the high expression of heat shock proteins could impart thermo-tolerance by protecting structural proteins and enzymes. Thus, these traits could be considered for breeding programs to develop resistant cultivars under a changing climate.

Computational modeling and functional characterization of a GgChi: A class III chitinase from corms of Gladiolus grandiflorus.

The present study describes the predicted model and functional characterization of an endochitinase (30 kDa) from corms of Gladiolus grandiflorus. ESI-QTOF-MS generated peptide showed 96% sequence homology with family 18, Class III acidic endochitinase of Gladiolus gandavensis. Purified G. grandiflorus chitinase (GgChi) hydrolyzed 4-methylumbelliferyl ß-d-N,N',N''-triacetylchitotriose substrate showing specific endochitinase activity. Since no structural details of GgChi were available in the Protein Data Bank (PDB), a homology model was predicted using the coordinate information of Crocus vernus chitinase (PDB ID: 3SIM). Ramachandran plot indicated 84.5% in most favored region, 14.8% in additional and 0.6% in generously allowed region while no residue in disallowed region. The predicted structure indicated a highly conserved (ß/α)8 (TIM barrel) structure similar to the family 18, class III chitinases. The GgChi also showed sequence and structural homologies with other active chitinases. The GgChi (50 µg/disc) showed no antibacterial activity, but did provide mild growth inhibition of phytopathogenic fungus Fusarium oxysporum at a concentration of 500 µg/well Similarly, insect toxicity bioassays of GgChi (50 µg) against nymphs of Bemisia tabaci showed 14% reduction in adult emergence and 14% increase in mortality rate in comparison to control values. The GgChi (1.5 mg) protein showed significant reduction in a population of flour beetle (Tribolium castaneum) after 35 days, but lower reactivity against rice weevil (Sitophilus oryzae). The results of this study provide detai.led insight on functional characterization of a family 18 class III acidic plant endochitinase.

Crystal Structure of Mistletoe Lectin I (ML-I) from Viscum album in Complex with 4-N-Furfurylcytosine at 2.85 Å Resolution.

BACKGROUND: Viscum album (the European mistletoe) is a semi-parasitic plant, which is of high medical interest. It is widely found in Europe, Asia, and North America. It contains at least three distinct lectins (i.e. ML-I, II, and III), varying in molecular mass and specificity. Among them, ML-I is in focus of medical research for various activities, including anti-cancer activities. To understand the molecular basis for such medical applications, a few studies have already addressed the structural and functional analysis of ML-I in complex with ligands. In continuation of these efforts, we are reporting the crystal structure of ML from Viscum album in complex with the nucleic acid oxidation product 4-N-furfurylcytosine (FC) refined to 2.85 Å resolution. FC is known to be involved in different metabolic pathways related to oxidative stress and DNA modification. METHODS: X-ray suitable hexagonal crystals of the ML-I/FC complex were grown within four days at 294 K using the hanging drop vapor diffusion method. Diffraction data were collected up to a resolution of 2.85 Å. The ligand affinity was verified by in-silico docking. RESULTS: The high-resolution structure was refined subsequently to analyze particularly the active site conformation and a binding epitope of 4-N-furfurylcytosine. A distinct 2Fo-Fc electron density at the active site was interpreted as a single FC molecule. The specific binding of FC is achieved also through hydrophobic interactions involving Tyr76A, Tyr115A, Glu165A, and Leu157A of the ML-I A-chain. The binding energy of FC to the active site of ML-I was calculated as well to be -6.03 kcal mol-1. CONCLUSION: In comparison to other reported ML-I complexes, we observed distinct differences in the vicinity of the nucleic acid base binding site upon interaction with FC. Therefore, data obtained will provide new insights in understanding the specificity, inhibition, and cytotoxicity of the ML-I A-chain, and related RIPs.