Apoplastic Proteins Research Articles

Verticillium longisporum infection affects the leaf apoplastic proteome, metabolome, and cell wall properties in Arabidopsis thaliana.

Verticillium longisporum (VL) is one of the most devastating diseases in important oil crops from the family of Brassicaceae. The fungus resides for much time of its life cycle in the extracellular fluid of the vascular system, where it cannot be controlled by conventional fungicides. To obtain insights into the biology of VL-plant interaction in the apoplast, the secretome consisting of the extracellular proteome and metabolome as well as cell wall properties were studied in the model Brassicaceae, Arabidopsis thaliana. VL infection resulted in increased production of cell wall material with an altered composition of carbohydrate polymers and increased lignification. The abundance of several hundred soluble metabolites changed in the apoplast of VL-infected plants including signalling and defence compounds such as glycosides of salicylic acid, lignans and dihydroxybenzoic acid as well as oxylipins. The extracellular proteome of healthy leaves was enriched in antifungal proteins. VL caused specific increases in six apoplast proteins (three peroxidases PRX52, PRX34, P37, serine carboxypeptidase SCPL20, α-galactosidase AGAL2 and a germin-like protein GLP3), which have functions in defence and cell wall modification. The abundance of a lectin-like, chitin-inducible protein (CILLP) was reduced. Since the transcript levels of most of the induced proteins were not elevated until late infection time points (>20 dpi), whereas those of CILLP and GLP3 were reduced at earlier time points, our results may suggest that VL enhances its virulence by rapid down-regulation and delay of induction of plant defence genes.

Comparative evaluation of extraction methods for apoplastic proteins from maize leaves

Proteins in the plant apoplast are essential for many physiological processes. We have analysed and compared six different infiltration solutions for proteins contained in the apoplast to recognize the most suitable method for leaves and to establish proteome maps for each extraction. The efficiency of protocols was evaluated by comparing the protein patterns resolved by 1-DE and 2-DE, and revealed distinct characteristics for each infiltration solution. Nano-LC-ESI-Q-TOF MS analysis of all fractions was applied to cover all proteins differentially extracted by infiltration solutions and led to the identification of 328 proteins in total in apoplast preparations. The predicted subcellular protein localisation distinguished the examined infiltration solutions in those with high or low amounts of intracellular protein contaminations, and with high or low quantities of secreted proteins. All tested infiltration solution extracted different subsets of proteins, and those implications on apoplast-specific studies are discussed.

Apoplastic superoxide production and peroxidase activity by intact and excised axenically grown seedling roots of sunflower

Excised and cold-preincubated sunflower seedling roots were compared with intact non-preincubated roots to test the effect of the injury stress and cold preincubation on the oxidative burst measured as apoplastic superoxide (O (2) (.-) ) generation and exocellular peroxidase (ECPOX) activity. Preincubated excised or intact roots released into the medium apoplastic proteins with peroxidase activity. Intact and excised roots responded to methyl jasmonate by an immediate oxidative burst that could not be induced by salicylic acid; both phytohormones also induced a slight and slow O (2)(.-) generation and ECPOX activity on excised roots, when added to the cold preincubation medium. The results with cyanide, azide, SHAM (ECPOX inhibitors) and diphenylene iodonium (inhibitor of trans-plasma membrane NAD(P)H-oxidases (NOX)-respiratory burst oxidase homologue in plants (RBOH), the trans-plasmamembrane nicotinamide adenine dinucleotide phosphate oxidase) are consistent with the hypothesis that different systems may be the origin of O (2) (.-) in intact and excised roots; ECPOX was an important component of them in both, together with NOX-RBOH in intact roots, but in excised roots the last one was replaced by an oxidase sensitive to the same inhibitors as the alternative mitochondrial oxidase. According to our hypothesis, these results could be explained if the electron flux would be deviated to different interconnected plasma membrane-redox systems, with different terminal oxidases, activated by different effectors or stresses.

Moving copper in plants

Moving copper in plants

A protease activity–depleted environment for heterologous proteins migrating towards the leaf cell apoplast

Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of proteases from several families along the secretory pathway, including A1 and A22 Asp proteases; C1A and C13 Cys proteases; and S1, S8 and S10 Ser proteases. In vitro protease assays confirmed the presence of various proteases in N.benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by ∼45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys proteases, had no impact on the apoplast proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway.

Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen

Pollen grains play important roles in the reproductive processes of flowering plants. The roles of apoplastic proteins in pollen germination and in pollen tube growth are comparatively less well understood. To investigate the functions of apoplastic proteins in pollen germination, the global apoplastic proteins of mature and germinated Arabidopsis thaliana pollen grains were prepared for differential analyses by using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) saturation labeling techniques. One hundred and three proteins differentially expressed (p value≤0.01) in pollen germinated for 6h compared with un-germination mature pollen, and 98 spots, which represented 71 proteins, were identified by LC–MS/MS. By bioinformatics analysis, 50 proteins were identified as secreted proteins. These proteins were mainly involved in cell wall modification and remodeling, protein metabolism and signal transduction. Three of the differentially expressed proteins were randomly selected to determine their subcellular localizations by transiently expressing YFP fusion proteins. The results of subcellular localization were identical with the bioinformatics prediction. Based on these data, we proposed a model for apoplastic proteins functioning in pollen germination and pollen tube growth. These results will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth.

Identification of NaCl stress-responsive apoplastic proteins in rice shoot stems by 2D-DIGE

Plants have evolved sophisticated systems to cope with adverse environmental conditions such as cold, drought, and salinity. Although a number of stress response networks have been proposed, the role of plant apoplast in plant stress response has been ignored. To investigate the role of apoplastic proteins in the salt stress response, 10-day old rice plants were treated with 200mM NaCl for 1, 6 or 12h, and the soluble apoplast proteins of rice shoot stems were extracted for differential analysis, compared with untreated controls, by 2-D DIGE saturation labeling techniques. One hundred twenty-two significantly changed spots were identified by LC-MS/MS, and 117 spots representing 69 proteins have been identified. Of these proteins, 37 are apoplastic proteins according to the bioinformatic analysis. These proteins are mainly involved in the processes of carbohydrate metabolism, oxido-reduction, and protein processing and degradation. According to their functional categories and cluster analysis, a stress response model of apoplastic proteins has been proposed. These data indicate that the apoplast is important in plant stress signal reception and response.

Comparative Proteomics Analysis of the Root Apoplasts of Rice Seedlings in Response to Hydrogen Peroxide

BackgroundPlant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H2O2) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H2O2-responsive proteins in the apoplast of rice seedling roots.Methodology/Principal Findings14-day-old rice seedlings were treated with low concentrations (300 and 600 µM) of H2O2 for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H2O2 were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H2O2 conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H2O2-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H2O2 stress.Conclusions/SignificanceThe present study is the first apoplast proteome investigation of plant seedlings in response to H2O2 and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H2O2 response.

Induction of pathogenesis-related proteins in intercellular fluid by cadmium stress in barley (Hordeum vulgareL.) — A proteomic analysis

The effect of cadmium on protein expression in the aerial parts of barley (Hordeum vulgare L. cv. ‘Mandolina’) seedlings was investigated by proteomic analysis of leaf apoplast proteins. Dramatic changes were observed in the protein pattern of intercellular washing fluid from Cd-treated (0–300 μM) barley leaves both by 1D- and 2D-PAGE. By mass spectrometric (MALDI-TOF and/or LC-MS/MS) analysis of induced proteins PR1 proteins, certain 1-3-glucanases (PR2), chitinases (PR3), members of the chitin binding PR4 family, a rich set of thaumatin-like proteins (PR5) and two PR17 proteins were identified, indicating that a general plant defence response, inducing massive secretion of pathogenesis-related proteins (PR) into the extracellular space, is an important part of the Cd-induced stress reactions. Although systemic induction of PR proteins is probably important for an adequate plant response to cadmium stress, many of these proteins are known to have an allergenic potential and as such present a health risk to plant eaters, even when the heavy metal concentration in the given plant organ is low.

Apoplast proteome reveals that extracellular matrix contributes to multistress response in poplar.

BackgroundRiverine ecosystems, highly sensitive to climate change and human activities, are characterized by rapid environmental change to fluctuating water levels and siltation, causing stress on their biological components. We have little understanding of mechanisms by which riverine plant species have developed adaptive strategies to cope with stress in dynamic environments while maintaining growth and development.ResultsWe report that poplar (Populus spp.) has evolved a systems level "stress proteome" in the leaf-stem-root apoplast continuum to counter biotic and abiotic factors. To obtain apoplast proteins from P. deltoides, we developed pressure-chamber and water-displacement methods for leaves and stems, respectively. Analyses of 303 proteins and corresponding transcripts coupled with controlled experiments and bioinformatics demonstrate that poplar depends on constitutive and inducible factors to deal with water, pathogen, and oxidative stress. However, each apoplast possessed a unique set of proteins, indicating that response to stress is partly compartmentalized. Apoplast proteins that are involved in glycolysis, fermentation, and catabolism of sucrose and starch appear to enable poplar to grow normally under water stress. Pathogenesis-related proteins mediating water and pathogen stress in apoplast were particularly abundant and effective in suppressing growth of the most prevalent poplar pathogen Melampsora. Unexpectedly, we found diverse peroxidases that appear to be involved in stress-induced cell wall modification in apoplast, particularly during the growing season. Poplar developed a robust antioxidative system to buffer oxidation in stem apoplast.ConclusionThese findings suggest that multistress response in the apoplast constitutes an important adaptive trait for poplar to inhabit dynamic environments and is also a potential mechanism in other riverine plant species.

An Effector-Targeted Protease Contributes to Defense against Phytophthora infestans and Is under Diversifying Selection in Natural Hosts

Since the leaf apoplast is a primary habitat for many plant pathogens, apoplastic proteins are potent, ancient targets for apoplastic effectors secreted by plant pathogens. So far, however, only a few apoplastic effector targets have been identified and characterized. Here, we discovered that the papain-like cysteine protease C14 is a new common target of EPIC1 and EPIC2B, two apoplastic, cystatin-like proteins secreted by the potato (Solanum tuberosum) late blight pathogen Phytophthora infestans. C14 is a secreted protease of tomato (Solanum lycopersicum) and potato typified by a carboxyl-terminal granulin domain. The EPIC-C14 interaction occurs at a wide pH range and is stronger than the previously described interactions of EPICs with tomato defense proteases PIP1 and RCR3. The selectivity of the EPICs is also different when compared with the AVR2 effector of the fungal tomato pathogen Cladosporium fulvum, which targets PIP1 and RCR3, and only at apoplastic pH. Importantly, silencing of C14 increased susceptibility to P. infestans, demonstrating that this protease plays a role in pathogen defense. Although C14 is under conservative selection in tomato, it is under diversifying selection in wild potato species (Solanum demissum, Solanum verrucosum, and Solanum stoliniferum) that are the natural hosts of P. infestans. These data reveal a novel effector target in the apoplast that contributes to immunity and is under diversifying selection, but only in the natural host of the pathogen.

Wintersweet accumulates apoplastic chitinase with a dual role in petals

Most monocotyledons like cereals accumulate antifreeze proteins in the apoplast during cold acclimation, but it is still uncertain whether dicotyledons do. Here we report the isolation and characterisation of a 33-kD apoplastic chitinase extracted from the corolla of wintersweet (Chinmonanthus praecox communis L.), which was purified using successive column chromatography on Phenyl-Sepharose, DEAE-Sepharose, and CM-Sepharose. Antifreezing activity of chitinase was confirmed in terms of the formation of bipyramidal ice crystals and high thermal-hysteresis values. Interestingly, chitinase was also found to affect germination of fungal spores of four major plant pathogens. From these data, we hypothesize that, under natural conditions, wintersweet as one of the overwintering dicotyledons also accumulates apoplastic antifreeze proteins like monocotyledons. To our knowledge, this is the first report on the isolation of dicotyledon apoplastic chitinase with high-level antifreeze and antifungal activities.

Disease development and PR-protein activity in wheat ( Triticum aestivum) seedlings treated with plant extracts prior to leaf rust ( Puccinia triticina) infection

The potential to control leaf rust ( Puccinia triticina; pathotype UVPt9) in vivo in susceptible (Thatcher) and resistant (Thatcher/ Lr15) near-isogenic wheat ( Triticum aestivum) lines by foliar applications of crude plant leaf extracts from Tulbaghia violacea and Agapanthus africanus, as well as a commercially available natural product, ComCat ®, and a Lupinus albus seed suspension (SS) was investigated. In vitro activities of the PR-proteins, β-1,3-glucanase, chitinase and peroxidase were quantified in these two wheat lines under both non-infected and infected conditions. Although foliar application of the different plant extracts accelerated the activities of all three enzymes in resistant infected wheat plants, it was only the A. africanus extract that increased the in vitro activities of the three apoplastic pathogenesis-related proteins significantly in both susceptible and resistant wheat cultivars, whether uninfected or infected. The latter resulted in the reduction of pustule and necrotic lesion formation in the susceptible and resistant wheat lines, respectively. Foliar treatment of wheat seedlings with either ComCat ® or SS had no effect on pustule and necrotic lesion formation. The A. africanus crude extract also had a significant in vitro inhibitory effect on both leaf rust spore germination and subsequent germ tube development.

Identifying secreted proteins of Marssonina brunnea by degenerate PCR

Marssonina brunnea is an important fungal pathogen of the Populus genus. To further our understanding of the pathogenesis of M. brunnea, we initiated a proteome-level study of the fungal secretome. Using de novo peptide sequencing by MS/MS, we obtained peptide sequences for 32 protein spots. Four proteins were identified by sequence homology to conserved proteins in public databases using MS-driven BLAST. To identify additional protein spots, we combined a degenerate PCR method, based on the Consensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) method, and a rapid amplification of cDNA ends method to clone the full-length cDNA fragments encoding the proteins identified in the gel. Using this method, we cloned the full-length cDNA fragments encoding 11 M. brunnea-specific proteins. This method provides an efficient approach to identification of species-specific proteins of non-sequenced organisms. Furthermore, we analyzed the expression patterns of these genes during infection. We found that most of the identified secreted proteins could be induced in artificial medium after hyphae entered poplar apoplast spaces. We propose that for the host-specialized M. brunnea, the elongation of hyphae has evolved closely with the secretion of apoplastic proteins.

Potential Role for Purple Acid Phosphatase in the Dephosphorylation of Wall Proteins in Tobacco Cells

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as alpha-xylosidase and beta-glucosidase. The dephosphorylation and phosphorylation of recombinant alpha-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of alpha-xylosidase and beta-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.

Cold-regulated proteins with potent antifreeze and cryoprotective activities in spruces ( Picea spp.)

Antifreeze proteins (AFPs) were obtained from intercellular spaces of spruce needles Picea abies (L.) Karst. and Picea pungens Engelm. by vacuum infiltration with ascorbic acid, followed by centrifugation to recover the infiltrate. As shown by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), apoplastic proteins are accumulated in these spruce species as a group of 5–9 polypeptide bands. These proteins have a molecular mass of 7–80 kDa. The spruce AFPs have the ability to modify the growth of ice and thermal hysteresis, TH, caused by these AFPs was close to 2.0 °C at a concentration of 400 μg/ml. The antifreeze activity of proteins from these winter-hardy coniferous species showed a positive correlation with the concentration of proteins after cold acclimation of needle tissues. Apoplastic proteins from winter spruce needles exhibited antifreeze activity, whereas no such activity was observed in extracts from summer needles. When we examined the possible role of spruce AFPs in cryoprotection, we found that lactate dehydrogenase, LDH, activity was higher after freezing in the presence of AFPs compared with bovine serum albumin. Amino-terminal sequence comparisons indicated that a 27-kDa protein from both P. abies and P. pungens was similar to some pathogenesis-related proteins namely chitinases, also from conifer species. These results show that spruces produce AFPs that are secreted into the apoplast of needles. The accumulation of AFPs in extracellular spaces caused by seasonal cold acclimation during winter indicates that these proteins may play a role in the acquisition of freezing tolerance of needle cells in coniferous species.

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.

Identification of an apoplastic protein involved in the initial phase of salt stress response in rice root by two-dimensional electrophoresis.

The apoplast of plant cells, which carries out multiple functions in plant metabolism and signaling, is not only a barrier but also the linker between the environment and the protoplast. To investigate the role of apoplastic proteins in the salt stress response, 10-d-old rice (Oryza sativa) plants were treated with 200 mM NaCl for 1, 3, or 6 h, and the soluble apoplast proteins were extracted for differential analysis compared with untreated controls using two-dimensional electrophoresis. Ten protein spots that increased or decreased significantly in abundance were identified by mass spectrometry. These proteins included some well-known biotic and abiotic stress-related proteins. Among them, an apoplastic protein, with extracellular domain-like cysteine-rich motifs (DUF26), O. sativa root meander curling (OsRMC), has shown drastically increased abundance in response to salt stress during the initial phase. OsRMC RNA interference transgenic rice has been generated to assess the function of OsRMC in the salt stress response. The results show that knocking down the expression level of OsRMC in transgenic rice led to insensitive seed germination, enhanced growth inhibition, and improved salt stress tolerance to NaCl than in untransgenic plants. These results indicate that plant apoplastic proteins may have important roles in the plant salt stress response.

Influence of environmental pH modulation on efficiency of apoplastic PR proteins during Fusarium culmorum– wheat seedling interaction

Variation of apoplastic pH by Fusarium culmorum and its influence on the production, activity and isoenzymes patterns of the pathogenesis‐related (PR) proteins β1,3‐glucanase, chitinase and peroxidase enzymes were detected in apoplastic fluids (AFs) from infected wheat seedlings. The time course in the 24–48 h interval post infection was characterized by an increase in activity and isoenzymatic differential induction of the selected PR proteins and by a concomitant rise of apoplastic pH. Chitinase attained maximum activity at pH 8·0 in the case of inoculated seedlings. Optimal β1,3‐glucanase activity in the pH range 6·0–8·0, was observed at pH 7·0. Peroxidase was strongly affected by pH, with enzyme activity having a maximum rate at pH 6·0 and thereafter rapidly declining at higher pH. Maximum peroxidase activity paralleled the appearance of the complete isoenzymatic pattern. In order to investigate the biological role of PR proteins in AFs, the in vitro antifungal activity was evaluated. In the interval 0–6 h, pH of macroconidia suspensions rose up to 7·2. AFs revealed inhibitory activity against germinating macroconidia of F. culmorum by decreasing the germination efficiency of macroconidia apical compartments, while this effect was compensated by an increased germination capacity of middle compartments. Present results suggest that during infection of wheat seedlings by F. culmorum the pH modulation favours host colonization by enhancing the activity of pectin lyase, and simultaneously inhibits the capacity of the host to oppose the pathogen by interfering with peroxidases which represent an important component of the defence arsenal.

Phloem sap of tomato plants contains a DIR1 putative ortholog

Arabidopsis thaliana defective in induced resistance 1 (At-DIR1) has been characterized as a protein responsible for the generation or transmission of the still unknown signal involved in systemic acquired resistance. This acidic apoplastic protein is a member of the family of lipid transfer proteins and was detected in vascular fluids. To our knowledge, no DIR1-like protein has been described in other plant species. Hence, we have performed data mining to identify a putative ortholog of DIR1 in tomato. This strategy allowed the detection of a few gene products displaying sequence similarity to At-DIR1 whose structural features were further analysed in silico. The best match (unigene SGN-327306) encoded a protein with an acidic pI, a peculiar characteristic of DIR1 among lipid transfer proteins, and was hence selected as a putative tomato ortholog of At-DIR1. This sequence, named Le-DIR1, served for the design of a specific antigenic peptide and the generation of polyclonal antibodies. The antiserum anti-Le-DIR1 recognized a peptide of the expected size (7kDa) in phloem sap of tomato plants, hence confirming the existence of the predicted protein in vascular fluids. This result supports the notion of the existence of common systemic acquired resistance (SAR) signaling molecules in different species.