The assessment of enriched apoplastic extracts using proteomic approaches

In plant tissues the extracellular environment or apoplast, incorporating the cell wall, is a highly dynamic compartment with a role in many important plant processes including defence, development, signalling and assimilate partitioning. Soluble apoplast proteins from Arabidopsis thaliana, Triticum aestivum and Oryza sativa were separated by two‐dimensional electrophoresis. The molecular weights and isoelectric points for the dominant proteins were established prior to excision, sequencing and identification by matrix‐assisted laser‐desorption ionisation time of flight mass spectrometry (MALDI ‐ TOF MS). From the selected spots, 23 proteins from O. sativa and 25 proteins from A. thaliana were sequenced, of which nine identifications were made in O. sativa (39%) and 14 in A. thaliana (56%). This analysis revealed that: (i) patterns of proteins revealed by two‐dimensional electrophoresis were different for each species indicating that speciation could occur at the level of the apoplast, (ii) of the proteins characterised many belonged to diverse families reflecting the multiple functions of the apoplast and (iii), a large number of the apoplast proteins could not be identified indicating that the majority of extracellular proteins are yet to be assigned. The principal proteins identified in the aqueous matrix of the apoplast were involved in defence, i.e. germin‐like proteins or glucanases, and cell expansion, i.e. β‐D‐glucan glucohydrolases. This study has demonstrated that proteomic analysis can be used to resolve the apoplastic protein complement and to identify adaptive changes induced by environmental effectors.     

Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins

Xylem plays a major role in plant development and is considered part of the apoplast. Here, we studied the proteome of Brassica oleracea cv Bartolo and compared it to the plant cell wall proteome of another Brassicaceae, the model plant Arabidopsis thaliana. B. oleracea was chosen because it is technically difficult to harvest enough A. thaliana xylem sap for proteomic analysis. We studied the whole proteome and an N-glycoproteome obtained after Concanavalin A affinity chromatography. Altogether, 189 proteins were identified by LC-MS/MS using Brassica EST and cDNA sequences. A predicted signal peptide was found in 164 proteins suggesting that most proteins of the xylem sap are secreted. Eighty-one proteins were identified in the N-glycoproteome, with 25 of them specific of this fraction, suggesting that they were concentrated during the chromatography step. All the protein families identified in this study were found in the cell wall proteomes. However, proteases and oxido-reductases were more numerous in the xylem sap proteome, whereas enzyme inhibitors were rare. The origin of xylem sap proteins is discussed. All the experimental data including the MS/MS data were made available in the WallProtDB cell wall proteomic database.     

A peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-elicited defense

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.     

Digging deeper into the plant cell wall proteome

The proteome of the plant cell wall/apoplast is less well characterized than those of other subcellular compartments. This largely reflects the many technical challenges involved in extracting and identifying extracellular proteins, many of which resist isolation and identification, and in capturing a population that is both comprehensive and relatively uncontaminated with intracellular proteins. However, a range of disruptive techniques, involving tissue homogenization and subsequent sequential extraction and non-disruptive approaches has been developed. These approaches have been complemented more recently by other genome-scale screens, such as secretion traps that reveal the genes encoding proteins with N-terminal signal peptides that are targeted to the secretory pathway, many of which are subsequently localized in the wall. While the size and complexity of the wall proteome is still unresolved, the combination of experimental tools and computational prediction is rapidly expanding the catalog of known wall-localized proteins, suggesting the unexpected extracellular localization of other polypeptides and providing the basis for further exploration of plant wall structure and function.     

The cytosolic, cell surface and extracellular proteomes of the biotechnologically important soil bacterium Corynebacterium efficiens YS-314 in comparison to those of Corynebacterium glutamicum ATCC 13032

Reference maps of the cytosolic, cell surface and extracellular proteome fractions of the amino acid-producing soil bacterium Corynebacterium efficiens YS-314 were established. The analysis window covers a pI range from 3 to 7 along with a molecular mass range from 10 to 130 kDa. After second-dimensional separation on SDS-PAGE and Coomassie staining, computational analysis detected 635 protein spots in the cytosolic proteome fraction, whereas 76 and 102 spots were detected in the cell surface and extracellular proteomes, respectively. By means of MALDI-TOF-MS and tryptic peptide mass fingerprinting, 164 cytosolic proteins, 49 proteins of the cell surface and 89 extracellular protein spots were identified, representing in total 177 different proteins. Additionally, reference maps of the three cellular proteome fractions of the close phylogenetic relative Corynebacterium glutamicum ATCC 13032 were generated and used for comparative proteomics. Classification according to the Clusters of Orthologous Groups of proteins scheme and abundance analysis of the identified proteins revealed species-specific differences. The high abundance of molecular chaperones and amino acid biosynthesis enzymes in C. efficiens points to environmental adaptations of this recently discovered amino acid-producing bacterium.     

Stabilization of cell wall proteins in Bacillus subtilis: a proteomic approach

Even though cell wall proteins of Bacillus subtilis are characterized by specific cell wall retention signals, some of these are also components of the extracellular proteome. In contrast to the majority of extracellular proteins, wall binding proteins disappeared from the extracellular proteome during the stationary phase and are subjected to proteolysis. Thus, the extracellular proteome of the multiple protease-deficient strain WB700 was analyzed which showed an increased stability of secreted WapA processing products during the stationary phase. In addition, stabilization of the WapA processing products was observed also in a sigD mutant strain which is impaired in motility and cell wall turnover. Next, we analyzed if proteins that can be extracted from B. subtilis cell walls are stabilized in the WB700 strain as well as in the sigD mutant. Thus, the cell wall proteome of B. subtilis wild type was defined showing most abundantly cell wall binding proteins (CWBPs) resulting from the WapA and WprA precursor processing. The inactivation of extracellular proteases as well as SigmaD caused an increase of CWBP105 and a decrease of CWBP62 in the cell wall proteome. We conclude that WapA processing products are substrates for the extracellular proteases which are stabilized in the absence of sigD due to an impaired cell wall turnover.     

Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors

A proteomic approach has been applied to investigate changes in the extracellular matrix of Arabidopsis thaliana cell suspension cultures following treatments with two fungal pathogen elicitors, chitosan and extracts of Fusarium moniliforme. The oxidative burst and induction of glutathione S-transferase were used as markers for induction of the pathogen defence response. Changes in the cell wall and culture filtrate proteome were profiled. Proteins whose abundance changed reproducibly were analysed via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS). An increase in the level of two classical cell wall proteins (a putative endochitinase and a polygalacturonase inhibiting protein) and two novel proteins (a putative receptor-like protein kinase and a probable apospory-associated protein) were seen at 24 hours following elicitation. The level of an unknown protein and a hypothetical protein, which has some homology to serine carboxypeptidases, were decreased at 24 hours post-elicitation. In the culture filtrate extracts, we identified two pathogen elicitor responsive proteins, a xyloglucan endo-1,4-beta-D glucanases (XEG) and a peroxidase. Using a combination of two-dimensional polyacrylamide gel electrophoresis, immunoblotting with a phosphotyrosine-specific antibody, and MALDI-TOF MS we discovered that spots that represent putative lectin receptor-like kinase, a putative endochitinase and a XEG possess phosphorylated tyrosine residues. The identification of phosphorylated bona fide cell wall proteins and a putative extracellular receptor-like kinase with no transmembrane domain implicate the existence of an extracellular phosphorylation network which could be involved in intercellular communication.     

Apoplastic ascorbate metabolism and its role in the regulation of cell signalling

The apoplast has crucial functions in plant biology. It comprises all the compartments beyond the plasmalemma, including the cell wall. As the reservoir of information on the biotic and abiotic environment surrounding the cell and a major conduit of information between cells, the apoplast has functions in stress perception and the subsequent appropriate control of growth and defence. The oxidative burst phenomenon, caused by environmental challenges and pathogen attack in particular, oxidises the apoplast. Ascorbic acid (AA), the major and probably the only antioxidant buffer in the apoplast, becomes oxidised in these conditions. The apoplastic enzyme ascorbate oxidase (AO) also regulates the reduction/oxidation (redox) state of the apoplastic ascorbate pool. We propose that a key function of the oxidative burst and of AO is to modify the apoplastic redox state in such a way as to modify receptor activity and signal transduction to regulate defence and growth.     

Defence reactions in the apoplastic proteome of oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> var. <Emphasis Type="Italic">napus</Emphasis>) attenuate <Emphasis Type="Italic">Verticillium longisporum</Emphasis>growth but not disease symptoms

Background

Verticillium longisporum is one of the most important pathogens of Brassicaceae that remains strictly in the xylem during most stages of its development. It has been suggested that disease symptoms are associated with clogging of xylem vessels. The aim of our study was to investigate extracellular defence reactions induced by V. longisporum in the xylem sap and leaf apoplast of Brassica napus var. napus in relation to the development of disease symptoms, photosynthesis and nutrient status.

Results

V. longisporum (strain VL43) did not overcome the hypocotyl barrier until 3 weeks after infection although the plants showed massive stunting of the stem and mild leaf chlorosis. During this initial infection phase photosynthetic carbon assimilation, transpiration rate and nutrient elements in leaves were not affected in VL43-infected compared to non-infected plants. Proteome analysis of the leaf apoplast revealed 170 spots after 2-D-protein separation, of which 12 were significantly enhanced in response to VL43-infection. LS-MS/MS analysis and data base searches revealed matches of VL43-responsive proteins to an endochitinase, a peroxidase, a PR-4 protein and a β-1,3-glucanase. In xylem sap three up-regulated proteins were found of which two were identified as PR-4 and β-1,3-glucanase. Xylem sap of infected plants inhibited the growth of V. longisporum.

Conclusion

V. longisporum infection did not result in drought stress or nutrient limitations. Stunting and mild chlorosis were, therefore, not consequences of insufficient water and nutrient supply due to VL43-caused xylem obstruction. A distinct array of extracellular PR-proteins was activated that might have limited Verticillium spreading above the hypocotyl. In silico analysis suggested that ethylene was involved in up-regulating VL43-responsive proteins.

     

Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure

Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.     

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.     

Expression of a Trichoderma reesei β-1,4 endo-xylanase in tall fescue modifies cell wall structure and digestibility and elicits pathogen defence responses

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H₂O₂ production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H₂O₂.     

Proteomic analysis of Salmonella enterica serovar typhimurium isolated from RAW 264.7 macrophages: identification of a novel protein that contributes to the replication of serovar typhimurium inside macrophages

To evade host resistance mechanisms, Salmonella enterica serovar Typhimurium (STM), a facultative intracellular pathogen, must alter its proteome following macrophage infection. To identify new colonization and virulence factors that mediate STM pathogenesis, we have isolated STM cells from RAW 264.7 macrophages at various time points following infection and used a liquid chromatography-mass spectrometry-based proteomic approach to detect the changes in STM protein abundance. Because host resistance to STM infection is strongly modulated by the expression of a functional host-resistant regulator, i.e. natural resistance-associated macrophage protein 1 (Nramp1, also called Slc11a1), we have also examined the effects of Nramp1 activity on the changes of STM protein abundances. A total of 315 STM proteins have been identified from isolated STM cells, which are largely housekeeping proteins whose abundances remain relatively constant during the time course of infection. However, 39 STM proteins are strongly induced after infection, suggesting their involvement in modulating colonization and infection. Of the 39 induced proteins, 6 proteins are specifically modulated by Nramp1 activity, including STM3117, as well as STM3118-3119 whose time-dependent abundance changes were confirmed using Western blot analysis. Deletion of the gene encoding STM3117 resulted in a dramatic reduction in the ability of STM to colonize wild-type RAW 264.7 macrophages, demonstrating a critical involvement of STM3117 in promoting the replication of STM inside macrophages. The predicted function common for STM3117-3119 is biosynthesis and modification of the peptidoglycan layer of the STM cell wall.     

Proteomics of ionically bound and soluble extracellular proteins in Medicago truncatula leaves

A large proportion of the apoplast proteome resides in the intercellular fluid (IF) or is ionically bound (IB) to the wall matrix. A combined analysis of IF and IB proteins of the Medicago truncatula leaf apoplast was performed. 2-DE analyses demonstrated the reproducible presence of 220 IF and 84 IB proteins in the apoplast. These two protein populations were largely distinct; 22 proteins could be spatially matched, but MALDI-TOF/TOF analyses suggested a considerably smaller number had common identities. MALDI-TOF/TOF characterisation identified 81 distinct proteins. Analyses of selected IF proteins (45) indicated 17 distinct proteins with mainly defence-related functions, whereas analyses of IB proteins (70) identified 63 distinct proteins of diverse natures, including proteins of non-canonical natures. The presence of non-canonical proteins in IB extracts is discussed in the light of evidence supporting a low level of contamination of purified walls from symplastic proteins. This work indicates that IB and IF proteins are functionally distinct fractions of the apoplast. The data obtained complements earlier studies of the Medicago proteome and therefore will be useful in future studies investigating the role of apoplastic proteins in plant processes.     

Changes in the tobacco leaf apoplast proteome in response to salt stress

The apoplast of plant cells is a dynamic compartment involved in many processes, including maintenance of tissue shape, development, nutrition, signalling, detoxification and defence. In this work we used Nicotiana tabacum plants as a model to investigate changes in the soluble apoplast composition induced in response to salt stress. Apoplastic fluid was extracted from leaves of control plants and plants exposed to salt stress, using a vacuum infiltration procedure. Two-dimension electrophoretic analyses revealed about 150 polypeptide spots in the pH range of 3.0 to 10.0, in independent protein extracts, with a high level of reproducibility between the two sample sets. Quantitative evaluation and statistical analyses of the resolved spots in treated and untreated samples revealed 20 polypeptides whose abundance changed in response to salt stress. Mass spectroscopic peptide separation and sequencing was used to identify polypeptides affected by salt stress. While the levels of some proteins were reduced by salt-treatment, an enhanced accumulation of protein species known to be induced by biotic and abiotic stresses was observed. In particular, two chitinases and a germin-like protein increased significantly and two lipid transfer proteins were expressed entirely de novo. Some apoplastic polypeptides, involved in cell wall modifications during plant development, remained largely unchanged. The significance of these components is discussed in the context of stress responses in plants.     

Extracellular matrix proteome of chickpea (Cicer arietinum L.) illustrates pathway abundance, novel protein functions and evolutionary perspect

The extracellular matrix (ECM) or cell wall is a dynamic system and serves as the first line mediator in cell signaling to perceive and transmit extra- and intercellular signals in many pathways. Although ECM is a conserved compartment ubiquitously present throughout evolution, a compositional variation does exist among different organisms. ECM proteins account for 10% of the ECM mass, however, comprise several hundreds of different molecules with diverse functions. To understand the function of ECM proteins, we have developed the cell wall proteome of a crop legume, chickpea (Cicer arietinum). This comprehensive overview of the proteome would provide a basis for future comparative proteomic efforts for this important crop. Proteomic analyses revealed new ECM proteins of unknown functions vis-à-vis the presence of many known cell wall proteins. In addition, we report here evidence for the presence of unexpected proteins with known biochemical activities, which have never been associated with ECM.     

Differential proteomic analysis of apoplastic proteins during initial phase of salt stress in rice

The plant cell apoplast, which consists of all the compartments beyond the plasma membrane, is implicated in a variety of functions during plant growth and development as well as in plant defence responses to stress conditions. To evaluate the role of apoplastic proteins in initial phase of salt stress, a 2-DE based differential proteomics approach has been used to identify apoplastic salt response proteins. Six salt response proteins have been identified, among them, an apoplastic protein OsRMC, which belongs to cysteine-rich repeat receptor like protein kinase subfamily but without the kinase domain, has shown drastically increased abundance in response to salt stress during the initial phase. Our results show, OsRMC negative regulates the salt tolerance of rice plants. These results indicated that plant apoplastic proteins may have important role in plant salt stress response signal pathway.Key words: rice, apoplast, proteomic, salt stress, receptor-like protein kinase, OsRMC     

Enemy at the gates: traffic at the plant cell pathogen interface

The plant apoplast constitutes a space for early recognition of potentially harmful non-self. Basal pathogen recognition operates via dynamic sensing of conserved microbial patterns by pattern recognition receptors or of elicitor-active molecules released from plant cell walls during infection. Recognition elicits defence reactions depending on cellular export via SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex-mediated vesicle fusion or plasma membrane transporter activity. Lipid rafts appear also involved in focusing immunity-associated proteins to the site of pathogen contact. Simultaneously, pathogen effectors target recognition, apoplastic host proteins and transport for cell wall-associated defence. This microreview highlights most recent reports on the arms race for plant disease and immunity at the cell surface.