Identification by 2-D DIGE of apoplastic proteins regulated by oligogalacturonides in Arabidopsis thaliana

Oligogalacturonides (OGs) are elicitors of plant defence responses released from the homogalacturonan of the plant cell wall during the attack by pathogenic micro-organisms. The signalling pathway mediated by OGs remains poorly understood, and no proteins involved in their signal perception and transduction have yet been identified. In order to shed light into the molecular pathways regulated by OGs, a differential proteomic analysis has been carried out in Arabidopsis. Proteins from the apoplastic compartment were isolated and their expression compared between control and OG-treated seedlings. 2-D gels and difference in gel electrophoresis (DIGE) techniques were used to compare control and treated proteomes in the same gel. The analysis of subcellular proteomes from seedlings allowed the identification of novel and low abundance proteins that otherwise remain masked when total cellular extracts are investigated. The DIGE technique showed to be a powerful tool to overcome the high interexperiment variation of 2-D gels. Differentially expressed apoplastic proteins were identified by MS and included proteins putatively involved in recognition as well as proteins whose PTMs are regulated by OGs. Our findings underscore the importance of cell wall as a source of molecules playing a role in the perception of pathogens and provide candidate proteins involved in the response to OGs.     

Effect of manganese toxicity on the proteome of the leaf apoplast in cowpea

Excess manganese (Mn) supply causes formation of visible brown depositions in the cell walls of leaves of cowpea (Vigna unguiculata), which consist of oxidized Mn and oxidized phenols. Because oxidation of Mn and phenolic compounds in the leaf apoplast was proposed to be catalyzed by apoplastic peroxidases (PODs), induction of these enzymes by Mn excess was investigated. POD activity increased upon prolonged Mn treatment in the leaf tissue. Simultaneously, a significant increase in the concentration of soluble apoplastic proteins in "apoplastic washing fluid" was observed. The identity of the released proteins was systematically characterized by analysis of the apoplast proteome using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. Some of the identified proteins exhibit sequence identity to acidic PODs from other plants. Several other proteins show homologies to pathogenesis-related proteins, e.g. glucanase, chitinase, and thaumatin-like proteins. Because pathogenesis-related-like proteins are known to be induced by various other abiotic and biotic stresses, a specific physiological role of these proteins in response to excess Mn supply remains to be established. The specific role of apoplastic PODs in the response of plants to Mn stress is discussed.     

Absolute protein quantification by LC/MS(E) for global analysis of salicylic acid-induced plant protein secretion responses

The plant cell wall is a dynamic cellular compartment consisting of a complex matrix of components that can change dramatically in response to environmental stresses. During pathogen attack, for instance, a wide spectrum of proteins that participate in various sequential processes involved in plant defense is secreted into the cell wall. In this study, a mass spectrometry, data-independent acquisition approach known as LC/MS (E) was used to assess temporal changes in the cell wall proteome in response to different levels of an endogenous inducer of plant disease defense responses, salicylic acid (SA). LC/MS (E) was used as a label-free method that enabled simultaneous protein identification and absolute femtomole quantification of each protein secreted into the extracellular matrix. A total of 74 secreted proteins were identified, 63 of which showed increased specific secretion in response to SA. A majority of this induced secretion occurred within 2 h of treatment, indicating that many proteins are involved in the early stages of plant defenses. We also identified a number of apparently nonclassically secreted proteins, suggesting that, as in many nonplant systems, Golgi/ER-independent mechanisms exist for plant protein secretion. These results provide new insight into plant apoplastic defense mechanisms and demonstrate that LC/MS (E) is a powerful tool for obtaining both relative and absolute proteome-scale quantification that can be applied to complex, time- and dose-dependent experimental designs.     

Changes in the apoplastic pH are involved in regulation of xyloglucan breakdown of azuki bean epicotyls under hypergravity conditions

Hypergravity inhibited elongation growth of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls by decreasing the mechanical extensibility of cell walls via the increase in the molecular mass of xyloglucans [Soga et al. (1999) Plant Cell Physiol. 40: 581]. Here, we report that the pH value of the apoplastic fluid in epicotyls increased from 5.8 to 6.6 by hypergravity (300 x g) treatment. When the xyloglucan-degrading enzymes extracted from cell walls of the 1 x g control epicotyls were assayed in buffer at pH 6.6 and 5.8, the activity at pH 6.6 was almost half of that at pH 5.8. In addition, when enzymically active cell wall preparations obtained from 1 x g control epicotyls were autolyzed in buffer at pH 5.8 and 6.6 and then xyloglucans were extracted from the autolyzed cell walls, the molecular mass of xyloglucans incubated at pH 5.8 decreased during the autolysis, while that at pH 6.6 did not change. Thus, the xyloglucans were not depolymerized by autolysis at the pH value (6.6) observed in the hypergravity-treated epicotyls. These findings suggest that in azuki bean epicotyls, hypergravity decreases the activities of xyloglucan-degrading enzymes by increasing the pH in the apoplastic fluid, which may be involved in the processes of the increase in the molecular mass of xyloglucans, leading to the decrease in the cell wall extensibility.     

Review article. Apoplastic barriers in roots: chemical composition of endodermal and hypodermal cell walls

Based on the characterization of the chemical composition of endodermal and hypodermal cell walls isolated from seven monocotyledonous and three dicotyledonous plant species, a model of the composition of apoplastic barriers in roots is proposed. Depending on the species, endodermal and hypodermal cell walls of roots contained varying amounts of the biopolymers suberin, lignin, cell wall proteins, and carbohydrates. Although analysis of the chemical composition of these apoplastic barriers of roots is now possible, it is pointed out that conclusions from these data concerning the functional properties of these cell walls can not easily be drawn. However, in analogy to suberized periderms it is argued that the suberin should play a role in establishing an apoplastic transport barrier in roots, albeit not a perfect barrier. Furthermore, due to the combined occurrence of suberin, lignin and cell wall proteins it is argued that endodermal and hypodermal cell walls also have an important function as barriers towards pathogens. Finally, it is pointed out that additional experimental approaches combining the investigation of transport properties and of the chemical composition of apoplastic transport barriers in roots are necessary before the function of endodermal and hypodermal cell walls in roots can be fully understood.     

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.     

Monoclonal antibody analysis of Perkinsus marinus extracellular products

The protozoan oyster parasite Perkinsus marinus releases a complex set of extracellular products (ECP) during in vitro culture. These products have been previously implicated in parasite virulence, and their expression can be altered by medium supplementation with oyster tissue homogenate. Little is known regarding ECP function, regulation, or mechanism of storage and release. Perkinsus marinus ECP were purified from a protein-free medium and used to produce a panel of five monoclonal antibodies. Several of the antibodies recognised series of proteins implying that the ECP may originate from comparatively few parental molecules. The ECP are secreted by several pathways, including the release of one product from an external cell layer, and two other products from two morphologically distinct intracellular compartments. Antibodies against separate epitopes on one protein provided information about possible protein structure. A sandwich ELISA format allowed sensitive quantification of that protein and showed significantly reduced protein expression in oyster tissue homogenate supplemented cultures. Immunopurification allowed tandem mass spectroscopic amino acid sequencing of that protein. Another antibody was used to characterise the P. marinus cell wall. This antibody specifically bound to trophozoite and tomont walls, and was used to investigate the morphological and antigenic changes in these walls during Ray's fluid thioglycollate medium-induced formation of hypnospores. It was also used to confirm that oyster tissue homogenate supplementation could induce formation of hypnospores. This antibody labeled P. marinus cells in fixed oyster tissue in a species-specific manner.     

Proteinase-inhibitor synthesis in tomato plants: Evidence for extracellular deposition in roots through the secretory pathway

The cellular and subcellular localization of proteinase Inhibitors I and II proteins, synthesized in transgenic tomato (Lycopersicon esculentum L.) plants from chimeric genes regulated by the 35S promoter, was investigated by immunocytochemical techniques. Newly synthesized inhibitor proteins were deposited in the cell vacuoles as in wild-type plants, but were also secreted into the cell walls of outer epidermal and secretory cells of the root cap. The Na ionophore monensin increased the levels of proteinase inhibitors found in rough endoplasmic reticulum, Golgi cisternae and in the cell walls of transgenic plants, supporting a role for the secretory pathway in the sorting and targeting of Inhibitor I and II proteins. The two inhibitor proteins were detected by Western-blot analysis in water-washes obtained from roots of transgenic tomato seedlings, confirming their extracellular presence. Wild-type tomato plants exhibited the presence of Inhibitor I and II proteins in the external cell walls, using silver-enhanced immunogold labelling, but not by Western-blot analysis. The extracellular Inhibitor I from transgenic plant roots migrated in electrophoretic gels with a slightly different apparent mass than the Inhibitor I isolated from tomato leaf vacuoles, indicating that specific structural features of this inhibitor protein have been altered during or after extracellular deposition. The presence of extracellular inhibitors in roots may help provide protection for the growing meristems against insects or microorganisms present in the soil.Abbreviations CaMV cauliflower mosaic virus - TEM transmission electron microscope Transmission electron microscopy was performed at the Electron Microscopy Center (EMC) of Washington State University. The authors thank the EMC staff for their technical advice and collaboration. We also thank Greg Wichelns for growing our plants and Greg Pearce, Scott Johnson, and Martha L. Orozco for their advice and technical help. The work was supported in part by the Washington State College of Agriculture and Home Economics Project No. 1791 and National Science Foundation grants Nos. DCB-8702538 and DCB-8608594.     

Extracellular sunflower proteins: evidence on non-classical secretion of a jacalin-related lectin

Extracellular proteins from sunflower seedlings were analyzed by electrophoresis followed by peptide mass fingerprinting. Tentative identification revealed novel proteins for this crop. A significant number of those proteins were not expected to be extracellular because they lacked the typical signal peptide responsible for secretion. In silico analysis showed that some members of this group presented the characteristic disordered structures of certain non-classical and leaderless mammalian secretory proteins. Among these proteins, a putative jacalin-related lectin (Helja) with a mannose binding domain was further isolated from extracellular fluids by mannose-affinity chromatography, thus validating its identification. Besides, immunolocalization assays confirmed its extracellular location. These results showed that a lectin, not predicted to be secreted in strict requirement of the N-terminal signal peptide, occurs in a sunflower extracellular compartment. The implications of this finding are discussed.     

2‐DE proteome maps for the leaf apoplast of Nicotiana benthamiana

We provide 2‐D gel reference maps for the apoplastic proteome of Nicotiana benthamiana leaves infiltrated or not with the bacterial gene vector Agrobacterium tumefaciens. About 90 proteins were analyzed by LC‐MS/MS for identification and function assignment. We show, overall, an effective response of the plant to agroinfiltration involving a specific, cell wall maintenance‐independent up‐regulation of defense protein secretion. The proteome maps described should be a useful tool for systemic studies on plant–pathogen interactions or cell wall metabolism. They also should prove useful for the monitoring of secreted recombinant proteins and their possible pleiotropic effects along the cell secretory pathway of N. benthamiana leaves used as an expression platform for clinically useful proteins.     

Bioelicitors Induce Association of Defence Enzymes with Cell Walls of Lycopersicum esculentum

Cell walls of plants are complex structures impregnated with various proteins having wide array of functions. In this study, twenty‐eight proteins isolated from tomato cell walls were subjected to MALDI‐TOF MS followed by mass peak analysis using ORIGIN 6 software. The mass peaks subjected to MASCOT and ProFound databases for peptide mass fingerprinting led to the identification of 9 protein domains. These proteins were further classified according to their functions. Fruit extracts of A. indica could elicit induction, localization and functioning of peroxidase (POX) and polyphenol oxidase (PPO) and their isoenzymes in cell walls of Lycopersicum esculentum (tomato) against Pseudomonas syringae pv. tomato. The results revealed the possible involvement of cell wall‐bound proteins in defence of plants against the invading pathogens. A number of novel isoenzymes of both POX and PPO were found to be located in the cell walls of the plants treated with neem extract. Neem extract can induce accumulation and binding of isoenzymes to cell walls. These isoenzymes could possibly protect host plants against the invading pathogens.     

Copper Uptake by Ryegrass Seedlings; Contribution of Cell Wall Adsorption

Net copper uptake by cellulose discs, isolated root cell walls,and by live and dead roots of whole ryegrass seedlings, werestudied using ⁶⁴Cu as a tracer. Uptake by cellulose discs stoppedafter around 10 h while uptake by isolated root cell walls continuedfor up to 50 h. An initial fast phase of uptake consisting predominantlyof cell wall adsorption was similar in live and dead tissuefor up to 19 h. A slower phase of uptake continued for up to50 h, greater in live than in dead tissue, the slower phaseof uptake in live tissue consisting of both a living and a deadcomponent. Based on these results, an alternative to the desorptionmethod for estimating the apoplastic contribution to total copperuptake is presented. Time-course studies with seedlings givena variety of growing solution/uptake solution regimes, and therelationship between copper uptake and external copper concentration,for short (4.8 h) and long (42.4 h) term uptakes, suggest thatdiffering contributions of cell wall adsorption and symplasmicabsorption may be responsible for differing effects of externalcopper concentration on uptake being expressed by the same tissue.Water flux had little effect on total uptake of copper althougha possible effect on absorption could not be ruled out. Key words: Copper uptake, cell wall adsorption, ryegrass seedlings     

Cell wall oxalate oxidase modifies the ferulate metabolism in cell walls of wheat shoots

Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide.     

Changes in ascorbic acid levels in apoplastic fluid during growth of pine hypocotyls. Effect on peroxidase activities associated with cell walls

The apoplastic fluid of pine ( Pinus pinaster Aiton) hypocotyls contains ascorbic acid (AA) and dehydroascorbic acid (DHA). The amounts of ascorbic and dehydroascorbic acids were in the nmol (g fresh weight)⁻¹ range and decreased with the hypocotyl age as well as along the hypocotyl axis. The ratio AA/(AA+DHA) also decreased with the hypocotyl age and along the hypocotyl. Both ascorbic oxidase and peroxidase activity against ascorbic acid showed very low activity not only in the apoplastic fluid but also in the fractions ionically and covalently bound to the cell walls. However, the peroxidase activity in the three abovementioned fractions was strongly increased in the presence of ferulic acid. That stimulation effect increased with the hypocotyl age and from the apical towards the basal region of the hypocotyls of 10-day-old seedlings. Furthermore, the oxidation of ferulic acid by apoplastic and ionically- and covalently-bound peroxidases was inhibited by ascorbic acid as long as ascorbate was available. A regulatory role of apoplastic ascorbic acid levels in the formation of dehydrodiferulic bridges between wall polysaccharides catalysed by cell wall peroxidases and thus in the cell wall stiffening during plant growth is proposed.     

Analysis of Putative Apoplastic Effectors from the Nematode,Globodera rostochiensis,and Identification of an Expansin-Like Protein That Can Induce and Suppress Host Defenses

The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.     

Iron deficiency-induced secretion of phenolics facilitates the reutilization of root apoplastic iron in red clover

Phenolic compounds are frequently reported to be the main components of root exudates in response to iron (Fe) deficiency in Strategy I plants, but relatively little is known about their function. Here, we show that removal of secreted phenolics from the root-bathing solution almost completely inhibited the reutilization of apoplastic Fe in roots of red clover (Trifolium pratense). This resulted in much lower levels of shoot Fe and significantly higher root Fe compared with control and also resulted in leaf chlorosis, suggesting this approach stimulated Fe deficiency. This was supported by the observation that phenolic removal significantly enhanced root ferric chelate reductase activity, which is normally induced by plant Fe deficiency. Furthermore, root proton extrusion, which also is normally increased during Fe deficiency, was found to be higher in plants exposed to the phenolic removal treatment too. These results indicate that Fe deficiency-induced phenolics secretion plays an important role in the reutilization of root apoplastic Fe, and this reutilization is not mediated by proton extrusion or the root ferric chelate reductase. In vitro studies with extracted root cell walls further demonstrate that excreted phenolics efficiently desorbed a significant amount of Fe from cell walls, indicating a direct involvement of phenolics in Fe remobilization. All of these results constitute the first direct experimental evidence, to our knowledge, that Fe deficiency-induced secretion of phenolics by the roots of a dicot species improves plant Fe nutrition by enhancing reutilization of apoplastic Fe, thereby improving Fe nutrition in the shoot.     

A proteomic approach to apoplastic proteins involved in cell wall regeneration in protoplasts of Arabidopsis suspension-cultured cells

To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.     

Identification of head and neck squamous cell carcinoma biomarker candidates through proteomic analysis of cancer cell secretome

Protein biomarker discovery for early detection of head and neck squamous cell carcinoma (HNSCC) is a crucial unmet need to improve patient outcomes. Mass spectrometry-based proteomics has emerged as a promising tool for identification of biomarkers in different cancer types. Proteins secreted from cancer cells can serve as potential biomarkers for early diagnosis. In the current study, we have used isobaric tag for relative and absolute quantitation (iTRAQ) labeling methodology coupled with high resolution mass spectrometry to identify and quantitate secreted proteins from a panel of head and neck carcinoma cell lines. In all, we identified 2,472 proteins, of which 225 proteins were secreted at higher or lower abundance in HNSCC-derived cell lines. Of these, 148 were present in higher abundance and 77 were present in lower abundance in the cancer-cell derived secretome. We detected a higher abundance of some previously known markers for HNSCC including insulin like growth factor binding protein 3, IGFBP3 (11-fold) and opioid growth factor receptor, OGFR (10-fold) demonstrating the validity of our approach. We also identified several novel secreted proteins in HNSCC including olfactomedin-4, OLFM4 (12-fold) and hepatocyte growth factor activator, HGFA (5-fold). IHC-based validation was conducted in HNSCC using tissue microarrays which revealed overexpression of IGFBP3 and OLFM4 in 70% and 75% of the tested cases, respectively. Our study illustrates quantitative proteomics of secretome as a robust approach for identification of potential HNSCC biomarkers. This article is part of a Special Issue entitled: An Updated Secretome.