Identification of Ethylene-Mediated Protein Changes during

Identification of Ethylene-Mediated Protein Changes during Nodulation in Medicago truncatula Using Proteome Analysis Joko Prayitno,† Nijat Imin,† Barry G. Rolfe,† and Ulrike Mathesius*,‡ ARC Centre of Excellence for Integrative Legume Research, Genomic Interactions Group, Research School of Biological Sciences, The Australian National University, Canberra ACT 0200, Australia, and ARC Centre of Excellence for Integrative Legume Research, School of Biochemistry and Molecular Biology, The Australian National University, Canberra ACT 0200, Australia Received May 30, 2006

Ethylene has been hypothesised to be a regulator of root nodule development in legumes, but its molecular mechanisms of action remain unclear. The skl mutant is an ethylene-insensitive legume mutant showing a hypernodulation phenotype when inoculated with its symbiont Sinorhizobium meliloti. We used the skl mutant to study the ethylene-mediated protein changes during nodule development in Medicago truncatula. We compared the root proteome of the skl mutant to its wildtype in response to the ethylene precursor aminocyclopropane carboxylic acid (ACC) to study ethylenemediated protein expression in root tissues. We then compared the proteome of skl roots to its wildtype after Sinorhizobium inoculation to identify differentially displayed proteins during nodule development at 1 and 3 days post inoculation (dpi). Six proteins (pprg-2, Kunitz proteinase inhibitor, and ACC oxidase isoforms) were down-regulated in skl roots, while three protein spots were up-regulated (trypsin inhibitor, albumin 2, and CPRD49). ACC induced stress-related proteins in wildtype roots, such as pprg-2, ACC oxidase, proteinase inhibitor, ascorbate peroxidase, and heat-shock proteins. However, the expression of stress-related proteins such as pprg-2, Kunitz proteinase inhibitor, and ACC oxidase, was down-regulated in inoculated skl roots. We hypothesize that during early nodule development, the plant induces ethylene-mediated stress responses to limit nodule numbers. When a mutant defective in ethylene signaling, such as skl, is inoculated with rhizobia, the plant stress response is reduced, resulting in increased nodule numbers. Keywords: ethylene • Medicago truncatula • model legume • nodulation • protein identification • sickle mutant • Sinorhizobium meliloti

1. Introduction Symbiotic nitrogen fixation between legumes and nitrogenfixing soil bacteria, generically called rhizobia, occurs in specific root and stem tissues called nodules. Nodule formation is a complex process involving signal exchanges between the host plant and its symbiotic partner. The successful symbiotic nitrogen fixation comprises two events that operate simultaneously to form functional nodules: (i) the initiation of infection threads from root tips and their growth toward dividing cells in the cortex and (ii) the initiation of nodule primordia from cortical cell divisions and their subsequent development leading to mature nodules.1 The host plant internally regulates both events through local and systemic mechanisms to maintain a certain number of nodules on the * To whom correspondence should be addressed. Tel: 61 2 6125 2840. Fax: +61 2 6125 0313. E-mail: [email protected]. † Research School of Biological Sciences, The Australian National University. ‡ School of Biochemistry and Molecular Biology, The Australian National University.

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root system. As a result, the progression of most infection threads and the maturation of most nodule primordia on the roots are arrested, allowing limited numbers of functional nodules on the root. Ethylene, a gaseous phytohormone, plays a role in the regulation of nodule development. Several studies have shown that Rhizobium inoculation leads to a temporal induction of ethylene production,2,3 and that exogenous ethylene can reduce nodule numbers.4 Similarly, an inhibitor of ethylene biosynthesis, L-R-aminoethoxyvinyl-glycine (AVG), was found to increase nodule numbers in alfalfa and Medicago truncatula.5,6 Using in situ hybridization, Heidstra et al.7 demonstrated that aminocyclopropane carboxylic acid (ACC) oxidase, the enzyme catalyzing the conversion of ACC to ethylene, is highly expressed opposite the phloem poles following Rhizobium inoculation where it was suggested to inhibit nodule initiation and thus determine the radial position of nodule initiation. A mutant of M. truncatula defective in ethylene perception has been isolated based on the lack of ethylene responses in a triple response assay.8 This mutant, sickle (skl), is insensitive 10.1021/pr0602646 CCC: $33.50

 2006 American Chemical Society

Ethylene-Mediated Changes in Nodulation in M. truncatula

to ethylene and has an approximately 10-fold increase in nodule numbers relative to its wild-type.8 Its hypernodulation and ethylene-insensitive phenotypes are determined by a single recessive allele, most similar to the ein2 gene of Arabidopsis.8 EIN2 is an important component of ethylene signaling, which directly regulates the transcription factors EIN3 and EIN3-like.9 In skl, the regulation of radial positioning of nodule primordia is lacking, resulting in the formation of nodule primordia across the circumference of the root.10 Likewise, the number of sustained infection threads in skl is increased.8 These results demonstrate that the host plant controls the progression of Rhizobium infection using endogenous ethylene. Together, ethylene has an effect on both events of nodulation, which is associated with the regulation of nodule meristems,7 and the control of rhizobial infection.11 It has been suggested that the increase of nodule numbers and sustained infection thread growth in skl could be caused by the inhibition of plant defense.8 However, so far no molecular details about the action of ethylene during nodulation is known. Here, we used a proteomic approach to identify proteins differentially regulated by ethylene and differentially regulated during the early stages of nodule development in M. truncatula wild-type and the ethylene-insensitive skl mutant. To achieve this goal, we carried out two sets of experiments. First, wildtype and skl seedlings were grown in the presence and absence of ACC, and their root protein profiles were investigated by 2-DE. This experiment aimed at identifying proteins inducible by ethylene. Second, wild-type and skl seedlings were inoculated with the symbiont Sinorhizobium meliloti, and the protein changes were analyzed at 1 and 3 days post inoculation (dpi). These time points were chosen because they reflect stages of early infection and nodule initiation, respectively, the two nodulation events known to be negatively regulated by ethylene. We then examined whether proteins that were changed by ethylene were also affected by rhizobia in wild-type. We hypothesised that the expression patterns of these proteins should not be affected by ethylene or inoculation in the skl mutant. Here, we report the identification of these differentially displayed proteins. Our results suggest that the hypernodulation phenotype of the skl mutant is a result of a reduction in plant stress or defense responses following inoculation.

2. Materials and Methods 2.1. Plant and Bacterial Growth Conditions. Seeds of the skl mutant were obtained from D. R. Cook.8 Seeds of M. truncatula cv Jemalong A17 were used as the wild-type. Seeds were scarified and surface-sterilized with 6.25% (v/v) sodium hypochlorite for 15 min. After six washes with sterile water, seeds were spread on nitrogen-free Fåhraeus agar medium12 containing 0.8% agar. Seeds were incubated in dark-cold condition (4 °C) for 2 days to break their dormancy, and then germinated in the dark at 28 °C overnight. Seedlings with similar root length were selected and transferred to 15-cm Petri dishes containing Fåhraeus agar medium. For the ACC experiment, the medium was supplemented with 10 µM ACC, where appropriate, and the seedlings were incubated vertically in the growth chamber with a light intensity of 90 µmol m-2 s-1, and 16 h of light per day at 20 °C for 1 and 3 days. To reduce light intensity around roots, pieces of dark-thick paper were placed at the lower half between the plates. For the nodulation experiment, the seedlings were incubated for 2 days in the growth chamber with similar conditions to the ACC experiment, then transferred to fresh Fåhraeus plates and incubated in the

research articles same growth chamber again. The seedlings were inoculated with 5 µL of diluted bacterial suspension 24 h later. S. meliloti strain 1021 was grown in liquid Bergensen’s Modified Medium (BMM)13 at 28 °C overnight and diluted with sterile water to an OD600 of 0.1-0.2 or approximately 107 cells mL-1 (as tested by plate count). As a control, the roots were flood-inoculated with an equivalent amount of diluted BMM. 2.2. Protein Extraction. For the ACC experiment, root segments comprising whole roots (from the base of the hypocotyl to the root tip) were harvested from seedlings at 1 and 3 days of ACC treatment. For the nodulation experiment, root segments representing the nodulation zone (within 1 cm above and below the root tip at the time of inoculation) were harvested without the root tip. Between 100 and 200 root segments were used for each of three biological repeats. The root proteins were extracted as described previously using a TCA-acetone precipitation method.14 2.3. 2-DE and Gel Staining. 2-DE was done as previously described.14 The first dimension IEF was carried out on 24 cm Immobiline Dry Strips with linear pH gradients from 4 to 7 (GE Healthcare, Castle Hill, Australia). For analytical gels, 150 µg of total protein samples was loaded at the anode end of the Multiphor II horizontal electrophoresis system (GE Healthcare). For preparative gels, 1000 µg of total protein was loaded in the same way. The second dimension SDS-PAGE was done using precast Excel Gels with a 12-14% acrylamide gradient (GE Healthcare). The proteins on analytical gels were visualized by silver staining as described previously.14 All staining steps were done using 250 mL of solutions in glass trays on an orbital shaker (70 rpm) at room temperature. Colloidal Coomassie staining was carried out according to Imin et al.15 2.4. Image Analysis. Silver-stained gels were scanned at 600 dots per inch resolution using a UMAX Astra 2400-S scanner (UMAX Techologies, Freemont, CA). All spot detection and spot comparisons were made using Image Master 2D Platinum version 5.0 (GE Healthcare). The relative molecular mass of proteins was determined by the comigration of protein standards (Low Molecular Weight Markers, GE Healthcare) using Image Master Software. 2.5. Experimental Design and Statistical Analysis. To reduce gel to gel variation, the spot volumes were normalized by calculating their relative value to total spot volumes (% spot volume/total spot volumes of all proteins on the gel) using Image Master software. The treatments tested in this study were (1) the effect of genotype (wild-type or skl), (2) the effect of treatment (control, ACC, or Sinorhizobium inoculation), and (3) the interaction of ACC or Sinorhizobium inoculation with the genotypes. Three biological repeats were done for each treatment at each time point for spot comparisons. For comparison of the genotype or treatment effects, data from the same genotype or the same treatment were pooled to give a total of six biological repeats. Each time point was analyzed separately. Data were tested for normality by Shapiro-Wilk test, and when the data were not normally distributed, a nonparametric test (Kruskal-Wallis test and Mann-Whitney test) was used to test for significant differences of the treatment. Homogeneity of the variance was tested using Levene’s test, and data were subjected to square root transformation when the variance among treatments were not homogeneous. For normally distributed data with homogeneous variance, twoway analysis of variance (ANOVA) was used to test significant difference within treatments at P < 0.05. The least significant difference (LSD) was used as a post hoc test when the twoJournal of Proteome Research • Vol. 5, No. 11, 2006 3085

research articles way ANOVA showed significance. All statistical analyses were carried out using SPSS version 13.0. 2.6. Trypsin Digestion and Mass Spectrometry. Protein spots were manually excised from Coomassie-stained gels using sterile scalpel blades. The gel pieces were destained in 50% 25 mM ammonium bicarbonate, pH 7.8, 50% acetonitrile, dehydrated with 100% acetonitrile, and digested with 8 µL of 15 ng/ µL sequencing grade modified trypsin (Promega, Madison, WI) in 25 mM NH4HCO3, pH 7.8, for 16 h at 37 °C. The resulting peptides were acidified with 8 µL of 1% trifluoroacetic acid (TFA) and extracted by sonication. The peptides were purified using C18 reversed-phase ZipTips (Millipore Corp., Bedford) following the manufacturer’s instruction. Peptides were eluted with 70% acetonitrile in 0.1% TFA for MALDI-TOF analysis, or with 50% methanol in 0.1% TFA for LC-MS analysis. For MALDI-TOF analysis, a 1 µL sample aliquot was spotted onto a sample plate, which was pre-spotted with 1 µL of matrix (8 mg/mL R-cyano-4-hydroxycinnamic acid in 70% (v/v) acetonitrile and 1% TFA) and allowed to air-dry. MALDI-TOF analysis was performed with an Applied Biosystems 4700 Proteomics Analyzer (at the Australian Proteome Analysis Facility, Sydney, Australia) or Applied Biosystem 4800 Proteomics Analyzer (at the John Curtin School of Medical Research, The Australian National University, Canberra, Australia) with TOF/TOF optics in MS mode. The spectra were acquired in reflectron mode over the m/z range of 800-3500 Da. The instrument was then switched to MS/MS mode where the 25 strongest peptides from the MS scan were isolated and fragmented, and their mass and intensities were measured. A near point external calibration was applied and gave a typical mass accuracy of ∼50 ppm or less. For LC-MS/MS analysis, a LCQ Deca XP Plus ProteomeX Workstation (Thermo Finnigan, San Jose, CA) was used. Sample (10 µl) was loaded onto the Biobasic SCX ion exchange column (Thermo Electron Corp., Bellefonte, PA), and peptides were eluted successively with 20% and 40% NH4Cl in 0.1% formic acid. The eluate was further separated on a Biobasic C-18 reverse-phase column (100 × 0.18 mm; 5 µm) (Thermo Electron Corp., Bellefonte, PA) with a 5%-80% gradient of acetonitrile in 0.1% formic acid over 60 min. This setup enabled good separation of peptides even if a protein spot contained several proteins. The eluted peptides were subjected to electrospray ionization with a 3 kV nanospray voltage. Full scan spectra were acquired over the m/z range of 300-2000, followed by MS/MS scan for the three most intense ions. 2.7. Database Searches. Mass spectra and ion data generated by MALDI-TOF MS/MS were used to search for protein identification against the M. truncatula EST database (MtGI; http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species) medicago) using Mascot Daemon version 2.1.0 software program (Matrix Science), which at the time of analysis (February 2006) contained 36 878 entries. Mass spectra and ionic data from LC-MS (in .raw format) were converted to .dta files using Bioworks version 3.1 software program (ThermoFinnigan). All .dta files obtained from one sample were concatenated using a Perl script written in ActiveState Perl, as recommended for data searches using Mascot. This Perl script was downloaded from http://www.matrixscience.com/help/instruments_xcalibur.html. For peptide matching, a maximum of one miscleavage per peptide and peptide modifications by oxidation of methionine and carbamidomethylation of cysteine were allowed. The peptide mass tolerance and ion mass (MS/MS) accuracy used for peptide matching were 100 ppm and 0.4 Da, 3086

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Figure 1. Effect of ACC on root length of wild-type (A17) and skl (skl) plants grown under light conditions at 3 days after treatment. Values are average of 100 seedlings ( SD. A star indicates significant difference according to Student’s t-test (P < 0.01) to the control treatment.

respectively, and when no matches were found, the peptide mass tolerance was increased to 500 ppm. The confidence of peptide matches was based on the significant value of the Mowse score, the mass accuracy, the number of peptide matches, and the percentage of sequence coverage. For the nodulation experiment, the S. meliloti genome database (http:// bioinfo.genopoletoulouse.prd.fr/annotation/iANT/bacteria/ rhime/) was also used to find possible matches of differentially expressed proteins to proteins of bacterial origin.

3. Results 3.1. The Effect of Ethylene and S. meliloti Inoculation on Root and Nodulation Phenotypes. To test the sensitivity of the wild-type and skl mutant to ethylene under our growth conditions, seedlings were grown in the presence and absence of 10 µM ACC. As shown in Figure 1, the skl roots were insensitive to 10 µM ACC under light conditions during a 3-day treatment, whereas the wild-type showed a significant reduction in root growth under those conditions. We used similar conditions to grow the plants for 2-D gel analysis. A time-course assay was done to examine the different stages of early nodule development in wild-type and skl. Twenty roots each were inoculated at the zone of emerging root hairs, which is most susceptible to nodule initiation, and examined microscopically over a 5-day period. After 1 day, root hair curling could be observed in the wild-type and skl. Two days after inoculation, the first cortical cell divisions could be detected in approximately 50% of the roots, and after 3 days, more than 90% roots of the wild-type and the skl mutant showed extensive cortical cell divisions or small nodule primordia, the first stage of a forming nodule. After 5 days, most roots showed advanced nodule primordia (data not shown). These results were similar to those reported previously for the skl mutant.8,10 We chose a 1 and 3 day time point for our subsequent proteomic experiments, because these time points reflected the earliest stages of infection and early nodule development, respectively, which are both affected by ethylene. 3.2. Comparison of Protein Profiling between Wild-Type and sickle in ACC Experiment. To compare protein profiles of wild-type and skl roots in the absence and presence of ACC, the roots were harvested at 1 and 3 days after treatment, and their proteins were analyzed by 2-D gel electrophoresis. Image analysis detected approximately 2000 spots in each gel within the pH range of 4-7 and a size range of 8-120 kDa, with high reproducibility between gels (Figure 2). The expression level of 22 spots was significantly changed (P < 0.05) between wild-


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Figure 2. Representative 2-DE protein map of control skl roots harvested at 3 days after treatment. Protein spot changes when compared to control wild-type roots or to ACC treatment are marked with numbers and listed in Table 1. Picture is taken from a silver-stained gel.

Figure 3. Highlight of some differentially displayed proteins in wild-type (A17) and skl roots in response to ACC at 3 days of treatment.

type and skl, either in response to genotype or in response to ACC treatment. The selected parts of gels showing differentially displayed wild-type and skl root proteins in the absence (control) and presence of ACC are highlighted in Figure 3. Examination of the genotype effect (wild-type vs skl) showed that seven spots (s01, s02, s03, s13, s46, s56, and s58) were significantly (P < 0.05) down-regulated, and four spots (s39, s41, s43, and s60) were significantly up-regulated in skl roots across time points (Table 1). Of seven proteins down-regulated in skl, three (s01, s02, and s03) belonged to the pprg-2 protein family, one was identified as Kunitz proteinase inhibitor (s13), two proteins (s56 and s58) were identified as ACC oxidase, and one (s46) could not be identified (Table 2). Four proteins up-regulated in skl were albumin 2 (s39), trypsin inhibitor (s60), CPRD49 (s43), and an unidentified protein (s41). The albumin 2 protein was significantly upregulated in skl from 1 day of incubation, while the other two proteins were up-regulated at 3 days of incubation. Furthermore, the relative expression of albumin 2 (s39) in skl was also increased from 2.5- to 5.5-fold when the plants were incubated for 3 days. ACC treatment significantly (P < 0.05) increased the expression of 14 proteins in wild-type roots, while it had no significant

effect on protein expression in skl roots (Table 1). Statistical analysis showed that none of the changes were due to the ACC treatment effect alone, but all were significantly altered as an interaction of ACC treatment with genotype. This was consistent with the insensitivity of skl to ethylene. Of these 14 proteins, two proteins were pprg-2 proteins (s01 and s03), four proteins were identified as L-ascorbate peroxidase (s04, s17, s63, and s68), and two proteins were heat-shock proteins (HSP, s104, and s105). Although the Mowse score of spot s104 was low (40), and its identity was obtained by matching the ionic mass of one peptide to the database (Table 2), previous identification of this spot resulted in the same match.14 Three proteins were identified as glyceraldehyde-3-phosphatase dehydrogenase (GAPDH, s22), ACC oxidase (s58), and vacuolar H+-ATPase subunit A (s62). Three other proteins (s16, s47, and s48) were unidentified (Table 2). 3.3. Effect of S. meliloti Inoculation on Protein Expression in Wild-Type and skl Roots. To compare protein profiles of inoculated wild-type and skl roots, 3-day-old seedlings were inoculated with S. meliloti, and the position of the root tip at the time of inoculation was marked as RT0. The root segments representing the nodulation zone without root tips (10 mm above and 4-10 mm below RT0) were harvested at 1 and 3 days after inoculation. The expression of 18 protein spots was significantly changed (P < 0.05) in wild-type and skl roots in response to either genotype or S. meliloti inoculation (Figure 4, Table 3). Of these 18 protein spots, 14 proteins could be identified (Table 4). Differential displays of some spots are highlighted in Figure 5. Examination of the genotype effect in this experiment showed that five proteins (s01, s02, s13, s56, and s58), identified as pprg-2 protein, Kunitz proteinase inhibitor, and ACC oxidase, were down-regulated in skl roots, consistent with results of the ACC experiment. However, these proteins were down-regulated in skl roots at 3 dpi (Table 3). Three proteins (s39, s43, and s60), identified as CPRD49, albumin 2, and trypsin inhibitor, respectively, and one unidentified spot (s41) were significantly up-regulated in skl roots, again similar to the ACC experiment. Journal of Proteome Research • Vol. 5, No. 11, 2006 3087

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Table 1. Quantification of Differentially Displayed Proteins of Wild-Type and skl Roots in Response to ACC at 1 and 3 days after Treatment

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Table 1. (Continued)

a Details of protein identifications are listed in Table 2. b Values are displayed as %Vol (spot volume/total volume, in %). Values are the average ( SD from three biological repeats. Treatments having the same lowercase letter are not significant (P > 0.05). Statistical test for normality, homogeneity, transformation, and mean separation are described in Materials and Methods. Empty bars, control wild-type; diagonal-line bars, wild-type plus ACC; filled bar, skl; stipple bars, skl plus ACC. c H%V is the highest average %Vol of each protein in the graph, n ) 3. d P value of the genotype effect (wild-type vs skl), n ) 6. When the genotype effect is significant, the fold change of skl relative to wild-type is given in parentheses. ND ) not determined. e P value of the ACC treatment effect (control vs ACC treatment), n ) 6. When the effect is significant, the fold change of skl relative to wild type is given in parentheses. ND ) not determined.

Inoculation with S. meliloti induced the expression of seven proteins (s10, s22, s27, s37, s49, s50, and s75) in both wildtype and skl roots (Table 3). These seven proteins were not detectable in uninoculated wild-type and skl roots. Of these seven proteins, one protein (s22) was previously identified as GAPDH (Table 2). Searches against the M. truncatula EST database to identify the six other proteins produced no significant match to any M. truncatula proteins. To test the possibility that changes in protein expression after Sinorhizobium inoculation were due to the presence of S. melilotiderived proteins, we conducted a search for all differentially displayed proteins shown in Table 3 against S. meliloti genomic database, resulting in five matches to S. meliloti proteins. These five proteins were consistently present on the gels of inoculated wild-type and skl roots but not on gels of uninoculated roots. Two of these proteins (s37 and s75) were identified as AcfC proteins, two of them (s49 and s50) were transporter proteins, and one (s22) was a hypothetical signal peptide (Table 4). Most likely, spot s22 contained two proteins, one of plant and one of bacterial origin, which may have not separated on the 2-DE gels. We then tested for statistically significant interactions between Sinorhizobium inoculation and genotype effect. Sinorhizobium inoculation significantly increased the expression of ACC oxidase (s58) in wild-type roots at 3 dpi, but not in skl roots (Table 3). The expression of this ACC oxidase was also higher in control wild-type roots than in control skl roots. Furthermore, the expression of two pprg-2 proteins (s01 and s03) and Kunitz proteinase inhibitor (s13) was significantly

lower in inoculated skl than in inoculated wild-type, whereas their expression was similar in control wild-type and skl roots. 3.4. Proteins Differentially Expressed in the ACC and Nodulation Experiments. To examine the possible involvement of proteins in both ethylene signaling and the early nodule development, results from the ACC experiment and the nodulation experiment were compared. Protein spots showing differential display in the ACC and nodule experiments were classified into three classes: (1) spots changed only in the ACC experiment, (2) spots differentially expressed only in the nodulation experiment, and (3) spots changed in both the ACC and nodulation experiments. In each class, differentially displayed spots were further categorized in response to genotype effect, treatment effect, and the interactions of treatment effect with a particular genotype. Results of this classification are shown in Figure 6. 3.4.1. Changes in Response to ACC: Overall, there were no proteins differentially displayed only in response to ACC treatment. However, 11 proteins, including a vacuolar H+ATPase subunit A, GAPDH, two heat-shock proteins, four ascorbate peroxidase isoforms, and three unidentified proteins were specifically up-regulated in wild-type, but not in skl, in response to ACC. 3.4.2. Changes in Response to Nodulation: Five proteins, all of bacterial origin, and two unidentified proteins were newly induced in wild-type and skl only in response to S. meliloti inoculation. Protein spot s22 was assigned to two categories. Its expression was induced by ACC in wild-type roots and by inoculation in both wild type and skl roots. Because this spot was identified as plant- and bacterial-protein origin (Tables 2 Journal of Proteome Research • Vol. 5, No. 11, 2006 3089

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Table 2. The Identity of Differentially Displayed Proteins of Wild-Type and skl Roots in Response to ACC Treatment at 1 and 3 days of Treatment spot no.a

protein identity

observed Mr (kDa)/pI

accession no.

MOWSE score

matched peptide

s01 (Mt77)

16/4.84

Pprg-2

TC106314

237

s02 (Mt78)

17/4.88

Pprg-2

TC106352

268

s03 (Mt79) s04 (Mt359)

16/4.98

Pprg-2

TC106314

61

25/5.08

L-ascorbate

TC106425

205

s13

18/5.05

Kunitz proteinase inhibitor-1

TC106781

75

s17

24/4.78

L-ascorbate

TC106425

75

s22 (Mt265) s39

10/4.59

TC106518

127

26/6.37

Glyceraldehyde-3-phosphate dehydrogenase Albumin 2

TC101004

258

s43

29/5.88

CPRD49

TC100585

93 64

s56

36/6.00

ACC oxidase

TC94542

82

s58

36/6.02

ACC oxidase

TC94542

117

s60

23/4.51

Trypsin inhibitor

TC107914

348

s62

26/4.87

vacuolar H+-ATPase subunit A

TC59090

123

s63 (Mt215)

25/4.96

L-ascorbate

peroxidase

TC106425

249

s68

24/4.83

L-ascorbate

peroxidase

TC106425

222

s104 (Mt177) s105 s16 s41 s46 s47 s48

41/4.80

Heat shock cognate protein 70

TC100389

40

TNFVLHKb LSIVEDGKb DADEIVPKb AIEGYVLANPDYb GDATLSDAVRDETKb VISAAQSVEIVEGNGGPGTIKKb LDAVDEANFGYNYSLVGGTGLDESLEKb TDFVLHKb DADEIIPKb GDAVLSEAVRb GDAVLSEAVREETKb VEFETNIVAGSDGGSIVKb VIPAAQSVEIVEGNGGPGTIKb TNFVLHK GDATLSDAVR EDKPEPPPEGR ALLSDPVFRPLVEK HQAELAHGANNGLDIAVR YAADEDAFFADYAEAHQK AMGLSDQDIVALSGGHTIGAAHK LVPTENDPFR GVIFTTDEVDIEFVK TGDSECPVTVLQDFSEVVR EDKPEPPPEGR ALLSDPVFRPLVEK HQAELAHGANNGLDIAVR YAADEDAFFADYAEAHQK LVSWYDNELGYSTR GILGYTEDDVVSTDFIGDTR ITFTPGR EVYLFR SGYINAVFR ILHGPVFVR DINSGFTCFR IDYGTNSLVQIIR GLQVVDDIFPKb TMPVEFGEDSPFDPIHPLDPTKb AIDLWSLIQR DQPALAIVYFGGNDSHLPHPSGLGPHVR GLEAVQTEVKb FVFEDYMNLYARb FVFEDYMNLYAR AHTDAGGIILLFQDDK DSNGNPIFFSSR FYVKPSIFGAAGGGVK FSTDAEIFIDLISTDTSR VDIVFPEKPECAESSK LVFCPTFTAPPGLCHDIGR WLLIEDDFPRPWVGIGGIEDYIGK ESEYGYVR VGNDNLIGEIIR ISADVYIPR EDKPEPPPEGR ALLSDPVFRPLVEK HQAELAHGANNGLDIAVR YAADEDAFFADYAEAHQK AMGLSDQDIVALSGGHTIGAAHK EDKPEPPPEGR ALLSDPVFRPLVEK HQAELAHGANNGLDIAVR YAADEDAFFADYAEAHQK AMGLSDQDIVALSGGHTIGAAHK NALENYAYNMR

39/4.73 14/4.50 27/6.38 35/5.84 13/4.76 13/4.84

Heat shock cognate protein 70 Unidentified Unidentified Unidentified Unidentified Unidentified

TC100389

39

NALENYAYNMR

a

peroxidase

peroxidase

sequence coverage (%)

62

43

11 39

20 17

10 22

13 15 7 9 49

14 39

39

2 2

Spots previously identified in Mathesius et al.14 are shown in parentheses. b Spots identified by LC-MS. Otherwise spots were identified by MALDI-TOF-

MS.

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Figure 4. Representative 2-DE protein map of inoculated skl roots harvested at 3 dpi. Protein spot changes when compared to wildtype roots or to the uninoculated treatment are marked with numbers and listed in Table 3. Picture is taken from silver-stained gel.

and 4), this spot consisted of at least two different proteins. One unidentified protein was also induced by S. meliloti inoculation. 3.4.3. Changes in Response to Genotype: Five protein spots, pprg-2, albumin 2, an unidentified protein, CPRD-49, and trypsin inhibitor, were differentially displayed only in response to genotype, but not in response to any treatment. In addition, nine proteins were differentially affected by genotype in both the nodulation and the ACC experiments. These proteins were two pprg-2 isoforms, Kunitz proteinase inhibitor, albumin 2, an anther specific protein, CPRD-49, two ACC oxidase isoforms, and a trypsin inhibitor. 3.4.4. Changes in Response to Both ACC and Nodulation: Four proteins (two pprg-2 and two ACC oxidase isoforms) showed differential changes in wild-type and skl roots when treated with ACC and when inoculated with S. meliloti, suggesting that these proteins are regulated by ethylene during nodulation.

4. Discussion Our hypothesis was that ethylene mediates some of the protein expression changes in response to rhizobia, and that expression of those proteins would only change in the wildtype but not the skl mutant. The ethylene-insensitive skl mutant did not respond to the ethylene precursor ACC in the proteomic analysis, confirming the physiological assays. Overall, our results showed that more proteins differed between the genotypes than in response to the treatments. We identified 11 proteins that were differentially affected in wild-type and skl in response to both ACC and nodulation, and these proteins are candidates for ethylene-affected proteins involved in the early stages of nodule development. Their biological functions and potential roles in nodulation are discussed below. 4.1. ACC Oxidase, a Component in Ethylene Synthesis, Is Regulated during Nodulation. ACC oxidase (ACO) is an enzyme catalyzing the conversion of ACC to ethylene.16 Therefore, it is expected that its expression in wild-type roots is increased when treated with ACC. Our results showed that the expression of ACO (s56 and s58) in the skl mutant was lower than in wild-type (Table 1). Likewise, Sinorhizobium inoculation increased the expression of ACO (spot s58) to 1.7-fold in wild-type roots at 3 dpi (Table 3), but not in skl roots. These

research articles results are consistent with the increase of ethylene synthesis following inoculation.2,3,17 Because the skl mutant is defective in ethylene perception downstream of ACO action, these results suggest that a feedback mechanism regulates the expression of ACO, possibly to maintain the hormonal homeostasis. In tomato and Arabidopsis, ACO is encoded by a gene family. These genes show differential expression during plant growth and development, and respond differentially to various external stimuli.18 Two ACO genes in Arabidopsis are ethylene-inducible,19 suggesting that the feedback mechanism in ethylene synthesis is a common mechanism to control internal ethylene levels. 4.2. Expression Pattern of pprg-2 Proteins Suggests an Ethylene-Mediated Response during Early Stages of Nodulation. We showed that the expression of pprg-2 proteins in untreated skl roots was lower than in wild-type roots at 1 and 3 days. In addition, ACC treatment increased pprg-2 expression in wild-type at 1 day of incubation, but not in skl roots (Table 1), suggesting that ethylene has a positive effect on the expression of pprg-2. PR-10 proteins, to which pprg-2 is related, are induced by exogenous salicylic acid (SA), jasmonic acid (JA)20 and abscisic acid (ABA),21 S. meliloti inoculation,22 wounding,23 and by abiotic stresses.24 Therefore, pprg-2 proteins may be involved in a general plant response to biotic and abiotic stress. A successful symbiosis is thought to require a reduction of defense responses by the host plant.25,26 In the Sinorhizobium-alfalfa symbiosis, the plant regulates the extent of infection by initiating a defense mechanism, which aborts most of the infection threads.27 In skl, many infection threads proceed to grow into the inner cortical cells, suggesting that ethylene is involved in defense responses that regulate the number of infections.8 Our results suggest that pprg-2 could be one of the mediators of that defense response. 4.3. The Regulation of a Proteinase Inhibitor by Ethylene during Nodulation. The expression of Kunitz proteinase inhibitor (spot s13) was reduced in skl roots at 3 days of incubation (ACC experiment) or at 1 and 3 dpi (nodulation experiment). In contrast, the expression of another proteinase inhibitor (PI), trypsin inhibitor (spot s60), was increased in skl roots at 3 days of incubation or at 3 dpi (Tables 1 and 3). These results suggest that ethylene induces different expression patterns among PIs in roots. PIs are part of the plant’s defense system against insect and pathogen attacks.28 PIs are induced by pathogen invasion, wounding, ABA, jasmonic acid (JA), ethylene, auxin, and the small peptide systemin.29-31 O’Donnell et al.29 showed that both ethylene and JA signals were required to induce a PI gene expression in response to wounding, while ethylene treatment alone was not able to induce its expression. It is not yet established if the skl mutation affects JA signaling; however, the lack of effect in the expression of spot s13 and s60 upon treatment with ACC in either wild-type or skl roots may indicate that ethylene itself is not the only cause for the up- and down-regulated expression of these proteins in skl. Consistent with this view, the expression level of Kunitz proteinase inhibitor (spot s13) in inoculated skl roots was significantly lower than that in inoculated wild-type roots at 1 and 3 dpi (Table 3), while its expression in control skl roots was similar to that in control wild-type roots. Therefore, the expression of spot s13 is possibly regulated by the interaction of ethylene and an unknown mechanism during nodule development. 4.4. Ascorbate Peroxidase and Heat-Shock Proteins Are ACC-Inducible. Our results demonstrated that four spots Journal of Proteome Research • Vol. 5, No. 11, 2006 3091

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Table 3. Differentially Displayed Proteins of Inoculated Wild-Type and skl Roots at 1 and 3 days Post Inoculation (dpi)

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Ethylene-Mediated Changes in Nodulation in M. truncatula Table 3. (Continued)

a Details of protein identifications are listed in Table 4. b Values are displayed as %Vol (spot volume/total volume, in %). Values are the average ( SD from three biological repeats. Treatments having the same lowercase letter are not significant (P > 0.05). Statistical test for normality, homogeneity, transformation, and mean separation are described in Materials and Methods. Empty bars, control wild-type; diagonal-line bars, inoculated wild-type; filled bar, skl; stipple bars, inoculated skl. c H%V is the highest %Vol of each protein in the graph. d P value of the genotype effect (wild-type vs skl), n ) 6. When the genotype effect is significant, the fold change of skl relative to wild-type is given in parentheses. ND ) not determined. e P value of the inoculation effect (control vs inoculation treatment), n ) 6. When the inoculation effect is significant, the fold change of skl relative to wild-type is given in parentheses. ND ) not determined.

Table 4. The Identity of Differentially Displayed Proteins in Inoculated Wild-Type and skl Roots at 1 and 3 dpia spot no.

observed Mr (kDa)/pI

s22

10/4.59

s37

accession no.

MOWSE score

Hypothetical signal peptide

SMc01418

133

28/4.57

AcfC, S. meliloti

SMc02156

118

s49

35/4.47

ABC transporter binding protein, S.meliloti

SMc02171

77

s50

35/4.52

Transmembrane periplasmic binding protein, S. meliloti

SMc02378

98

s75

29/4.66

AcfC, S. meliloti

SMc02156

111

s10 s27 s46

12/4.52 9/4.62 35/5.84

Unidentified Unidentified Unidentified

a

protein identity

matched peptide

MGDVTGDGVKGEWDVARP NWPPFMVEGDAAAEGAYSIVER LEDVAAFR LYGAGGPDTAIR SNIVAFEQGSGASFK IVVTEGAGVANTSGTGVWEDIAGR TDTNPFFVK IVGHDITNGNEEGGR VAFLDLTPSQPSVDVLR GEPDIAPEGWVDLLPDVVNR YIVDANPDIK ELFPDPEDPSK AYGAEAAGFTLVDTGSAAGLDGSIAK DAQDGVIR LYGAGGPDTAIR FEDLLAEGTK IVVTEGAGVANTSGTGVWEDIAGR LEDVAAFR

sequence coverage (%)

32 22

12 20

23

The proteins were identified by MALDI MS/MS, and the data were matched against the S. meliloti database.

Figure 5. Highlight of some differentially displayed proteins in inoculated wild-type (A17) and skl roots at 3 days postinoculation.

identified as cytosolic ascorbate peroxidase (APX) were significantly induced by ACC at 3 days incubation (Table 1). In contrast, the expression of APX was similar in all treatments

of the ACC experiment at 1 day of incubation, and all treatments of the nodulation experiment either at 1 or 3 dpi. APX plays a key role in the breakdown of intracellular reactive oxygen species (ROS) such as O2- and hydrogen peroxide, which are generated in plants during plant growth and development, photosynthesis, hormonal action, nodule development, and by a variety of biotic and abiotic stress.32 In Arabidopsis, the cytosolic APX1 enzyme is a crucial component of ROS-scavenging mechanisms. A mutant lacking this enzyme, KO-Apx1, has an increased level of H2O2 because ROS-scavenging mechanisms are collapsed.33 Our results suggest that (1) ethylene induces ROS production in wild-type, which subsequently increases the expression level of APX, and the presence of intermediate processes may explain the existence of a lag phase response at 1 day of incubation in wild-type; (2) under nonstress growth conditions, the abundance of APX in skl roots Journal of Proteome Research • Vol. 5, No. 11, 2006 3093

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Figure 6. Venn diagram of differentially displayed proteins in the ACC experiment and the nodulation experiment. Spot changes in each experiment are classified into genotype effect, treatment effect (ACC treatment or S. meliloti inoculation), and the interaction of genotype and treatment. Proteins up-regulated by ACC treatment in wild-type, but not in skl, are indicated by vA. Proteins down-regulated by ACC treatment in skl relative to ACC-treated wild-type are indicated by VSACC. Proteins downregulated by S. meliloti inoculation in skl relative to inoculated wild-type are indicated by VSin. Proteins up- or down-regulated in skl vS and VS, respectively. Proteins induced by S. meliloti inoculation irrespective of genotype are indicated by x.

is similar to wild-type, possibly because ROS production is not affected by the skl mutation; and (3) M. truncatula plants may use ROS-scavenging mechanism other than APX to reduce ROS level during early nodule development, because the expression level of APX in inoculated plants is similar to control plants. Previous reports showed that the peroxidase gene, RIP1, was up-regulated between 12 and 24 h postinoculation in response to Nod factors, the rhizobial signals necessary to induce a nodule.34 The involvement of ethylene in inducing APX activity and expression has been demonstrated in chickpea (Cicer arietinum) and rice.35,36 High nitrate concentrations increased the activity of APX in chickpea nodules.36 The increased activity of APX in chickpea nodules was reduced to control levels (without nitrate treatment) by aminoethoxyvinylglycine (AVG), an ethylene biosynthesis inhibitor, indicating that ethylene mediates the nitrate-induced APX activity.36 In rice, ethylene induced the transcript level of OsAPX1/2 in leaves as early as 3 h after treatment.35 The expression of spot s22, identified as GAPDH, was induced by ACC at 3 days of incubation (Table 1). GAPDH catalyses the conversion of glyceraldehyde-3-phosphate to 1,3diphosphate-glycerate in the glycolysis and fermentation pathway. The transcript level of this enzyme is increased rapidly at 24 h in response to anaerobic and submergence stress.37,38 Since the expression of this enzyme is not detected in skl roots when treated with ACC (Table 1), this result suggest that the expression of GAPDH may be regulated by ethylene under stress conditions. Two other proteins showing an increased abundance in wildtype roots after ACC treatment were spots s104 and s105, identified as heat-shock protein (HSP). The expression of these proteins spots was not increased in inoculated wild-type, 3094


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suggesting that HSPs are not regulated during nodule development. HSPs comprise a diverse group of proteins, and are highly conserved in eukaryotes. HSPs are involved in repair and degradation processes during stress-induced responses, and their expression is induced by a variety of stresses and is also developmentally regulated.39 4.5. Skl Mutation Affects the Expression of Albumin 2 and CPRD49. Two spots up-regulated in skl roots were identified as albumin 2 (spot s39) and CPRD49 (spot s43). Albumin 2 is a water-soluble storage protein present in the seeds of dicotyledonous plants and is a member of a superfamily of seed storage proteins called prolamins that are involved in plant defense.40 The increase of albumin 2 expression in skl roots indicates that this protein may have a broader function in plants and that its expression is regulated by ethylene signaling. A BLAST search to find any sequences with homology of CPRD49 showed that this protein has a conserved domain similar to isoamyl acetate hydrolase of yeast. Isoamyl acetate is a volatile ester produced by yeast during beer fermentation. Volatile esters are produced and emitted by vegetative, floral, and fruit parts of plants during growth and in response to stress or insect infestation.41 As the expression of CPRD49 protein is increased in skl, this result suggests that the expression of CPRD49 protein is negatively regulated by ethylene. 4.6. Identification of Rhizobial Proteins. The identification of proteins of rhizobia inside nodules in M. truncatula and Melilotus alba (white sweet clover) has been reported previously.42,43 In our study, four spots from rhizobia present in wildtype and skl roots following Sinorhizobium inoculation were bacterial periplasmic membrane protein. An accessory colonization factor, AcfC (spots s37 and s75) contains an ABC-type periplasmic domain. Bacterial periplasmic membrane proteins are mostly involved in transport processes and represent the largest group of bacterial proteins identified in nodules.44 Bacterial periplasmic transport systems use membrane-bound complexes and substrate-bound, periplasmic binding proteins to transport a wide variety of substrates, such as amino acids, peptides, sugars, vitamins, and inorganic ions. The expression level of protein spot s49 (ABC-transporter) was increased in skl roots. This result may reflect the higher level of bacterial infection in skl roots than in wild-type.8

5. Concluding Remarks We have demonstrated that ACC induces stress-related proteins in wild-type roots, such as pprg-2 protein, ACC oxidase, proteinase inhibitor, ascorbate peroxidase, and heatshock proteins. The expression of stress-related proteins, such as pprg-2, Kunitz proteinase inhibitor, and ACC oxidase,was down-regulated in inoculated skl roots. This indicates that these stress-related proteins are regulated by ethylene during nodule development. Since the skl mutant has increased nodule numbers and infections, we propose that down-regulated expression of these proteins allows higher numbers of infections and nodules to form. We hypothesize that during early nodule development, the plant induces ethylene-mediated stress-responses to limit nodule numbers. When a mutant defective in ethylene signals, such as skl, is inoculated with rhizobia, the plant’s stress-response is reduced, resulting in increased nodule numbers.

Acknowledgment. We thank Charles Hocart and Carolyn McKinlay at the Research School of Biological Sciences Mass Spectrometry Facility ANU for LC-MS analysis; Peter Milburn

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and the Australian Cancer Research Foundation at the Biomolecular Resource Facility, John Curtin School of Medical Research, ANU, and the Australian Proteome Analysis Facility at Macquarie University for MALDI TOF-TOF analysis. This work was supported by the Australian Research Council through the ARC Centre of Excellence for Integrative Legume Research (CE0348212). U.M. was supported by a Research Fellowship from the Australian Research Council (DP0557692).

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Identification of Ethylene-Mediated Protein Changes during

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