Associating Wound-Related Changes in the Apoplast Proteome of Medicago with Early Steps in the ROS Signal-Transduction Pathway Nelson C. Soares,† Rita Francisco,† Jesus Maria Vielba,†,# Caˆndido Pinto Ricardo,†,‡ and Phil A. Jackson*,† Instituto de Tecnologia Quı´mica e Biolo´gica, Oeiras, Portugal, and Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisbon, Portugal Received November 2, 2008
Early wound-related changes in the leaf apoplast proteome of Medicago truncatula have been characterized by 2-DE and MALDI-TOF/TOF and the differential expression of 28/110 extracellular proteins could be reproducibly observed 6 h after wounding. Wounding induced an initial (0-30 min) burst of O2-, followed by a later (3-6 h) production of O2- and H2O2. The infiltration of 5 µM DPI e 3 min after wounding inhibited both phases of the oxidative burst and suppressed wound-regulated changes in 9/28 extracellular proteins. DPI infiltrated 15 min after wounding only partially inhibited early O2production and was ineffective in suppressing wound-related changes in these proteins. This strongly suggests that in wounded Medicago, rapid O2- is required for mobilizing the downstream (3-6 h), differential expression of several extracellular proteins. Further studies with DPI and exogenous sources of ROS supported the regulation of these proteins within early, wound-related ROS-signaling events. The study forms the basis for associating wound-related changes in the apoplast proteome with ROSdependent and ROS-independent pathways. Proteins mobilized within the ROS-dependent pathway were largely ionically bound to cell walls and included SODs, peroxidases and germin-like proteins, suggesting their involvement within wound-activated, ROS regulatory loops. Keywords: Medicago truncatula • extracellular protein • cell wall protein • wounding • reactive oxygen species (ROS) • Apoplast proteome • MALDI-TOF/TOF
Introduction Mechanical wounding represents a major threat to plants, affecting gaseous exchange, intercellular solute dynamics,1 the rate of photosynthesis2 and can provide sites for pathogen invasion. The response to wounding includes the modification of damaged sites to establish a more effective and regulated interaction with the external environment. This includes the release of intercellular fluid (IF), or guttation fluid at the wound site, thereby transporting several extracellular defense proteins to the cut site to prevent pathogen ingress.3 In the case of latex producing plants, such as Carica papaya and Hevea brasiliensis, guttation fluid contains latex for wound-sealing.4 Other plant species can effect a similar response to cell wall penetration by aphids by utilizing a material comprised of pectinaceous polysaccharides.5 In the majority of plant species, however, the process of wound-sealing includes an extensive modification of the apoplast proximal to the wound site, including suberization,6 * To whom correspondence should be addressed. Dr. Phil A. Jackson, Instituto de Tecnologia Quı´mica e Biolo´gica, Apartado 127, 2781-901, Oeiras, Portugal. Tel: 00351-214469634. E-mail:
[email protected]. † Instituto de Tecnologia Quı´mica e Biolo´gica. # Present address: Instituto de Investigaciones Agrobiolo´gicas de Galicia, Apartado 122, 15080 Santiago de Compostela, Spain. ‡ Instituto Superior de Agronomia.
2298 Journal of Proteome Research 2009, 8, 2298–2309 Published on Web 02/26/2009
lignification,7 the deposition of cutin8 and the cross-linking of cell wall matrix polymers.9 This process involves many apoplastic proteins related with the perception and transduction of various environmental signals in cooperation with the plasma membrane, leading to alterations to the cell wall biosynthetic machinery as well as the mobilization of apoplastic proteins involved in extracellular matrix (ECM) modification and defense.10,11 The apoplast can be a major site of reactive oxygen species (ROS) accumulation during a variety of stress conditions, and apoplastic ROS can coordinate the expression of genes in other subcellular locations.12,13 It is therefore likely that the apoplast hosts ROS regulatory proteins, either through their direct role in ROS metabolism, or within processes related with ROS perception and regulatory loops. Recent evidence has also indicated that ATP and protein phosphorylation events are normal extracellular occurrences in plants, implying the extracellular matrix contains intermediates of phosphorylation cascades employed in response to environmental stimuli such as wounding.14,15 Despite the important role the cell wall plays in stress responses and the large number of secreted proteins involved, the characterization of apoplastic proteins within wounding responses has received little attention to date. Many studies have identified the jasmonic acid (JA) pathway as a major pathway for the regulation of wound-responses in plants. Increases in jasmonates and activation of components 10.1021/pr8009353 CCC: $40.75
2009 American Chemical Society
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Environmental Stress and Adaptation of the JA pathway are often reported as early wound responses,16-18 and the interaction of the JA pathway with ethylene, salicylic acid and abscisic acid pathways (Ribot et al.19 and references therein), suggests that wound-activation of the JA pathway is potentially complex, permitting specific woundresponses to mechanical wounding or to the action of specialist or generalist insect herbivores.20 Conversely, ROS signaling pathways in plant wounding responses have been less studied. The rapid initiation of ROS production in response to wounding has been documented in some species,21,22 but is thought not to occur in others, including Medicago.23 In some species, wound-related production of ROS can be a multiphasic process, as in potato,6 or biphasic as in ryegrass24 and Arabidopsis.25 Such biphasic ROS productions are reminiscent of the oxidative burst which occurs following avirulent pathogen attack (reviewed in Lamb and Dixon26) where ROS production is initiated within minutes and terminates after 0.5-1 h, followed by a more sustained production 3-6 h later.27,28 In other species, both local and systemic H2O2production have been detected only during later (3-6 h) phases of the wound-response.23,29 The ROS generated in response to wounding can be used directly as antimicrobial agents,26 and to cross-link ECM components30,31 to impede cell wall lysis by pathogen enzymes.32 However, ROS can also act as both local and diffusible signal molecules for the differential regulation of genes.26 In Arabidopsis, wounding and wound-related ROS production leads to the activation of the MAP kinase pathway, involving MAPK3, MAPK6 and OXI1 kinase. This occurs within 5-30 min after wounding,33 which is coincident or prior to the woundactivation of the octadecanoid pathway leading to JA synthesis.34 Several dicots may also employ ROS signaling in later phases of wounding. In wounded tomato leaves, H2O2 produced from NADPH oxidase operates as a secondary messenger in a JA-dependent pathway. Here, JA is required to activate early signaling genes (0.5-2 h) including polygalacturonase, which in its turn indirectly activates NADPH oxidase-mediated H2O2 production via its liberation of wall galacturonides.35 The H2O2 is thought to activate a later subset (4-6 h) of defensive genes. Microarray studies in Arabidopsis have identified genes associated with36 or targeted by ROS-activated signal pathways,37,38 and emphasize that different conditions of stress can provoke the formation of different ROS at different intracellular locations, both of which could provide essential cues for the development of specific, downstream molecular responses.38,39 However, this information is not easily translatable to other, less characterized species, with which it remains difficult to predict the genes targeted by ROS signaling pathways during the response to particular forms of stress. Stress-related changes in the apoplast proteome have been characterized in several species, including after elicitation,15,40 salt-stress,41 excess manganese42 and boron deficiency.43 However, changes in the apoplast proteome in response to wounding have not been studied in any detail, and stress-related changes in the apoplast proteome have yet to be associated with specific signal-transduction pathways. Here, we report a proteomic study of the changes in the Medicago leaf apoplast during the early response to wounding. A NADPH inhibitor, diphenyleneiodonium chloride (DPI) and exogenous sources of O2- and H2O2 were utilized to probe the relationship that changes in the extracellular proteome have with wound-related ROS-signaling. We report that wounding
O2-
and signals generated during the first 3 min after wounding have a substantial impact on the composition of the leaf extracellular proteome. Many of the wall proteins targeted by ROS-dependent signaling can be directly related with ROS metabolism, suggesting a regulatory role for these proteins in later stages of the oxidative burst in wounded plants.
Materials and Methods Plant Material. Medicago truncatula cv. Jemalong seeds were placed on water-soaked filter paper for 48 h and germinated seedlings were then planted in a substrate composed of sand/ peat/soil (1:1:1) and grown in a 19/25 °C, night/day cycle with a photoperiod of 12 h and light intensity of 250 µmol m-2 s-1. Leaves were harvested for extraction after 42-45 days. Protein Extraction. As a control, plant leaves were detached from the plant and immediately frozen in liquid nitrogen before homogenization. Leaf wounding employed a scalpel blade to transversally section the leaf into two halves of approximately equal lengths, and both were used for subsequent protein extraction. Wounded leaf-halves were then vacuum-infiltrated with deionized water for 3 periods of 20 s before being placed on water-dampened tissue and left under light (250 µmol m-2 s-1) at 25 °C for 6, 12, 24, or 72 h. Alternatively, wounded leaves were infiltrated with Menadione (1 mM), glucose (5 mM) plus glucose oxidase (5 µg/mL), and/or DPI (5-100 µM). Ionically bound (IB) proteins and soluble proteins of the intercellular fluid (IF) were extracted as described previously.44 Extracts were processed using the 2-DE Cleanup Kit (GE Healthcare, NJ) as per the manufacturer’s instructions, resuspended in a small volume of IEF buffer and stored at -70 °C until analysis. The Detection of Hydrogen Peroxide and Superoxide in Leaves. Wounded leaves were placed on water-dampened tissue and left under light (250 µmol m-2 s-1) at 25 °C for 3-360 min. For the detection of H2O2, leaves were infiltrated with 5 mM 3,3 diaminobenzidine (DAB; Sigma), at pH 3.8. For the detection of O2-, leaves were infiltrated with 6 mM nitroblue tetrazolium (NBT; Fluka); in all cases, the results presented were based on 3 replicates. Inhibitory effects of diphenyleneiodonium chloride (DPI; Sigma) on H2O2 or O2- accumulation were tested by co-infiltration of 5 µM DPI with DAB or NBT solutions, respectively. NBT/DAB staining and inhibitory effects of DPI were also tested with whole leaves obtained by excision from the plant at the base of the petiole and placing the petiole in DAB/NBT solutions in the presence or absence of 100 µM DPI. NBT or DAB staining was revealed after the immersion of leaves in 60 °C ethanol (96%) for 10 min to remove chlorophyll. Images were captured with an Olympus DP11 digital acquisition device from an Olympus bifocal (SZ60) or Olympus BH2RFCA microscope. Two-Dimensional Gel Electrophoresis (2-DE) and Protein Identification by MALDI-TOF/TOF. Protein concentration in the extracts was determined according to Bradford et al.45 after addition of 0.1 M HCl as suggested by Ramagli et al.46 For isoelectric focusing (IEF), the IPGphor system was used (GE Healthcare) with 3-10 nonlinear (NL) pH gradient strips (IPG strips, GE Healthcare, Sweden). Proteins were solubilized in 8 M urea, 2% (w/v) CHAPS, 40 mM DTT and 0.5% (v/v) IPG buffer (3-10 NL (GE Healthcare, Sweden)). IEF was carried out at 30 V for 12 h, followed by 250 V for 1 h, 500 V for 1.5 h, 1000 V for 1.5 h, a gradient to 8000 V over 1.5 h and maintenance at 8000 V for a further 4 h, all at 20 °C. Prior to the second dimension (SDS-PAGE), the focused IPG strips were equiliJournal of Proteome Research • Vol. 8, No. 5, 2009 2299
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brated for 2-15 min in buffer containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS and a trace amount of Bromophenol Blue. DTT at 1% (w/v) was added to the first equilibration step and 2.5% (w/v) iodoacetamide to the second. SDS-PAGE was performed on 12% or 15% polyacrylamide gels.47For analytical 2-DE gels, silver staining was performed according to Blum et al.48 and gels were loaded with 40 µg of total protein. Gels were scanned using the ImageQuant v3.3 densitometer (Molecular Dynamics) and were analyzed by the Image Master Platinum software v.5.0 (GE Healthcare). For preparative gels, MS-compatible silver staining49 was utilized and the gels were loaded with at least 150 µg of total protein. Selected spots were excised from the gels and destained with a solution containing 20% (w/v) sodium thiosulphate and 1% (w/v) potassium ferricyanide for 5 min. The supernatant was removed and the gel spots were washed twice with 25 mM ammonium bicarbonate in 50% (v/v) acetonitrile for 20 min. The gel spots were then washed in acetonitrile, dried in a Speed-Vac (Savant) and digested overnight with 20 µg/mL of trypsin in 25 mM ammonium bicarbonate at 37 °C. Tryptic peptides were passed through C18 Zip-Tips and mixed with 5 mg/mL of an R-cyano-4-hydroxycinnamic acid as matrix and subject to MALDI-TOF/TOF analysis (4700 Proteomics Analyzer, Applied Biosystems). Databases (Est-others, NCBInr, NRDB) were queried with either Mascot data files obtained from MALDI-TOF/TOF mass spectral data or the compiled partial de novo sequences obtained per spot, using mascot software available at www. matrixscience.com and MS-Blast50 at http://dove.emblheidelberg.de/Blast2, respectively. In case of Mascot results, monoisotopic masses were used to search the databases, allowing a peptide mass accuracy of 0.6 Da and one partial cleavage. Oxidation of methionine and carbamidomethyl modification of cysteine were considered. Statistical Analysis of Changes in Protein Spot Abundance after Wounding. Gels were scanned using the ImageQuant v3.3 densitometer (Molecular Dynamics) and were analyzed by the Image Master Platinum software v.5.0 (GE Healthcare). All statistical analyses were based on at least four biological replicates from independent experiments. To correct for gelgel variations in spot staining intensities, the sum of spot volumes in replicate gels were normalized to 100 and individual spot volumes were presented as a % of total spot volume. Only spots detected in at least three biological replicate gels were considered for further analysis. To test the statistical significance of changes in spot volumes, a paired Student’s t-test was applied, and levels of p e 0.05 were considered significant. To limit the identification of false significant changes, the data was then postfiltered with an intensity-dependent threshold fold-change. Threshold values of (2 SD were calculated from the variation in replicate data derived from 64 IB and 180 IF proteins reproducibly detected in control samples, as described in Results. Wound-regulated proteins (WRPs) corresponded to those displaying statistically significant changes in spot volume > threshold values after wounding. The distinction of ROSdependent and ROS-independent WRPs was based on the sensitivity their wound-regulation displayed to infiltration with 5 µM DPI. 2300
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Figure 1. Changes in the leaf cell wall proteome (ionically bound proteins) of M. truncatula 0-72 h after wounding. Two areas of the 2-DE gels (left and right panels) containing a high number of wound-responsive proteins were selected as representative examples. After 6 h, there is already some considerable quantitative differences visible in spots 28, 29, 36, 39, 40 (6 h left panel) and spots 2, 7, 10 (6 h right panel). Transient changes can be seen in, for example, spots 40 (6-12 h, left panels) and spots 4, 6 (12-24 h, right panels), as well as later changes, including quantitative differences in spots 5, 3, 9 (72 h, right panel) and the de novo appearances of spots 131 and 132 (72 h, right panel). Gels were loaded with 40 µg of total protein.
Results The Leaf Apoplast Proteome Undergoes Considerable Changes in Response to Wounding. The changes in leaf apoplastic proteins in response to leaf wounding were initially studied over 72 h by 2-DE. Six hours after wounding, the number of proteins that showed differential expression was already considerable, for example,spots 26, 28, 36, 39, 40 (Figure 1, 6 h left panel) and spots 2, 7, 10 (6 h right panel). Some transient quantitative differences were also observed in spot 40 (6-12 h, left panels) and spots 3, 4, and 6 (12-24 h, right panels), as well as later (post 12 h) changes including quantitative differences in spots 5 and 9 (72 h, right panel) and the de novo appearances of spots 131 and 132 (72 h, right panel). This indicates that the apoplast is an important site for the expression of leaf wound responses and that there exists a complex temporal regulation of the apoplastic proteome during this process. To investigate differentially regulated apoplastic proteins that could be more credibly associated with the earliest events following wounding, such as an initial oxidative burst, subsequent 2-DE comparisons were made between unwounded leaves and those that had been wounded for 6 h.
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Figure 2. Detection of H2O2 production in wounded Medicago leaves by DAB staining. (A-D) Time course assay of wound-induced H2O2 accumulation. (E) Inhibitory effects of 5 µM DPI on wound-induced H2O2 production. (F-H) Greater magnification of the wound site: (F) leaf decolorization immediately after wounding; (G) 6 h after wounding; (H) wounded leaf infiltrated with 5 µM DPI and decolorized 6 h after wounding. (I) Detached leaf supplied with DAB; (J) detached leaf supplied with DAB and 100 µM DPI. N.B. inhibition of leaf vein H2O2.
ROS Production in Response to Wounding. Changes in H2O2 and O2- in response to leaf wounding were assayed by histochemical studies with DAB and NBT, respectively. A time course assay of the development of the H2O2-DAB reaction product (up to 6 h) demonstrated that H2O2 accumulation could be first detected 1 h after wounding, particularly at the wound site. Staining was also evident in the mid-rib at the site of detachment and in the vascular tissues. In all cases, the staining intensified over 6 h after wounding (Figure 2A-D). DAB staining of veins was also evident in detached, but otherwise unwounded leaves (Figure 2I). In an attempt to inhibit H2O2 production in leaves, we have infiltrated leaves with a NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI) at 5 µM, immediately after wounding. This treatment reduced the accumulation of H2O2 at the wound site, but not in the veins (Figure 2E,H), which could be inhibited only at higher (100 µM) concentrations of DPI (Figure 2J). Considering DPI can inhibit different sources of H2O2, namely, NADPH oxidases and peroxidases, but with differing inhibitory efficacies,51,52 these results suggest that H2O2 production proximal to the wound site employs a different mechanism than that required for H2O2 in vascular tissues. A time course of O2- development showed that O2- can be detected as early as 3 min after wounding, where it accumulates strongly proximal to the wound site. This staining, as well as that in vascular tissues removed from the cut site, intensifies over 6 h (Figure 3A-H,L). The infiltration of 5 µM DPI within 3 min after wounding substantially reduced O2- production in both tissues over 6 h (Figure 3I,M). This inhibition was less effective if leaves were infiltrated 15 min after wounding (Figure 3J), indicating that later phases of the wound-induced oxidative burst require successful development of the early O2- production. The kinetics of the O2- production was examined more closely by infiltration of NBT at different times after wounding, then allowing staining to develop over 5 min before leaf decolorization. This permitted us to evaluate the level of O2production at specific times (Figure 3N-U). Our observations strongly suggest that O2- production is a biphasic process. An initial phase was characterized by high production of O2- within minutes, which substantially decreased over 30 min and was
undetectable after 1 h (Figure 3N-Q). A renewed production of O2- became detectable between 1-3 h after wounding (Figure 3R), followed by a decrease to negligible levels at 6 h (Figure 3U). The Rapid Production of O2- Is Required for the Regulation of Early Wound-Responsive Apoplastic Proteins. In a previous study, we described our 2-DE and MALDI-TOF/TOF analysis of the M. truncatula leaf apoplast proteome.44 Here, we wished to identify proteins differentially expressed within 6 h after wounding and to determine which could be related with early ROS signaling events. For this purpose, comparative analyses were made with apoplastic proteins extracted from control leaves (unwounded) and from leaves which had been infiltrated with 0 or 5 µM of the NADPH oxidase inhibitor, DPI, within 3, 6, or 15 min after wounding and left until 6 h. Six hours after wounding, changes in 28/110 proteins of the apoplast proteome could be reproducibly detected. These 28 proteins were therefore classified as wound-responsive proteins (WRPs). The infiltration of DPI e 3 min after wounding effectively suppressed the wound-related changes in 9 of these WRPs (Table 1 and Figure 4). The Infiltration of DPI after 6 min was not as effective in this suppression, and infiltration after 15 min produced no significant inhibition of woundrelated changes. This result is shown qualitatively for WRPs 44 and 65 in Figure 4. A larger selection of proteins with differing sensitivity to wounding and the application of DPI after 3 min are depicted in Figure 5. Considered together with the histochemical data obtained, these results indicate that the differential regulation of 9/28 WRPs depends on the successful development of a O2- burst within the first minutes after wounding. On the basis of this result, we have classified the 28 WRPS as either ROS-dependent (9 proteins) or ROSindependent (19 proteins; Table 1). A more complete description of the apoplast proteome and the sensitivities to wounding and DPI can be found in Table 2 within Supporting Information. In a further assay, higher DPI concentrations (5-100 µM) were infiltrated within 3 min after wounding and changes in spot intensities were assayed after 6 h. All concentrations suppressed the differential regulation of the 9 ROS-dependent Journal of Proteome Research • Vol. 8, No. 5, 2009 2301
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Figure 3. Detection of O2- production in wounded Medicago leaves by NBT staining. (A-H) Time course assay of wound-induced O2accumulation. (I and J) Inhibitory effects of 5 µM DPI on wound-induced O2- production: (I) infiltration with DPI within 3 min, (J) infiltration with DPI at 15 min. (K-M) Greater magnification of wound site: (K) leaf decolorization immediately after wounding, (L) decolorization 6 h after wounding, (M) wounded leaf infiltrated with 5 µM DPI and decolorized 6 h after wounding. (N-U) Kinetics of the O2accumulation. Wounded leaves were incubated for the period indicated before infiltration with NBT and decolorization after a further 5 min.
WRPs observed above, and no further suppression of woundrelated changes in the proteome could be detected (data not shown). To confirm the involvement of ROS in the regulation of these WRPs, we have infiltrated wounded leaves with DPI together with different exogenous sources of ROS: menadione, as a supply of O2-, and glucose oxidase + glucose, as a source of H2O2. Analyses were made with IB proteins, since this fraction contained 8/9 of the ROS-dependent WRPs identified. These analyses revealed that the supply of menadione apparently bypassed the DPI inhibition of 3 ROS-dependent WRPs (spots 7, 10, 62), whereas glucose-oxidase effectively reconstituted the wound responsiveness of 7 ROS-dependent WRPs (spots 7, 10, 26, 44, 50, 62, 65). The effect on the levels of WRPs 10, 44, and 65 is shown in Figure 6. A single ROS-dependent WRP (spot 2; ABC-a.a. type transporter) remained unresponsive to either source of ROS (results not shown). These results confirm that at least 7 WRPs are regulated in a ROS-dependent manner, but also indicate that H2O2 is implicated in the regulation of the majority of these proteins. Identifying ROS-Dependent and ROS-Independent Changes in Apoplastic Proteins of the Wounded Leaf. To test for significant differences in treatment versus control data, we have used Student’s paired t-test. However, such tests of significance can often produce false positives when applied to large data sets with a limited number of replicates. This has prompted the additional use of a fold-change threshold in a number of studies. Here, we calculated the variation of the control data set, so that a statistically relevant fold-change threshold corresponding to 2 SD of the control data set could be applied. Treatment-induced fold-changes of g2 SD can be expected to have e5% probability of belonging to the control population. 2302
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An analysis of the protein spot volume data of 64 IB and 180 IF proteins from four control data sets was used to estimate the variation in protein spot volumes inherent in our experimental system (sum of quantitative + biological variation). An initial analysis showed the variation to have a multiplicative error structure with increasing mean spot volume (data not shown). The data was therefore transformed logarithmically to approach a normal distribution by:
xi,j ) ln
svi,j svj
(1)
where svi,j is the replicate spot volume data i from spot j and svj is the calculated average spot volume of spot j. When plotted as a probability density function (F(x)), the data can be seen to follow a normal distribution (Figure 7A), and a KolmogorovSmirnov test of goodness of fit of the observed distribution to the theoretical, Gaussian distribution indicated that deviation was only significant at p e 0.005. However, further analysis demonstrated that a considerable number of low-abundance proteins display high variation. This can be seen most clearly when relative foldchanges in control spot volume (inherent variation) were calculated from all possible pairwise comparisons of replicate data for each spot and plotted against spot volume. The relationship of fold-change with spot volume is emphasized by calculating the local average fold-change for intervals along the x-axis (Figure 7B). The relationship described is one where greatest errors are obtained with spot volumes approaching the limit of quantitative resolution. Least
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Table 1. Wound-Responsive Proteins in the Leaf Apoplast of Medicago and Their Association with Early ROS Signallinga average spot volume ( SD
spot spot no.
2 4 7 10 11 12 26 27 28 36 37 39 40 43 44 46 48 49 50 62 65 68 69 72 73 81 82 87 89 93 100 101 113 116 118
homologue
accession no.c
ABC-a.a type transporter BAD04724 Plastocyanin CAA26709 Peroxidase precursor AAB41811 Superoxide dismutase AAC14127 Superoxide dismutase AAB05888 Lysine-rich wall structural XP_465138 protein Putative NAD-dependent CAD76101 oxidoreductase Sensory box histidine kinase AAR34625 Oxygen evolving enhancer AW559699 protein 1 ATP synthase beta AAK72818 Peroxidase AAB41811 S-Adesonylmethionine synthase Q5P2V5 Triophosphate isomerase AAT46998 Germin-like protein P94040 Germin-like protein AW776317* Oxygen evolving enhancer P12359 protein 1 Type I antifreeze protein AAR22529 Vacuolar sorting-associated CAA81876 protein ATP synthase (beta subunit) AAD46914 Unknown protein AAB41813 Peroxidase CAA62226 Oxygen evolving enhancer AW776405* protein 1 β-1,3-Glucanase Q9ZP12 β-1,3-Glucanase Q9ZP12 Class II Chitinase P29024 Class II Chitinase Q9SDY6 Not Identified Chitinase CAA64868 Not Identified β-1,3-Glucanase AF435088* Thaumatin-like protein O04364 Not Identified Not Identified Not Identified Not Identified -
classd
control
wounded
Student’s t testb DPI
cont. cont. wound vs wound vs dpi vs DPI
regulation up/ downe
ROS dep.f
T/B EPC OR OR OR S
1.99 ( 0.43 4.98 ( 1.23 3.74 ( 0.43 0.88 ( 0.16 0.39 ( 0.15 0.29 ( 0.21
3.77 ( 0.34 7.73 ( 0.85 1.58 ( 0.83 2.2 ( 0.22 0.46 ( 0.07 0.56 ( 0.26
0.7 ( 0.99 12.53 ( 0.41 3.33 ( 0.97 1.77 ( 0.19 0.27 ( 0.5 0.34 ( 0.21
0.010 0.001 0.010 0.010 -
0.001 0.05 -
0.010 0.050 0.050 0.01 -
Up Up Down Up Const. Const.
Yes No Yes Yes -
OR
0.85 ( 0.17
1.72 ( 0.2
NDg
0.001
-
-
Up
Yes
ID EPC
0.94 ( 0.23 1.2 ( 0.47
1.25 ( 0.41 0.8 ( 0.24 2.57 ( 0.96 3.27 ( 0.95
0.050
0.001
-
Const. Up No
0.050 0.050 0.001 0.010 0.001
0.050 0.001 Down No Const. 0.050 Down No 0.010 0.050 Down No Const. 0.001 0.001 Up Yes 0.001 Down No
EPC 2.23 ( 0.93 1.31 ( 0.11 OR 0.56 ( 0.34 0.44 ( 0.04 Misc 0.86 ( 0.22 0.35 ( 0.19 EPC 2.43 ( 0.46 0.52 ( 0.08 OR 9.96 ( 2.10 8.95 ( 2.69 OR 20.15 ( 2.94 34.79 ( 2.22 EPC 6.84 ( 0.05 0.53 ( 0.42
0.32 ( 0.22 0.37 ( 0.81 0.22 ( 0.9 0.16 ( 0.11 11 ( 0.63 11.39 ( 4.25 ND
D Misc
1.51 ( 0.22 1.54 ( 0.61
0.87 ( 0.23 1.08 ( 0.5 0.25 ( 0.14 ND
0.010
0.001
-
Const. Down No
EPC U OR EPC
0.54 ( 0.25 0.44 ( 0.32 0.93 ( 0.42 1.37 ( 0.95
1.17 ( 0.44 0.93 ( 0.22 2.51 ( 0.67 1.64 ( 1.57
ND 0.31 ( 0.08 1.32 ( 1.1 1.25 ( 0.58
0.050 0.050 0.010 -
-
0.010 0.010 -
Up Up Up Const.
Yes Yes Yes -
D D D D U D U D D U U U U
0.25 ( 0.06 0.04 ( 0.03 1.39 ( 0.24 4.48 ( 0.77 1.89 ( 0.21 3.74 ( 0.68 0.46 ( 0.05 1.03 ( 0.46 0.55 ( 0.05 0.03 ( 0.01 0.53 ( 0.03 0.18 ( 0.01 0.52 ( 0.04 0.28 ( 0.01 1.41 ( 0.14 3.99 ( 0.54 0.25 ( 0.06 0.04 ( 0.03 0.25 ( 0.07 0.04 ( 0.01 0.33 ( 0.06 0.74 ( 0.10 0.24 ( 0.07 0.86 ( 0.19 3.08 ( 0.57 10.93 ( 1.83
ND 4.49 ( 0.71 3.72 ( 0.53 1.39 ( 0.33 0.16 ( 0.08 0.34 ( 0.06 0.41 ( 0.03 3.10 ( 0.79 ND ND ND 1.05 ( 0.33 9.19 ( 1.02
0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050
0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050
-
Down Up Up Up Down Down Down Up Down Down Up Up Up
No No No No No No No No No No Yes No No
a Quantitation of the constitutively expressed proteins (11, 12, 27, 37, 43, 48 and 68) in control and treated leaves is shown for comparison. b Student’s t test of statistical significance of changes in spot volumes. Levels of p e 0.05 were considered significant. c Accession number for the homologue in NCBI database. Proteins identified by the Genebank accession number are identified by an asterisk. d The protein class abbreviated as follows: T/B, Transport/ binding; EPC, Energy production and conversion; OR, Oxidoreductase; S, Structural; ID, Interaction domain; Misc, Miscellaneous; D, Defense; U, Unknown. e Const., constitutive protein that remained unchanged with wounding; Up, proteins up-regulated after wounding; Down, those that were down-regulated after wounding. f No, wound-responsive proteins regulated in a ROS-independent manner; Yes, wound-responsive regulated in ROS dependent manner. g ND, not detectable.
squares regression was used to obtain the best fit of the data to the exponential decline: FC ) a · sv-b + c
(2)
where FC is the fold-change, sv is spot volume, a and b describe the parameters of the exponential decline and c is the basal level of error. Atypical behavior of low abundance spot proteins have also been reported by Gustafsson et al.53 and noted by Asirvatham et al.,54 and is also a characteristic of low-abundance gene copies in microarray studies.55 This atypical behavior indicates that defining a single threshold fold-change for the whole data
set would be misleading, since it would be both too permissive for low-abundance proteins and excessively high for proteins in the mid-high range of volumes. We have therefore chosen to utilize a spot intensity-dependent threshold fold-change in our studies, similar to that described by Mariani et al.55 Threshold fold-changes corresponding to 2 SD were therefore calculated from data pools derived from contiguous intervals over the range of spot volumes. As shown in Figure 7C, the derived threshold fold-change declines exponentially, and can be modeled by least-squares regression to fit eq 2 (parameters displayed in figure). This equation was used to determine the threshold foldchange treatments had to induce in control spot volumes Journal of Proteome Research • Vol. 8, No. 5, 2009 2303
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Figure 4. The effect of DPI on the expression of ionically bound WRPs, when infiltrated at different times after wounding. The figure shows areas of the 2-DE gels depicting selected WRPs (top two panels), and demonstrates how the application of 5 µM DPI within 3 min after wounding effectively suppresses some downstream (6 h) wound-related changes (middle panel). The inhibitory effects of DPI can be seen to be less effective after 6 min and completely ineffective after 15 min (bottom two panels). Gels were loaded with 40 µg of total protein.
to provide intensity changes significant at the 95% confidence level and which could be considered further. It is of interest that the application of the standard Student t-test alone indicated 32 WRPs and 14 ROS-dependent WRPs, whereas postfiltering of the data with the cutoff threshold fold-changes described above reduced this to 28 and 9, respectively (Table 1). MALDI-TOF/TOF Characterization of Wound-Responsive Proteins. The 2-DE studies revealed that from the 110 indicated apoplastic proteins, 82 remained constitutive, whereas 28 showed statistically significant differential regulation in response to wounding (Table 1). From these 28 proteins 21 showed homology with known proteins and one had homology with an unknown protein (spot no. 62). For the remaining proteins (spot nos. 82, 89, 101, 113, 116 and 118), although it was possible to obtain de novo sequence information from a good spectra, no significant similarity was obtained to any protein in the current databases. There was no obvious functional compartmentalization between WRPs and constitutive proteins, although it is worth 2304
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Figure 5. The identification of WRPs mobilized by ROS signals originating from the earliest phases of wounding. Depicted is a comparison of extracellular proteins extracted from unwounded leaves (Control), wounded leaves after 6 h (Wounded), and wounded leaves (6 h) infiltrated with 5 µM DPI immediately after wounding (Wounded + DPI). Proteins that remained unchanged with treatments were classified as constitutive (e.g., spots 29, 68, 48, 37 {left panel} and spots 6, 3, 1 {right panel}). WRPs that were insensitive to DPI were denominated ROS-independent WRPs (e.g., spots 28, 39, 36, 40, 46, 49 {left panel}). WRPs whose response to wounding could be suppressed by DPI were denominated ROS-dependent WRPs (e.g., spot 26 {left panel}; spots 7, 10 {right panel}). Selected subsections of silver stained 2-DE gels are shown above. All gels were loaded with 40 µg total protein.
noting that no WRPs were found among structural proteins, those containing interaction domains or glycoactive proteins (Figure 8). The proportion of IF proteins that suffered differential regulation with wounding was similar to that observed for IB proteins. Qualitatively however, most of the WRPs in the IF were either defense-related proteins or of unknown nature, whereas the analyses of IB proteins identified WRPs of diverse types, including proteins related with energy and conversion, oxidoreductases, transport/binding, miscellaneous and unknown (Figure 8). This is in agreement with our previous results, which describe a functional distinction between IF and IB proteins in M. truncatula.44 The analyses further revealed that, in the IF, the differential regulation of a single WRP was ROS-dependent, compared with 8 WRPs in the IB fraction. This suggests that the ECM, rather than intercellular spaces, is a main site targeted by ROS signaling during wounding in Medicago. With the exception of a peroxidase, all ROS-dependent WRPS were up-regulated (Table 1), indicating that ROS signaling functions in early wounding responses more as a protein inducer rather than a repressor. This is in agreement with observations reported in Arabidopsis where of the 175 nonredundant expressed sequences tags regulated by H2O2, 113 are induced and 62 are repressed by oxidative stress-related H2O2.37 Interestingly, whereas no oxidoreductases could be identified among the ROS-independent WRPs, a number of ROS-dependent WRPs could be related with ROS metabolism, including a SOD, a germin-like protein and two peroxidases (Table 1).
Environmental Stress and Adaptation Although constitutively expressed oxidoreductases (e.g., proteins 11, 37 and 43) might also contribute to the regulation of ROS levels during wounding, the ROS-dependent expression of WRPs with roles in ROS metabolism are of especial interest, since they can be specifically examined for their potential role in ROS-signaling and ROS-regulatory events in downstream phases of the wounding response.
Discussion ROS signaling pathways appear to play a central role in the coordination of plant responses to a wide variety of stress.23,24,56 In this report, the relationship between the early production of ROS and changes in the apoplast proteome has been examined in wounded M. truncatula leaves. Interestingly, in contrast to Arabidopsis or tomato, wound-related ROS signaling was reported to be inactive in Medicago and three other Fabaceae species.23 In agreement, Leitner et al.57 indicated that ROS generated in response to herbivorous attack of M. truncatula leaves were not produced by wounding alone. Here, we have demonstrated that M. truncatula does generate both H2O2 and O2- in response to wounding and that this occurred both proximal to the wound site and in surrounding leaf tissues (Figures 2 and 3). The kinetics of ROS production in response to wounding appears to differ substantially between species, varying from biphasic productions, such as in ryegrass24 to long-term and multiphasic productions, such as in potato.6 Our findings suggests Medicago employs a biphasic production of ROS in response to wounding: a first phase apparently predominated by O2- production that occurs within seconds after wounding and a later phase of both O2- and H2O2 production between 1 and 3 h later.
research articles We have utilized the application of DPI at different times after wounding to probe the relation of ROS production with wound-related changes in the apoplast proteome. The infiltration of DPI 15 min after wounding effectively inhibited phase II ROS production (1-6 h), but had no detectable effect on the wound-regulated changes in the apoplast proteome over 6 h. This therefore precludes a role for phase II production of O2and H2O2 in the regulation of these early WRPs. However, the infiltration of DPI within 3 min after wounding suppressed both phase I production of O2- and the differential regulation of 9/28 WRPs. This strongly suggests that a O2--driven signaling pathway is rapidly deployed in wounded Medicago and that this pathway coordinates the regulation of several WRPs within the early (3-6 h) wound response. Whether this regulation involves a direct interaction of O2- with downstream signaling components, or requires its conversion to other ROS intermediates, is currently unclear. However, a role for ROS in the differential regulation of a subset of WRPs was supported by the observation that inhibition of the wound response in 7/8 (ROS-dependent) IB WRPs by DPI could be bypassed by supplying exogenous sources of ROS. The wound surface can facilitate pathogen ingress, and therefore, wounded tissues require the rapid deployment of defense proteins to avoid infection and disease. We find that 13 IF proteins were differentially regulated in response to wounding and 7 were defense-related (Figure 8A). However, only a single IF protein of unknown function could be classified as a ROS-dependent WRP (protein 113, Table 1), suggesting that, in wounded Medicago, IF proteins are not major targets for regulation by ROS signaling pathways. In contrast, IB proteins of diverse natures were differentially regulated after wounding, and a higher proportion of these
Figure 6. The effect of menadione (M) or glucose oxidase + glucose (Glu-ox) on the expression of WRPs 10, 44, and 65 in DPI-treated wounded leaves. Both exogenous supplies of ROS bypass DPI inhibition of wound-responsive superoxide dismutase (WRP 10; left panels). The supply of glucose oxidase + glucose bypasses DPI inhibition of the wound-response in a germin-like protein (WRP 44) and a peroxidase (WRP 65), but menadione seems to be ineffective (middle and right panel columns). All gels were loaded with 40 µg total protein. Journal of Proteome Research • Vol. 8, No. 5, 2009 2305
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Figure 8. A functional classification of leaf apoplastic proteins in Medicago and their relationship with ROS signaling in response to wounding. The protein class was abbreviated as follows: D, Defense; EP&C, Energy Production and Conversion; GA, Glycoactive; ID, Interaction Domains; Misc, Miscellaneous; OR, Oxidoreductase; S, Structural; T/B, Transport/Binding; U, Unknown.
Figure 7. Calculating an intensity-dependent threshold foldchange to account for abnormally high variation in proteins with low spot volumes. (A) Variation in the log-normalized data set (bars) fitted to a Gaussian distribution (continuous line). (B) Variation in basal fold-change in the control data set (open gray diamonds) as a function of spot volume. A pairwise comparison of replicate data was used. Localized averages (black diamonds) were calculated and the best fit (black line) was sought by regression analysis to an exponential decline (eq 2). (C) Threshold fold-change as a function of spot volume. Threshold fold changes equivalent to 2 SD were calculated for intervals of spot volumes over the range indicated and the best fit (line) to an exponential decline (eq 2) was sought by regression analysis (parameters displayed in upper right-hand corner). 2306
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wound-responsive proteins of this fraction was ROS-dependent. Wound-responsive IBs contained several oxidoreductases (5) and those functionally associated with energy production and conversion (6; Figure 8B). Some of these proteins are not predicted to be classical secretory proteins but are of an unexpected nature. Previously, we have reported that e8% of proteins in apoplast extracts prepared under identical conditions to those utilized here are potentially symplastic contaminants,44 suggesting that at least some of these unexpected proteins are actually secreted to the apoplast. However, the presence of these proteins in apoplastic extracts remains a controversial subject, and requires additional confirmatory studies. We therefore place more emphasis on those WRPs which that can be described as classical extracellular proteins. A potentially interesting group of WRPs are IB oxidoreductases (5), all of which were additionally dependent on ROS production for differential expression. In Arabidopsis, at least 152 genes constitute a dynamic reactive oxygen gene network and are differentially coordinated to regulate ROS accumulation in a spatially controlled manner36 The variable composition of this network appears to reflect the changing requirements for redox maintenance and ROS signaling in development and the acclimation to stress.29,39 Relative to the symplasm, the apoplasm has been described as having relatively little antioxidative defense.58 Our results revealed that a considerable proportion of apoplastic proteins has potential roles in ROS
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Environmental Stress and Adaptation 44
metabolism (14%, 15 proteins) and of these, 5 were ROS dependent proteins, suggesting they constitute part of the Medicago reactive oxygen gene network related with redox control and signal regulation within the extracellular matrix. Although wound-related changes in other apoplastic oxidoreductases may contribute to the regulation of ROS, early changes in oxidoreductases regulated by ROS are of especial interest, since their functional relation with ROS metabolism is highly suggestive of their deployment as part of regulatory feed-back or feed-forward loops within later stages of the oxidative burst. Such oxidoreductases included two peroxidases, a SOD, a germin-like protein and a protein with homology to a NAD-dependent oxidoreductase. The up-regulated extracellular SOD could contribute to the regulation of wound-related changes in apoplastic O2-/H2O2 levels through the dismutation of O2- to H2O2.59 Similarly, the up-regulation of a germin-like protein, which can also present oxalate oxidase activity (oxo) or superoxide dismutase activity,60,61 may also contribute directly to later changes in ROS levels. Class III peroxidases (EC 1.11.1.7) have frequently been associated with wound responses62,63 and can utilize hydrogen peroxide for the peroxidation of many reducing substrates to reinforce cell walls through lignification,64 suberin synthesis65 or the cross-linking of extensins,31 which can confer cell resistance do pathogen attack.32 As such, these enzymes can represent a considerable sink for apoplastic H2O2. Conversely, under certain conditions, class III peroxidases can also contribute to the production of stress-related H2O2.52,66 Immunocytochemical studies to probe the potential sink/source relationship of these proteins with the oxidative burst in wounded leaves could therefore be informative. Relative to Arabidopsis, the relationship between MAPK pathways and ROS signaling in Medicago has received little attention to date. However, H2O2 was found to activate OMTK1 (oxidative stress-activated MAPKKK1) in Medicago sativa. Active OMTK1 is capable of interacting with and activating the MAPK, MMK3, suggesting the existence of a MAPK scaffold involved in transducing H2O2 signals.67 Additional potential components include the recently characterized MtRbohs (Medicago respiratory burst oxidase homologues; NADPH oxidases).68 Other stress-responsive components of MAPK pathways have been described in Medicago, including the wound-activated MAPK, SIMK, and its negative regulator, the protein phosphatase 2C, MP2C,69 although their relationship with early ROS signaling events remains unclear. This proteomic study has identified several wound-regulated proteins in the apoplast whose differential regulation can be classified as ROS-dependent or ROS-independent. This study therefore provides several potentially useful molecular markers with which to probe the relationship that known MAPKs and related components may have with early ROS signaling in wounded Medicago.
Acknowledgment. This work was partly financed by the Instituto de Biologia Experimental e Tecnolo´gica (Plant-IBET initiative). Nelson C. Soares acknowledges a fellowship (ref: SFRH/BD/6487/2001) from Fundac¸a˜o para a Cieˆncia e a Tecnologia. Supporting Information Available: Table of MALDITOF/TOF characterization of leaf apoplastic proteins of Medicago. This material is available free of charge via the Internet at http://pubs.acs.org.
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research articles
Environmental Stress and Adaptation oxidative burst in response to biotic stress in plants: A threecomponent system. J. Exp. Bot. 2002, 53, 1367–1376. (67) Nakagami, H.; Kiegerl, S.; Hirt, H. Omtk1, a novel mapkkk, channels oxidative stress signaling through direct mapk interaction. J. Biol. Chem. 2004, 279 (26), 26959–26966. (68) Lohar, D. P.; Haridas, S.; Gantt, J. S.; VandenBosch, K. A. A transient decrease in reactive oxygen species in roots leads to root hair
deformation in the legume-rhizobia symbiosis. New Phytol. 2007, 173 (1), 39–49. (69) Meskiene, I.; Baudouin, E.; Schweighofer, A.; Liwosz, A.; Jonak, C.; Rodriguez, P. L.; Jelinek, H.; Hirt, H. Stress-induced protein phosphatase 2c is a negative regulator of a mitogen-activated protein kinase. J. Biol. Chem. 2003, 278 (21), 18945–18952.
PR8009353
Journal of Proteome Research • Vol. 8, No. 5, 2009 2309
