Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum.
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ABSTRACT:
SUBMITTER: Guo Q
PROVIDER: S-EPMC8126025 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mesembryanthemum crystallinum (common ice plant) is a halophyte species that has adapted to extreme conditions. In this study, we cloned a McHB7 transcription factor gene from the ice plant. The expression of McHB7 was significantly induced by 500 mM NaCl and it reached the peak under salt treatment for 7 days. The McHB7 protein was targeted to the nucleus. McHB7-overexpressing in ice plant leaves through Agrobacterium-mediated transformation led to 25 times more McHB7 transcripts than the non-transformed wild type (WT). After 500 mM NaCl treatment for 7 days, the activities of superoxide dismutase (SOD) and peroxidase (POD) and water content of the transgenic plants were higher than the WT, while malondialdehyde (MDA) was decreased in the transgenic plants. A total of 1082 and 1072 proteins were profiled by proteomics under control and salt treatment, respectively, with 22 and 11 proteins uniquely identified under control and salt stress, respectively. Among the 11 proteins, 7 were increased and 4 were decreased after salt treatment. Most of the proteins whose expression increased in the McHB7 overexpression (OE) ice plants under high salinity were involved in transport regulation, catalytic activities, biosynthesis of secondary metabolites, and response to stimulus. The results demonstrate that the McHB7 transcription factor plays a positive role in improving plant salt tolerance.
| S-EPMC8232686 | biostudies-literature
Project description:The increased expression of McPIP2;1 (MipC), a root-specific aquaporin (AQP) from Mesembryanthemum crystallinum, under salt stress has suggested a role for this AQP in the salt tolerance of the plant. However, whether McPIP2;1 transports water or another solute and how its activity is regulated are so far unknown. Therefore, wild type (wt) or mutated McPIP2;1 protein was expressed in Xenopus laevis oocytes. Then, the osmotic water permeability (P(f)) of the oocytes membrane was assessed by hypotonic challenges. Selectivity of McPIP2;1 to water was determined by radiolabeled glycerol or urea uptake assays. Moreover, swelling and in vitro phosphorylation assays revealed that both water permeation and phosphorylation status of McPIP2;1 were significantly increased by the phosphorylation agonists okadaic acid (OA), phorbol myristate acetate (PMA), and 8-Br-cAMP, and markedly decreased by the inhibitory peptides PKI 14-22 and PKC 20-28, inhibitors of protein kinases A (PKA) and C (PKC), respectively. Substitution of Ser(123) or both, Ser(123) and Ser(282), abolished the water channel activity of McPIP2;1 while substitution of Ser(282) only partially inhibited it (51.9% inhibition). Despite lacking Ser(123) and/or Ser(282), the McPIP2;1 mutant forms were still phosphorylated in vitro, which suggests that phosphorylation may have a dual role on this AQP. Our results indicate that McPIP2;1 water permeability depends completely on Ser(123) and is positively regulated by PKA- and PKC-mediated phosphorylation. Regulation of the phosphorylation status of McPIP2;1 may contribute to control water transport through root cells when the plant is subjected to high salinity conditions.
| S-EPMC2878089 | biostudies-literature
Project description:One of the remarkable adaptive features of the halophyte Mesembryanthemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF) and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples identified 352 significantly differing metabolites (268 after correction for FDR). Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na(+) and Cl(-) ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggests large alterations in M. crystallinum epidermal bladder cells.
| S-EPMC4462104 | biostudies-literature
Project description:Salt stress impedes plant growth and development, and leads to yield loss. Recently, a halophyte species Mesembryanthemum crystallinum has become a model to study plant photosynthetic responses to salt stress. It has an adaptive mechanism of shifting from C3 photosynthesis to crassulacean acid metabolism (CAM) photosynthesis under stresses, which greatly enhances water usage efficiency and stress tolerance. In this study, we focused on investigating the morphological and physiological changes [e.g., leaf area, stomatal movement behavior, gas exchange, leaf succulence, and relative water content (RWC)] of M. crystallinum during the C3 to CAM photosynthetic transition under salt stress. Our results showed that in M. crystallinum seedlings, CAM photosynthesis was initiated after 6 days of salt treatment, the transition takes place within a 3-day period, and plants became mostly CAM in 2 weeks. This result defined the transition period of a facultative CAM plant, laid a foundation for future studies on identifying the molecular switches responsible for the transition from C3 to CAM, and contributed to the ultimate goal of engineering CAM characteristics into C3 crops.
| S-EPMC7090145 | biostudies-literature
Project description:BackgroundIce plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. However, no further functional characterization of these genes in host plant due to lack of easy and effective transformation protocols.ResultsTo establish efficient transformation system of ice plants, three types of ice plant materials, hypocotyl-derived callus, aseptically-grown seedlings and pot-grown juvenile plants, were used to develop Agrobacterium-mediated transformation protocols. The highest transient transformation efficiency was with 5-day-old ice plant callus co-incubated with an Agrobacterium tumefaciens at 2.5 × 109 cells mL-1 for 48 h. The 3-day-old ice plant seedlings with root tip removed were successfully infected with A. tumefaciens or A. rhizogenes, and obtained 85% and 33-100% transient transformation rates, respectively. The transient transformation assays in ice plant callus and seedlings demonstrated that the concentrations of Agrobacteria, the durations of co-incubation time, and the plant growth stages were three important factors affecting the transient transformation efficiencies. Additionally, pot-grown juvenile plants were syringe-injected with two A. rhizogenes strains A8196 and NCPPB 1855, to establish transformed roots. After infections, ice plants were grown hydroponically and showed GUS expressions in transformed roots for 8 consecutive weeks.ConclusionsOur Agrobacterium-mediated transformation protocols utilized hypocotyl-derived callus and seedlings as plant materials, which can be easily obtained in large quantity. The average successful transient transformation rates were about 2.4-3.0% with callus and 33.3-100.0% with seedlings. We also developed a rapid and efficient protocol to generate transgenic roots by A. rhizogenes infections without laborious and challenging tissue culture techniques. This protocol to establish composite ice plant system demonstrates excellent improvements in efficiency, efficacy, and ease of use over previous ice plant transformation protocols. These Agrobacterium-mediated transformation protocols can be versatile and efficient tools for exploring gene functions at cellular and organ levels of ice plants.
| S-EPMC6323063 | biostudies-literature
Project description:BackgroundMyalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a serious and complex physical illness that affects all body systems with a multiplicity of symptoms, but key hallmarks of the disease are pervasive fatigue and 'post-exertional malaise', exacerbation after physical and/or mental activity of the intrinsic fatigue and other symptoms that can be highly debilitating and last from days to months. Although the disease can vary widely between individuals, common symptoms also include pain, cognitive deficits, sleep dysfunction, as well as immune, neurological and autonomic symptoms. Typically, it is a very isolating illness socially, carrying a stigma because of the lack of understanding of the cause and pathophysiology.MethodsTo gain insight into the pathophysiology of ME/CFS, we examined the proteomes of peripheral blood mononuclear cells (PBMCs) by SWATH-MS analysis in a small well-characterised group of patients and matched controls. A principal component analysis (PCA) was used to stratify groups based on protein abundance patterns, which clearly segregated the majority of the ME/CFS patients (9/11) from the controls. This majority subgroup of ME/CFS patients was then further compared to the control group.ResultsA total of 60 proteins in the ME/CFS patients were differentially expressed (P < 0.01, Log10 (Fold Change) > 0.2 and < -0.2). Comparison of the PCA selected subgroup of ME/CFS patients (9/11) with controls increased the number of proteins differentially expressed to 99. Of particular relevance to the core symptoms of fatigue and post-exertional malaise experienced in ME/CFS, a proportion of the identified proteins in the ME/CFS groups were involved in mitochondrial function, oxidative phosphorylation, electron transport chain complexes, and redox regulation. A significant number were also involved in previously implicated disturbances in ME/CFS, such as the immune inflammatory response, DNA methylation, apoptosis and proteasome activation.ConclusionsThe results from this study support a model of deficient ATP production in ME/CFS, compensated for by upregulation of immediate pathways upstream of Complex V that would suggest an elevation of oxidative stress. This study and others have found evidence of a distinct pathology in ME/CFS that holds promise for developing diagnostic biomarkers.
| S-EPMC7512220 | biostudies-literature
Project description:Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC-MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC-MS/MS using a 1?×?150?mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.5% CV) using data from >2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16?h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16?h. The system identifies >30,000 phosphopeptides in 12?h and protein-protein or protein-drug interaction experiments can be analyzed in 20?min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC-MS/MS is suitable for a broad range of proteomic applications.
| S-EPMC6952431 | biostudies-literature
Project description:Mesembryanthemum crystallinum (common ice plant) is one of the facultative halophyte plants, and it serves as a model for investigating the molecular mechanisms underlying its salt stress response and tolerance. Here we cloned one of the homeobox transcription factor (TF) genes, McHB7, from the ice plant, which has 60% similarity with the Arabidopsis AtHB7. Overexpression of the McHB7 in Arabidopsis (OE) showed that the plants had significantly elevated relative water content (RWC), chlorophyll content, superoxide dismutase (SOD), and peroxidase (POD) activities after salt stress treatment. Our proteomic analysis identified 145 proteins to be significantly changed in abundance, and 66 were exclusively increased in the OE plants compared to the wild type (WT). After salt treatment, 979 and 959 metabolites were significantly increased and decreased, respectively, in the OE plants compared to the WT. The results demonstrate that the McHB7 can improve photosynthesis, increase the leaf chlorophyll content, and affect the TCA cycle by regulating metabolites (e.g., pyruvate) and proteins (e.g., citrate synthase). Moreover, McHB7 modulates the expression of stress-related proteins (e.g., superoxide dismutase, dehydroascorbate reductase, and pyrroline-5-carboxylate synthase B) to scavenge reactive oxygen species and enhance plant salt tolerance.
| S-EPMC9318261 | biostudies-literature
Project description:Understanding the molecular mechanisms that convey salt tolerance in plants is a crucial issue for increasing crop yield. The ice plant (Mesembryanthemum crystallinum) is a halophyte that is capable of growing under high salt conditions. For example, the roots of ice plant seedlings continue to grow in 140 mM NaCl, a salt concentration that completely inhibits Arabidopsis thaliana root growth. Identifying the molecular mechanisms responsible for this high level of salt tolerance in a halophyte has the potential of revealing tolerance mechanisms that have been evolutionarily successful. In the present study, deep sequencing (RNAseq) was used to examine gene expression in ice plant roots treated with various concentrations of NaCl. Sequencing resulted in the identification of 53,516 contigs, 10,818 of which were orthologs of Arabidopsis genes. In addition to the expression analysis, a web-based ice plant database was constructed that allows broad public access to the data. The results obtained from an analysis of the RNAseq data were confirmed by RT-qPCR. Novel patterns of gene expression in response to high salinity within 24 hours were identified in the ice plant when the RNAseq data from the ice plant was compared to gene expression data obtained from Arabidopsis plants exposed to high salt. Although ABA responsive genes and a sodium transporter protein (HKT1), are up-regulated and down-regulated respectively in both Arabidopsis and the ice plant; peroxidase genes exhibit opposite responses. The results of this study provide an important first step towards analyzing environmental tolerance mechanisms in a non-model organism and provide a useful dataset for predicting novel gene functions.
| S-EPMC4338230 | biostudies-literature
Project description:This study identified the constituents of purified flavonoid (PEF) isolated from Mesembryanthemum crystallinum and examined their inhibitory effects on low-density lipoprotein (LDL) oxidation and non-enzymatic glycosylation. More than 30 kinds of flavonoid compounds were identified in M. crystallinum, including tangeretin, nobiletin, farrerol, protocatechuic aldehyde, diosmin, and rutin. Moreover, tangeretin corresponds to approximately 51% of the total identified flavonoids. PEF had a low IC50 value for 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH·), hydroxyl radical (·OH), and superoxide anion free radical (O
| S-EPMC9521712 | biostudies-literature