Journal: BMC Plant Biology

Article Title: Transcript profiling of plastid ferrochelatase two mutants reveals that chloroplast singlet oxygen signals lead to global changes in RNA profiles and are mediated by Plant U-Box 4

doi: 10.1186/s12870-025-06703-7

Figure Lengend Snippet: Jasmonic acid biosynthesis and signaling are induced in fc2 under cycling light conditions. The impact of singlet oxygen ( 1 O 2 ) on jasmonic acid (JA) biosynthesis and signaling was assessed in constant light (24 h) and 16 h light/8 h dark diurnal (cycling) light conditions. ( a ) Schematic illustration of the JA biosynthesis pathway, with relative expression (compared to wt in the same light condition) of 11 key JA biosynthesis and signaling genes. Expression data is from the DESeq2 analysis of the included RNA-seq data set. Error bars in the graph represent standard error (ifcSE) estimated by DESeq2 (**, padj < 0.01; ***, padj < 0.001). In chloroplasts, Defective In Anther Dehiscence 1 (DAD1) catalyzes the conversion of galactolipids into α-linolenic acid (α-LeA), which is converted into 12-oxo-phytodienoic acid (OPDA) by a series of enzymes; 13-Lipoxygenase (LOX), Allene Oxide Synthase (AOS), and Allene Oxide Cyclase (AOC). Subsequently, OPDA is exported out of the chloroplast via the channel protein JASSY. Inside the peroxisome, OPDA is reduced by the OPDA reductase 3 (OPR3) and shortened in the carboxylic acid side chain by β-oxidation enzymes (ACX1) into JA. In the cytosol, Jasmonate Resistant 1 (JAR1) conjugates isoleucine to JA converting it into JA-Ile. Jasmonate Transporter 1 (JAT1) transports JA-Ile into the nucleus. In response to stressed conditions, JA-Ile attach with the COI1-SCF (coronatine insensitive1- Skp1-Cul1-F-box), to promote ubiquitination and degradation of jasmonic acid repressors JAZ and JAV1, thereby activating JA response genes. ( b ) Graph showing JA content (ng/g fresh weight (FW)) measured in whole rosettes from plants grown for 19 days constant (24 h) light conditions or 17 days in 24 h light conditions and two days of 16 h light/8 h dark diurnal (cycling) light conditions. Values are means ± SEM ( n = 3 biological replicates). Statistical analyses in b were performed using one-way ANOVA tests, and the different letters above the bars indicate significant differences within data sets determined by Tukey–Kramer post-tests ( P ≤ 0.05). Separate analyses were performed for the different light treatments, and the significance of cycling light treatment is denoted by letters with a prime symbol (ʹ). ( c ) Heatmap showing relative expression of 58 genes from gene ontology term “JA mediated signaling pathway” (GO:0009867). The blue and red colors correspond to low and high gene expression, respectively

Article Snippet: Polyclonal antibodies for NAD(P)H dehydrogenase subunit 45 (NDH45), Thylakoid Membrane Cytochrome B6 Protein (PetB), ATP Synthase Gamma Chain (AtpC), and Rubisco large subunit (RbcL) were purchased from PhytoAB (California, USA).

Techniques: Expressing, RNA Sequencing, Ubiquitin Proteomics, Gene Expression

Journal: Biochemical Journal

Article Title: A pipeline to evaluate inhibitors of the Pseudomonas aeruginosa exotoxin U

doi: 10.1042/BCJ20200780

Figure Lengend Snippet: ( A ) Left: His-tagged ExoU was purified from C43(DE3) E. coli . Immobilised metal affinity chromatography (IMAC) purified His-ExoU, including two major contaminant proteins, were resolved by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and identified by employing mass spectrometry. Right: His-ExoU was further purified to homogeneity by size-exclusion chromatography; 5 µg of His-ExoU was resolved and visualised by SDS–PAGE. ( B ) The hydrolysis of arachidonoyl Thio-PC substrate by ExoU was assessed in the presence of 10 µM of the indicated compound. To each reaction, ubiquitin and PIP 2 were added in order to allow induction of ExoU phospholipase activity. Experiments were performed in triplicate, the results represent means, and error bars represent standard deviations and representative profiles are shown of substrate conversion as a function of absorbance with the progression of time (QD = quinacrine dihydrochloride and OP = oleyoxylethyl phosphoryl choline). ( C ) Chemical structures of prospective compounds that are proposed to inhibit ExoU mediated toxicity in cells. IC 50 values are shown.

Article Snippet: Substrate arachidonoyl thio-phosphatidylcholine (ATPC) was purchased from Cayman Chemical as an ethanolic solution.

Techniques: Purification, Affinity Chromatography, Polyacrylamide Gel Electrophoresis, SDS Page, Mass Spectrometry, Size-exclusion Chromatography, Ubiquitin Proteomics, Activity Assay