Ascorbate deficiency influences the leaf cell wall glycoproteome in Arabidopsis thaliana

© 2014 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.The cell wall forms the first line of interaction between the plant and the external environment. Based on the observation that ascorbate-deficient vtc mutants of Arabidopsis thaliana have increased cell wall peroxidase activity, the cell wall glycoproteome of vtc2-2 was investigated. Glycoproteins were purified from fully expanded leaves by Concanavalin A affinity chromatography and analysed by liquid chromatography quadrupole time-of-flight mass spectrometry. This procedure identified 63 proteins with predicted glycosylation sites and cell wall localization. Of these, 11 proteins were differentially expressed between vtc2-2 and wild type. In particular, PRX33/34 were identified as contributing to increased peroxidase activity in response to ascorbate deficiency. This is the same peroxidase previously shown to contribute to hydrogen peroxide generation and pathogen resistance. Three fasciclin-like arabinogalactan proteins (FLA1, 2 and 8) had lower abundance in vtc2-2. Inspection of published microarray data shows that these also have lower gene expression in vtc1 and vtc2-1 and are decreased in expression by pathogen challenge and oxidative stresses. Ascorbate deficiency therefore impacts expression of cell wall proteins involved in pathogen responses and these presumably contribute to the increased resistance of vtc mutants to biotrophic pathogens.Hazara University (Mansehra, NWFP, Pakistan)Higher Education Commission (Pakistan)BBSRCExeter University Science Strategy Fun

The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana: further evidence for redox control of anthocyanin synthesis

PublishedArticleThis is the peer reviewed version of the following article: AGE, M., SULTANA, N., PASZKIEWICZ, K., FLORANCE, H. and SMIRNOFF, N. (2012), The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana: further evidence for redox control of anthocyanin synthesis. Plant, Cell & Environment, 35: 388–404. , which has been published in final form at http://dx.doi.org/10.1111/j.1365-3040.2011.02369. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Ascorbate and anthocyanins act as photoprotectants during exposure to high light (HL). They accumulate in Arabidopsis leaves in response to HL on a similar time-scale, suggesting a potential relationship between them. Flavonoids and related metabolites were identified and profiled by LC-MS/MS. The ascorbate deficient mutants vtc1, vtc2 and vtc3 accumulated less anthocyanin than wild-type during HL acclimation. In contrast, kaempferol glycoside accumulation was less affected by light and not decreased by ascorbate deficiency, while sinapoyl malate levels decreased during HL acclimation. Comparison of six Arabidopsis ecotypes showed a positive correlation between ascorbate and anthocyanin accumulation in HL. mRNA-Seq analysis showed that all flavonoid biosynthesis transcripts were increased by HL acclimation in wild-type. RT-PCR analysis showed that vtc1 and vtc2 were impaired in HL induction of transcripts of anthocyanin biosynthesis enzymes, and the transcription factors PAP1, GL3 and EGL3 that activate the pathway. Abscisic acid and jasmonic acid, hormones that could affect anthocyanin accumulation, were unaffected in vtc mutants. It is concluded that HL induction of anthocyanin synthesis involves a redox-sensitive process upstream of the known transcription factors. Because anthocyanins accumulate in preference to kaempferol glycosides and sinapoyl malate in HL, they might have specific properties that make them useful in high light acclimation.Biotechnology and Biological SciencesResearch Council (BBSRC)Exeter University ScienceStrategy FundHazara UniversityMansehra (NWFP) PakistanHigher Education Commission (Pakistan

Rapid HILIC-Z ion mobility mass spectrometry (RHIMMS) method for untargeted metabolomics of complex biological samples

INTRODUCTION: Recent advances in high-throughput methodologies in the ‘omics’ and synthetic biology fields call for rapid and sensitive workflows in the metabolic phenotyping of complex biological samples. OBJECTIVE: The objective of this research was to evaluate a straightforward to implement LC–MS metabolomics method using a commercially available chromatography column that provides increased throughput. Reducing run time can potentially impact chromatography and therefore the effects of ion mobility spectrometry to expand peak capacity were also evaluated. Additional confidence provided via collision cross section measurements for detected features was also explored. METHODS: A rapid untargeted metabolomics workflow was developed with broad metabolome coverage, combining zwitterionic-phase hydrophilic interaction chromatography (HILIC-Z) with drift tube ion mobility-quadrupole time-of-flight (DTIM-qTOF) mass spectrometry. The analytical performance of our method was explored using extracts from complex biological samples, including a reproducibility study on chicken serum and a simple comparative study on a bacterial metabolome. RESULTS: The method is acronymised RHIMMS for rapid HILIC-Z ion mobility mass spectrometry. We present the RHIMMS workflow starting with data acquisition, followed by data processing and analysis. RHIMMS demonstrates improved chromatographic separation for a selection of metabolites with wide physicochemical properties while maintaining reproducibility at better than 20% over 200 injections at 3.5 min per sample for the selected metabolites, and a mean of 13.9% for the top 50 metabolites by intensity. Additionally, the combination of rapid chromatographic separation with ion mobility allows improved annotation and the ability to distinguish isobaric compounds. CONCLUSION: Our results demonstrate RHIMMS to be a rapid, reproducible, sensitive and high-resolution analytical platform that is highly applicable to the untargeted metabolomics analysis of complex samples. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11306-022-01871-1

A low-molecular-weight alginate oligosaccharide disrupts pseudomonal microcolony formation and enhances antibiotic effectiveness

In chronic respiratory disease the formation of dense, 3-dimensional ‘micro colonies' by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial sputum (AS) medium was established to study the effects of low molecular weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates (n=3) and reference non-mucoid and mucoid multi-drug resistant (MDR) CF isolates (n=7). Bacterial growth, biofilm development and disruption were studied using cell-viability assays and image analysis using scanning electron- and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production (p10 μm) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 μg/ml) in AS medium versus. In contrast, in an established biofilm model in the AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (≥2%) treatment however, induced dose-dependent biofilm disruption (p0.2%; p<0.05) reductions in pseudomonal quorum sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung, and highlight a novel approach to treat MDR pseudomonal infections

Effects of Glyphosate and its Formulation, Roundup, on Reproduction in Zebrafish (Danio rerio)

This is an open access article that is freely available in ORE or from the publisher's web site. Please cite the published version.Copyright © 2014 American Chemical SocietyRoundup and its active ingredient glyphosate are among the most widely used herbicides worldwide and may contaminate surface waters. Research suggests both Roundup and glyphosate induce oxidative stress in fish and may also cause reproductive toxicity in mammalian systems. We aimed to investigate the reproductive effects of Roundup and glyphosate in fish and the potential associated mechanisms of toxicity. To do this, we conducted a 21-day exposure of breeding zebrafish (Danio rerio) to 0.01, 0.5, and 10 mg/L (glyphosate acid equivalent) Roundup and 10 mg/L glyphosate. 10 mg/L glyphosate reduced egg production but not fertilization rate in breeding colonies. Both 10 mg/L Roundup and glyphosate increased early stage embryo mortalities and premature hatching. However, exposure during embryogenesis alone did not increase embryo mortality, suggesting that this effect was caused primarily by exposure during gametogenesis. Transcript profiling of the gonads revealed 10 mg/L Roundup and glyphosate induced changes in the expression of cyp19a1 and esr1 in the ovary and hsd3b2, cat, and sod1 in the testis. Our results demonstrate that these chemicals cause reproductive toxicity in zebrafish, although only at high concentrations unlikely to occur in the environment, and likely mechanisms of toxicity include disruption of the steroidogenic biosynthesis pathway and oxidative stress.Natural Environment Research Counci

The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana: further evidence for redox control of anthocyanin synthesis

Ascorbate and anthocyanins act as photoprotectants during exposure to high light (HL). They accumulate in Arabidopsis leaves in response to HL on a similar timescale, suggesting a potential relationship between them. Flavonoids and related metabolites were identified and profiled by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The ascorbate-deficient mutants vtc1, vtc2 and vtc3 accumulated less anthocyanin than wild-type (WT) during HL acclimation. In contrast, kaempferol glycoside accumulation was less affected by light and not decreased by ascorbate deficiency, while sinapoyl malate levels decreased during HL acclimation. Comparison of six Arabidopsis ecotypes showed a positive correlation between ascorbate and anthocyanin accumulation in HL. mRNA-Seq analysis showed that all flavonoid biosynthesis transcripts were increased by HL acclimation in WT. RT-PCR analysis showed that vtc1 and vtc2 were impaired in HL induction of transcripts of anthocyanin biosynthesis enzymes, and the transcription factors PAP1, GL3 and EGL3 that activate the pathway. Abscisic acid (ABA) and jasmonic acid (JA), hormones that could affect anthocyanin accumulation, were unaffected in vtc mutants. It is concluded that HL induction of anthocyanin synthesis involves a redox-sensitive process upstream of the known transcription factors. Because anthocyanins accumulate in preference to kaempferol glycosides and sinapoyl malate in HL, they might have specific properties that make them useful in HL acclimation

Aligning extracted LC-MS peak lists via density maximization

Rapid improvements in mass spectrometry sensitivity and mass accuracy combined with improved liquid chromatography separation technologies allow acquisition of high throughput metabolomics data, providing an excellent opportunity to understand biological processes. While spectral deconvolution software can identify discrete masses and their associated isotopes and adducts, the utility of metabolomic approaches for many statistical analyses such as identifying differentially abundant ions depends heavily on data quality and robustness, especially, the accuracy of aligning features across multiple biological replicates. We have developed a novel algorithm for feature alignment using density maximization. Instead of a greedy iterative, hence local, merging strategy, which has been widely used in the literature and in commercial applications, we apply a global merging strategy to improve alignment quality. Using both simulated and real data, we demonstrate that our new algorithm provides high map (e.g. chromatogram) coverage, which is critically important for non-targeted comparative metabolite profiling of highly replicated biological datasets

Alternative splicing determines sensitivity of murine calcium-activated potassium channels to glucocorticoids

Large-conductance Ca2+- and voltage-activated potassium (BK) channels are important regulators of cellular excitability. Here, we present a patch-clamp electrophysiological analysis of splice-variant-specific regulation by the synthetic glucocorticoid dexamethasone (DEX) of BK channels consisting of cloned STREX or ZERO α-subunit variants expressed in human embryonic kidney (HEK 293) cells.STREX channels in isolated membrane patches were inhibited by protein kinase A (PKA) and this was blocked on pre-treatment of intact cells with DEX (100 nm) for 2 h.The effect of DEX required the synthesis of new mRNA and protein. Furthermore, it required protein phosphatase 2A (PP2A)-like activity intimately associated with the channels, as it was blocked by 10 nm okadaic acid but not by the specific protein phosphatase-1 inhibitor peptide PPI−2.ZERO variant channels that lack the STREX insert were activated by PKA but were not influenced by DEX. ZERO channels containing a mutant STREX domain (S4STREXA) were also activated by PKA. Importantly, DEX blocked PKA activation of S4STREXA channels in a PP2A-dependent manner.Taken together, the STREX domain is crucial for glucocorticoid regulation of BK channels through a PP2A-type enzyme. Moreover, glucocorticoids appear to induce a generic set of proteins in different types of cells, the actions of which depend on the expression of cell-specific targets