1,721,052 research outputs found
Large-scale proteome analysis of tomato fruit microsomes
No full textSolanum lycopersicum (tomato) is one of the most important
plant crops and a model system to study development and ripening
of fleshy fruit. The membrane system is very important for
all biological processes; for example, the endoplasmic reticulum
(ER) and the Golgi apparatus play a pivotal role in the secretion
of proteins and in the synthesis of the non-cellulosic portion of
the cell wall. The aim of this work was to characterize the membrane
proteome profile of tomato fruit at the ‘‘mature green’’ ripening
stage. Total microsomes were prepared and separated by
centrifugation through a iodixanol continuous gradient. The
organelle distribution pattern (plasma membrane, ER, Golgi,
chloroplast, nucleus) was determined using known markers of
these compartments. Proteins were identified using a combination
of 1-D SDS polyacrylamide gel electrophoresis and nanoLC-ESIMS/
MS. After electrophoresis, each lane was cut into 14 pieces,
digested with trypsin and analyzed by nano-HPLC coupled to an
Orbitrap mass spectrometer. More than 2400 different proteins
were identified, for which the Arabidopsis homologue was
searched and subjected to GO analysis to determine the represented
biological process, molecular function and subcellular
localization. GO term enrichment analysis confirmed the enrichment
in membrane proteins. This large scale proteomic analysis
provides a detailed reference map of the membrane proteome at
specific stages of tomato fruit development and a background for
comparison of physiological processes such as ripening or biotic
and abiotic stresses
Comprehensive analysis of the membrane phosphoproteome regulated by oligogalacturonides in Arabidopsis thaliana
Full text linkEarly changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity
Proteomic insights into oligogalacturonide signalling in plant defence and development
No full textOligogalacturonides (OGs) are fragments derived from the hydrolysis of plant cell wall homogalacturonan by fungal endo-polygalacturonases and are well known damage-associated molecular patterns (DAMPs). In Arabidopsis thaliana, perception of OGs triggers an intracellular signaling cascade that initiates defense against pathogens. OGs may also work as regulators of plant growth and development mainly through their antagonism with auxin (IAA). However, most of the mechanism by which the OG signal is transduced is not yet known.
In this study, quantitative phosphoproteomics was applied to investigate the initial signaling events specifically activated by OG signaling. Analysis of quantitative changes in the proteome and phosphoproteome, determined by dimethyl labelling and LC/MSMS analysis in a membrane-enriched fraction, revealed rapid phosphorylation changes, with no substantial changes in overall protein abundance after a 10 min treatment with OGs.
We also analyzed the changes in the nuclear proteome in response to IAA, OGs or a IAA/OG co-treatment, to identify regulatory elements that mediate the inhibition of the auxin-induced responses by OGs. The analysis of proteomic and transcriptomic data suggests that changes in abundance of nuclear proteins in response to OGs/IAA may arise not only from enhanced or reduced expression but also from post-translational modifications, degradation and/or translocation of proteins to different compartments. Some interesting candidates in OG/IAA antagonism have been identified and their functional role is under study
Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana
Full text linkEarly changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity
Phosphoproteomics of early oligogalacturonides signaling in Arabidopsis
No full textDuring the infection of a plant tissue by phytopathogenic fungi,
homogalacturonan, the main component of pectin, is broken down into fragments called oligogalacturonides (OGs) by the
action of fungal polygalacturonases. The interaction between polygalacturonases
and their inhibitors (polygalacturonase-inhibiting
proteins: PGIPs) in the plant cell wall favours the formation
of OGs with degree of polymerization from 10 to 15 that function
as signals for the activation of the plant innate immune
response. Although the effects of OGs in plant defence are well
recognised, the perception/transduction mechanisms of these elicitors
are still not completely described. By a 2D-DIGE approach
coupled with the quantitative phospho-specific stain ProQ Diamond
we studied the early events of phosphorylation in Arabidopsis
thaliana in response to OGs. Soluble proteins and prefractionated
total membranes were analyzed and the differential
phosphorylated proteins were identified by MALDI-ToF mass
spectrometry. Phosphorylation changes were detected in several
oxidative stress-related proteins that included heat shock proteins,
jasmonate-inducible jacalin proteins and H+-ATPases,
among which the vacuolar-H+ATPase encoded by DET3 gene.
Phosphorilation changes were also detected in plasma membrane
proteins like AtPcaP1, a plasma membrane-bounded protein
involved in the cross-talk between Ca+2 signaling and PtdInsPs
in the intracellular signaling pathway
Polygalacturonases, polygalacturonase-inhibiting proteins and pectic oligomers in plant-pathogen interactions
No full textPolygalacturonases (PGs) are produced by fungal pathogens during early plant infection and are believed to be important pathogenicity
factors. Polygalacturonase-inhibiting proteins (PGIPs) are plant defense proteins which reduce the hydrolytic activity of endoPGs and favor
the accumulation of long-chain oligogalacturonides (OGs) which are elicitors of a variety of defense responses. PGIPs belong to the
superfamily of leucine reach repeat (LRR) proteins which also include the products of several plant resistance genes. A number of evidence
demonstrates that PGIPs efficiently inhibit fungal invasion
Proteomic analysis of endoplasmic reticulum and golgi proteins during tomato fruit ripening.
No full textGene Encodes a Novel Functional Pectin Methylesterase Inhibitor Associated to Grape Berry Development
No full textPectin is secreted in a highly methylesterified form and partially de-methylesterified in the cell wall by pectin methylesterases (PMEs). PME activity is expressed during plant growth, development and stress responses. PME activity is controlled at the post-transcriptional level by proteins named PME inhibitors (PMEIs). We have identified, expressed and characterized VvPMEI1, a functional PME inhibitor of Vitis vinifera. VvPMEI1 typically affects the activity of plant PMEs and is inactive against microbial PMEs. The kinetics of PMEI-PME interaction, studied by surface plasmon resonance, indicates that the inhibitor strongly interacts with PME at apoplastic pH while the stability of the complex is reduced by increasing the pH. The analysis of VvPMEI1 expression in different grapevine tissues and during grape fruit development suggests that this inhibitor controls PME activity mainly during the earlier phase of berry development. A proteomic analysis performed at this stage indicates a PME isoform as possible target of VvPMEI1
The interaction between endopolygalacturonase from Fusarium moniliforme and PGIP from Phaseolus vulgaris studied by surface plasmon resonance and mass spectrometry
Full text linkA combination of surface plasmon resonance (SPR) and matrix-assisted laser-desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) was used to study the interaction between endopolygalacturonase (PG) from Fusarium moniliforme and a polygalacturonase-inhibiting protein (PGIP) from Phaseolus vulgaris. PG hydrolyses the homogalacturonan of the plant cell wall and is considered an important pathogenicity factor of many fungi. PGIP is a specific inhibitor of fungal PGs and is thought to be involved in plant defence against phytopathogenic fungi. SPR was used either to study the effect of the PG glycosylation on the formation of the complex with PGIP, and as a sensitive affinity capture of an interacting peptide from a mixture of PG fragments obtained by limited proteolysis. Mass spectrometry allowed to characterise the interacting peptide eluted from the sensor surface. Copyright © 2001 John Wiley & Sons, Ltd
- …
