Identification of chalcone synthase genes and their expression patterns reveal pollen abortion in cotton

Chalcone synthase (CHS) is a key enzyme and producing flavonoid derivatives as well play a vital roles in sustaining plant growth and development. However, the systematic and comprehensive analysis of CHS genes in island cotton (G. barbadense) has not been reported yet especially response to cytoplasmic male sterility (CMS). To fill this knowledge gap, a genome-wide investigation of CHS genes were studied in island cotton. A total of 20 GbCHS genes were identified and grouped into five GbCHSs. The gene structure analysis revealed that most of GbCHS genes consisted of two exons and one intron, and 20 motifs were identified. Twenty five pairs duplicated events (12 GbCHS genes) were identified including 23 segmental duplication pairs and two tandem duplication events, representing that GbCHS gene family amplification mainly owned to segmental duplication events and evolving slowly. Gene expression analysis exhibited that the GbCHS family genes presented a diversity expression patterns in various organs of cotton. Coupled with functional predictions and gene expression, the abnormal expression of GbCHS06, 10, 16 and 19 might be associated with pollen abortion of CMS line in island cotton. Conclusively, GbCHS genes exhibited diversity and conservation in many aspects, which will help to better understand functional studies and a reference for CHS research in island cotton and other plants.     

Establishment of a novel method for the identification of fertilization defective mutants in Arabidopsis thaliana

Plant reproduction is an extremely important phenomenon, as it is strongly associated with plant genetics and early development. Additionally, foundations of the reproductive system have direct implications on plant breeding and agriculture. Investigation of the functions of male and female gametophytes is critical since their fusion is required for seed formation. Although a large number of mutants have been generated to understand the functions of male and female gametophytes, only a small number of genes required for plant fertilization have been identified to date. This is because the screening method used previously required the dissection of siliques, and fertilization-specific mutants exhibiting semi-fertility (or ∼50% fertility) were difficult to identify. Here, we report a new efficient screening method for the identification of fertilization defective mutants in Arabidopsis thaliana using vanillin staining. This method is based on the pollen tube-dependent ovule enlargement morphology (POEM) phenomenon, which generates a partial seed coat within the ovule without fertilization. Using this method, we successfully identified 23 putative fertilization defective mutants in Arabidopsis.     

The chromosome-scale assembly of endive (Cichorium endivia) genome provides insights into the sesquiterpenoid biosynthesis

Endive (Cichorium endivia L.) is a leafy vegetable in the Asteraceae family. Sesquiterpene lactones (STLs) in endive leaves bring a bitter taste that varies between varieties. Despite their importance in breeding varieties with unique flavours, sesquiterpenoid biosynthesis pathways in endive are poorly understood. We assembled a chromosome-scale endive genome of 641 Mb with a contig N50 of 5.16 Mb and annotated 46,711 protein-coding genes. Several gene families, especially terpene synthases (TPS) genes, expanded significantly in the C. endivia genome. STLs biosynthesis-related genes and TPS genes in more bitter varieties have shown a higher level of expression, which could be attributed to genomic variations. Our results penetrate the origin and diversity of bitter taste and facilitate the molecular breeding of endive varieties with unique bitter tastes. The high-quality endive assembly would provide a reference genome for studying the evolution and diversity of Asteraceae.     

A novel KU70-mutant human leukemic cell line generated using CRISPR-Cas9 shows increased sensitivity to DSB inducing agents and reduced NHEJ activity

KU70 (XRCC6 gene in humans) is one of the proteins in the KU70-KU80 heterodimer which is the first component recruited to broken DNA ends during DNA double-strand break repair through nonhomologous end joining (NHEJ). Previous studies have shown that Ku70 deficient mouse cells are defective in NHEJ and V(D)J recombination. In contrast, heterozygous KU70 mutant human cell lines did not show any significant change in cell viability and sensitivity towards ionizing radiation. In this study, we used CRISPR-Cas9 technique to generate a KU70 mutant (heterozygous) human pre-B leukemic cell line (N6-KU70–2-DG). We observed that the N6-KU70–2-DG cells showed a prominent reduction in the expression of both KU70 mRNA and protein. The mutant cells showed reduced cell viability, increased sensitivity to DSB inducing agents such as ionizing radiation (IR) and etoposide, and increased number of unrepaired DSBs after exposure to IR. In addition, the mutant cells showed a reduction in the NHEJ activity and increased rate of microhomology mediated joining (MMEJ) activity. KU70 mutant cells also revealed enhanced level of senescence markers following irradiation. Thus, we report a novel KU70-mutant leukemic cell line (heterozygous) with reduced NHEJ, which is sensitive to DNA damaging agents, unlike the previously reported other KU heterozygous mutant cell lines.     

Genetic Code Expansion Through Quadruplet Codon Decoding

Noncanonical amino acid mutagenesis has emerged as a powerful tool for the study of protein structure and function. While triplet nonsense codons, especially the amber codon, have been widely employed, quadruplet codons have attracted attention for the potential of creating additional blank codons for noncanonical amino acids mutagenesis. In this review, we discuss methodologies and applications of quadruplet codon decoding in genetic code expansion both in vitro and in vivo.     

Unveiling pharmacological studies provide new insights on Mangifera longipes and Quercus gomeziana

Mangifera longipes and Quercus gomeziana both is an ethnomedicinally important Asian herb that has been known for numerous healing activity of tribal people. The present research aims to investigate the phytochemical analysis with in vitro, in vivo possibilities of the soluble ethanol extract of M. longipes root (EEMLR) and Q. gomeziana leaves (EEQGL) by an experimental approach. The plant extract of EEMLR and EEQGL was found secondary metabolites, notably steroids, glycosides, tannins, flavonoids, saponins, gums, and alkaloids. Additionally, the extract showed significant activity in antioxidant, antipyretic, anti-inflammatory, membrane stabilization, cytotoxic, thrombolytic, and analgesic activities while no response in antibacterial activity. Our findings reveal that soluble ethanol extract of EEMLR and EEQGL is safe, which can be an effective source for exploring new medicinal products. This research's outcomes may provide potentials for mitigating pyrexia, inflammation, pain, cellular toxicity, and coagulation.     

Widespread Arginine Phosphorylation in Staphylococcus aureus

Arginine phosphorylation was only recently discovered to play a significant and relevant role in the Gram-positive bacterium Bacillus subtilis. In addition, arginine phosphorylation was also detected in Staphylococcus aureus, suggesting a widespread role in bacteria. However, the large-scale analysis of protein phosphorylation, and especially those that involve a phosphoramidate bond, comes along with several challenges. The substoichiometric nature of protein phosphorylation requires proper enrichment strategies prior to LC-MS/MS analysis, and the acid instability of phosphoramidates was long thought to impede those enrichments. Furthermore, good spectral quality is required, which can be impeded by the presence of neutral losses of phosphoric acid upon higher energy collision–induced dissociation. Here we show that pArg is stable enough for commonly used Fe³⁺-IMAC enrichment followed by LC-MS/MS and that HCD is still the gold standard for the analysis of phosphopeptides. By profiling a serine/threonine kinase (Stk1) and phosphatase (Stp1) mutant from a methicillin-resistant S. aureus mutant library, we identified 1062 pArg sites and thus the most comprehensive arginine phosphoproteome to date. Using synthetic arginine phosphorylated peptides, we validated the presence and localization of arginine phosphorylation in S. aureus. Finally, we could show that the knockdown of Stp1 significantly increases the overall amount of arginine phosphorylation in S. aureus. However, our analysis also shows that Stp1 is not a direct protein-arginine phosphatase but only indirectly influences the arginine phosphoproteome.     

Conformational Transitions in Yeast Chorismate Mutase Important for Allosteric Regulation as Identified by Nuclear Magnetic Resonance Spectroscopy

Proteins fluctuate between different conformations in solution, and these conformational fluctuations can be important for protein function and allosteric regulation. The chorismate mutase from Saccharomyces cerevisiae (ScCM), a key enzyme in the biosynthesis of aromatic amino acids, is allosterically activated and inhibited by tryptophan and tyrosine, respectively. It was initially proposed that in the absence of effector, ScCM fluctuates between activated R and inhibited T conformations according to the Monod-Wyman-Changeux (MWC) model, although a more complex regulation pattern was later suggested by mutagenesis and kinetic data. Here we used NMR relaxation dispersion experiments to understand the conformational fluctuations on the microsecond-to-millisecond timescale that occur in ScCM. In the absence of allosteric effectors, ScCM did not exclusively exchange between T and R conformations, suggesting that the two-state MWC model is insufficient to explain conformational dynamics. Addition of tyrosine led to the quenching of much of the motion on this timescale, while new motions were identified in the presence of tryptophan. These new motions are consistent with conformational fluctuations into an alternative conformation that may be important for enzyme activity.     

Smilax china root extract as a novel Glucose- 6-phosphate dehydrogenase inhibitor for the treatment of hepatocellular carcinoma

A novel therapeutic strategy for cancer treatment is to target altered tumor metabolism. Glucose- 6-phosphate dehydrogenase (G6PD) has been recently discovered to be implicated in apoptosis and angiogenesis, making it an excellent target in cancer treatment. The current study aimed to screen the plant extracts library to find potent hits against G6PD through enzymatic assay. Protein expression was induced by IPTG and purified using Ni-NTA columns after transformation of the pET-24a-HmG6PD plasmid into E. coli BL21-DE3 strain. An enzymatic assay was established by using purified rG6PD protein, for the screening of G6PD inhibitors. Out of 46 plant extracts screened, the sixteen plant extracts have shown inhibitory activity against the G6PD enzyme. At doses from 1 to 4 µg/ml, this extract demonstrated concentration-dependent inhibition of G6PD with an IC₅₀ value of I.397 µg/ml. Moreover, the anticancer activity evaluation against HepG2 cells determined Smilax china as a potent inhibitor of cancer cells (IC₅₀ value of 16.017 μg/ml). The acute and subacute toxicities were not observed in mice with various concentrations (50, 100, 200 and 2000 mg/kg). Furthermore, to identify the compounds from Smilax china as G6PD inhibitors, a literature-based phytochemical investigation of Smilax china was conducted, and sixty compounds were docked against the NADP+ and G6P binding sites of G6PD. The results of this study showed that three compounds were Scirpusin A, Smilachinin and Daucosterol with MolDock Score of ?156.832, ?148.215, and ?145.733 respectively, against NADP+ binding site of G6PD. Conclusively, Smilax china root extract could be a safer drug candidate for the treatment of hepatocellular carcinoma.     

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ?stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase–substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.     

Structural analysis revealed a novel conformation of the NTRC reductase domain from Chlamydomonas reinhardtii

In plant chloroplasts, thiol regulation is driven by two systems. One relies on the activity of thioredoxins through their light dependent reduction by ferredoxin via a ferredoxin-thioredoxin reductase (FTR). In the other system, a NADPH-dependent redox regulation is driven by a NADPH-thioredoxin reductase C (NTRC). While the thioredoxin system has been deeply studied, a more thorough understanding of the function of this plant specific NTRC is desirable. NTRC is a single polypeptide harbouring a thioredoxin domain (Trx) at the C-terminus of a NADPH-dependent Thioredoxin reductase (TrxR). To provide functional and structural insights, we studied the crystal structure of the TrxR domain of the NTRC from Chlamydomonas reinhardtii (CrNTRC, Cre01.g054150.t1.2) and its Cys136Ser (C136S) mutant, which is characterized by the mutation of the resolving cysteine in the active site of the TrxR domain. Furthermore, we confirmed the role of NTRC as electron donor for 2-Cys peroxiredoxin (PRX) also in C. reinhardtii. The structural data of TrxR were employed to develop a scheme of action which addresses electron transfer between TrxR and Trx of NTRC and between NTRC and its substrates.     

Battling the biotypes of balsam: the biological control of Impatiens glandulifera using the rust fungus Puccinia komarovii var. glanduliferae in GB

Impatiens glandulifera, or Himalayan balsam, is a prolific invader of riverine habitats. Introduced from the Himalayas for ornamental purposes in 1839, this annual species has naturalised across Great Britain (GB) forming dense monocultures with negative affects across whole ecosystems. In 2006 a programme exploring biocontrol as an alternative control method was initiated and to date, two strains of the rust fungus Puccinia komarovii var. glanduliferae have been released. To better understand the observed differences in susceptibility of GB Himalayan balsam stands to the two rust strains, inoculation studies were conducted using urediniospores and basidiospores. Experiments revealed large variation in the susceptibility of stands to urediniospores of the two rust strains, with some resistant to both. Furthermore, the infectivity of basidiospores was found to differ, with some stands fully susceptible to the urediniospore stage, being immune to basidiospore infection. Therefore, before further rust releases at new sites, it is necessary to ensure complete compatibility of the invasive stands with both urediniospores and basidiospores. However, for successful control across GB it is essential that plant biotypes are matched to the most virulent rust strains. This will involve additional strains from the native range to tackle those biotypes resistant to the strains currently released.     

Human HSPB1 mutation recapitulates features of distal hereditary motor neuropathy (dHMN) in Drosophila

Distal hereditary motor neuropathies (dHMN) are a group of inherited peripheral nerve disorders characterized by length-dependent motor neuron weakness and subsequent muscle atrophy. Missense mutations in the gene encoding small heat shock protein HSPB1 (HSP27) have been associated with hereditary neuropathies including dHMN. HSPB1 is a member of the small heat shock protein (sHSP) family characterized by a highly conserved α-crystallin domain that is critical to their chaperone activity. In this study, we modeled HSPB1 mutant-induced neuropathies in Drosophila using a human HSPB1^S135F mutant that has a missense mutation in its α-crystallin domain. Overexpression of the HSPB1 mutant produced no significant defect in the Drosophila development, however, a partial reduction in the life span was observed. Further, the HSPB1 mutant gene induced an obvious loss of motor activity when expressed in Drosophila neurons. Moreover, suppression of histone deacetylase 6 (HDAC6) expression, which has critical roles in HSPB1 mutant-induced axonal defects, successfully rescued the motor defects in the HSPB1 mutant Drosophila model.     

Family-wide Characterization of Methylated DNA Binding Ability of Arabidopsis MBDs

13 MBD-containing genes (AtMBD1-13) have been identified in Arabidopsis thaliana so far, however, their DNA binding ability is still controversial. Here, we systematically measured the DNA binding affinities of these MBDs by ITC and EMSA binding assays, except for those of pseudogenes AtMBD3 and AtMBD13, and found that only AtMBD6 and AtMBD7 function as methylated DNA readers. We also found that the MBD of AtMBD5 exhibits very weak binding to methylated DNA compared to that of AtMBD6. To further investigate the structural basis of AtMBDs in binding to methylated DNA, we determined the complex structure of the AtMBD6 MBD with a 12mer mCG DNA and the apo structure of the AtMBD5 MBD. Structural analysis coupled with mutagenesis studies indicated that, in addition to the conserved arginine fingers contributing to the DNA binding specificity, the residues located in the loop1 and α1 are also essential for the methylated DNA binding of these MBDs in Arabidopsis thaliana, which explains why AtMBD5 MBD and the other AtMBDs display very weak or no binding to methylated DNA. Thus, our study here systematically demonstrates the DNA binding ability of the MBDs in Arabidopsis thaliana, which also provides a general guideline in understanding the DNA binding ability of the MBDs in other plants as a whole.     

Selective measurement of NAPE-PLD activity via a PLA1/2-resistant fluorogenic N-acyl-phosphatidylethanolamine analog

N-acyl-phosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase enzyme that converts NAPEs to bioactive N-acyl-ethanolamides. Altered NAPE-PLD activity may contribute to pathogenesis of obesity, diabetes, atherosclerosis, and neurological diseases. Selective measurement of NAPE-PLD activity is challenging, however, because of alternative phospholipase pathways for NAPE hydrolysis. Previous methods to measure NAPE-PLD activity involved addition of exogenous NAPE followed by TLC or LC/MS/MS, which are time and resource intensive. Recently, NAPE-PLD activity in cells has been assayed using the fluorogenic NAPE analogs PED-A1 and PED6, but these substrates also detect the activity of serine hydrolase-type lipases PLA₁ and PLA₂. To create a fluorescence assay that selectively measured cellular NAPE-PLD activity, we synthesized an analog of PED-A1 (flame-NAPE) where the sn-1 ester bond was replaced with an N-methyl amide to create resistance to PLA₁ hydrolysis. Recombinant NAPE-PLD produced fluorescence when incubated with either PED-A1 or flame-NAPE, whereas PLA₁ only produced fluorescence when incubated with PED-A1. Furthermore, fluorescence in HepG2 cells using PED-A1 could be partially blocked by either biothionol (a selective NAPE-PLD inhibitor) or tetrahydrolipstatin (an inhibitor of a broad spectrum of serine hydrolase-type lipases). In contrast, fluorescence assayed in HepG2 cells using flame-NAPE could only be blocked by biothionol. In multiple cell types, the phospholipase activity detected using flame-NAPE was significantly more sensitive to biothionol inhibition than that detected using PED-A1. Thus, using flame-NAPE to measure phospholipase activity provides a rapid and selective method to measure NAPE-PLD activity in cells and tissues.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.