Copyright ª Physiologia Plantarum 2007, ISSN 0031-9317
Physiologia Plantarum 130: 380–390. 2007
Differential Frankia protein patterns induced by phenolic extracts from Myricaceae seeds Emilie Bagnarol*, Jean Popovici, Nicole Alloisio, Joe¨lle Mare´chal, Petar Pujic, Philippe Normand and Maria P Fernandez Universite´ de Lyon, CNRS, UMR5557, Ecologie Microbienne, IFR 41, Universite´ Lyon1, Villeurbanne, F-69622, France
Correspondence *Corresponding author, e-mail:
[email protected] Received 7 September 2006; revised 30 November 2006 doi: 10.1111/j.1399-3054.2007.00875.x
Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the early symbiotic interactions between the bacterium Frankia and actinorhizal plants. Myricaceae, the most primitive actinorhizal family, was used as an experimental model to study specificity mechanisms because it includes species with either narrow or large specificity. Seed phenolic extracts from two Myricaceae species, Myrica gale, a narrow specificity host and Morella cerifera considered as a promiscuous host, were used to induce three Frankia strains ACN14a, M16467 and Ea112. The global protein pattern was altered by exposure to the plant extracts. The addition of 30 mg l21 of seed phenolic extracts provoked the inhibition of many protein biosynthesis whereas 1 and 10 mg l21 induced a global reprogramming of Frankia protein pattern. Changes in intensity of 115 spots in response to seed extracts were detected and analyzed by matrix-assisted laser desorption/ ionization time of flight mass spectrometry. Fifty proteins were efficiently identified with Frankia protein data banks deduced from the sequences of Frankia strains ACN14a and EaN1pec genomes. Differential proteins were involved in different metabolism pathways such as glycolysis and gluconeogenesis, transcription, fatty acids, carbohydrates, coenzymes and purines metabolisms. Chaperones biosynthesis and iron transport regulation, essential for nitrogen fixation, seem to be strain dependant. Several proteins possibly involved in the regulation of nodulation were also differentially expressed. The most obvious response was the upregulation of oxidative stress proteins such as FeSOD and Tellurium resistance proteins, suggesting a reorganization of Frankia metabolism to protect against host plant defense.
Introduction The actinorhizal symbiosis, materialized by nitrogenfixing nodules, results from the interaction between the actinobacterium Frankia and plants belonging to eight
dicotyledonous families collectively called ‘actinorhizal’ (Benson and Silvester 1993). The establishment of the symbiosis requires a number of interactions between the two organisms.
Abbreviations – 2DE, two-dimensional electrophoresis; AICAR, 5-amino-imidazole-4-carboxamide; BCA, bicinchoninic acid; CHAPS, 3-((3-Cholamidopropyl)dimethylammonio)-1-propanesulfonate; Chs, chalcone synthase; COG, cluster of orthologous genes; DTT, dithiothreitol; IEF, isoelectrofocusing; IPG, immobilized pH gradient; MALDI-TOF, matrix-assisted laser desorption/ ionization time of flight; PAA, phenylacetic acid; PAGE, polyacrylamide gel electrophoresis; Pal, phenylammonia lyase; RKIP, raf kinase inhibitor protein; SAICAR, 5#-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide; SDS, sodium dodecylsulfate; SOD, superoxide dismutase.
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Secondary metabolites synthesized via the phenylpropanoid metabolic pathway in plants are often involved in plant–microbes interactions, either as components of the plant defense mechanisms or as early symbiotic signals. In the Rhizobium/Legumes symbiosis, the recognition of host-derived flavonoids is the basic mechanism through which rhizobia interact specifically with their hosts. The bacterial factor (Nod factor) thereby synthesized is the signal that induces root hair deformation and the nodulation programme in the plant tissues. The role of plant phenolics in the actinorhizal symbiosis was addressed in several studies. Nodulation may be influenced by host-derived phenolics (cinnamic, benzoic and hydroxybenzoic acids) (Perradin et al. 1983) and by Alnus seed flavonoid-like compounds, identified as flavanone and isoflavanone (Benoit and Berry 1997). In the Casuarina glauca–Frankia symbiosis, Laplaze et al. (1999) revealed a cell-specific flavan biosynthesis and accumulation in C. glauca nodule lobes, creating a compartmentalization in the cortex. Genes like pal (phenylammonia lyase) and chs (chalcone synthase), involved in flavonoids biosynthesis are activated in Alnus glutinosa inoculated by Frankia (Hammad et al. 2003). Nevertheless, the molecular bases that control symbiosis initiation and specificity are still unknown. The lack of genetic tools to study Frankia has restricted progress in understanding the regulatory events occurring during the early steps of the symbiosis. Common features between actinorhizal and legume symbioses have been observed during the first steps of the infectious process. A similar root hair curling step is observed with both Frankia and Rhizobium prior to nodule formation, but the Frankia extracellular deforming factor seems to be structurally and functionally different from Rhizobium Nod factors (Ceremonie et al. 1998, 1999). Until now, Alnus (Betulaceae family), Casuarina (Casuarinaceae) and Datisca (Datiscaceae) species have been used as models to study the interactions between Frankia and its host plant. In this study, we used Myricaceae family as an experimental model to study symbiosis specificity because (i) Myricaceae contain a large qualitative and quantitative diversity of phenolic compounds; (ii) Frankia isolates from Myricaceae are available; and (iii) Myricaceae family includes species with either narrow or large specificity. Myrica gale, a narrow symbiotic specificity host, is exclusively nodulated by Alnus–Comptonia-infective strains [cluster 1 of Normand et al. (1996)], whereas the other Myrica species, now grouped into genus Morella (Huguet et al. 2001, 2004) are considered promiscuous hosts because they can be nodulated by both Alnus–Comptonia strains (cluster 1) and Elaeagnaceae-infective strains [cluster 3 of Normand et al. (1996)]. We hypothesized that this Physiol. Plant. 130, 2007
difference of host specificity might depend on a specific molecular dialogue between both partners. Myricaceae seed phenolics were tested on different Frankia strains varying in their host compatibility. We chose seed extracts instead of root exudates because of the difficulty to obtain sufficient quantity and reproducibility with root exudates (D’Arcy Lameta 1982, 1986, Maxwell and Phillips 1990). Moreover, the flavonoids that are active in Rhizobium nod genes induction have been identified from seeds and seed coats of Alfalfa (Hartwig et al. 1990, Hartwig and Phillips 1991), and flavonoid-like compounds from Alnus rubra seeds have been shown to influence nodulation of that host by Frankia (Benoit and Berry 1997). Two Frankia strains belonging to cluster 1, isolated from Alnus crispa and Morella pensylvanica and one strain belonging to cluster 3 isolated from Elaeagnus augustifolia were analyzed using a global proteomic approach. The proteome analysis technique and differential protein display with two-dimensional electrophoresis (2DE) has facilitated the analysis of the contribution of both partners during different plant/microbe interactions, pathogenic (Colditz et al. 2004) or symbiotic (Dainese-Hatt et al. 1999, Djordjevic 2004, Guerreiro et al. 1997, Hammad et al. 2001, Natera et al. 2000). In contrast to transcriptome analyses, the study of protein populations enables a more direct access to cell processes by monitoring the actual pattern of translated gene products. In this study, a combination of 2DE for the separation of complex mixtures of proteins and matrixassisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry for identification of protein by tryptic peptide mass fingerprinting was used. The recent publication of three Frankia whole genome sequences [ACN14a and CcI3 (cluster 1) and EAN1pec (cluster 3) strains] (Normand et al. 2007) has represented a crucial improvement for MALDI-TOF mass spectrometry analyses in this study. Frankia proteomes obtained under the different induction conditions were compared and the up- or downregulations of several metabolism pathways were discussed.
Materials and methods Plant extracts, bacterial strains and induction conditions Seeds from Morella cerifera (designated as Myrica cerifera ‘Pumila’ in the catalogue) were obtained from Sheffield’s Seed Co., Inc. (Locke, NY) and My. gale seeds were collected in December 2004 on the shore of Biscarosse lake, Bordeaux, France. My. gale air-dried fruits (500 g) were extracted by sonicating twice with 2 l of 381
methanol, filtered and evaporated to dryness, 52 g of residue were obtained. The same protocol was used to obtain 13 g of residue from 500 g of Mo. cerifera air-dried fruits. Each resulting residue was diluted in methanol before induction. Three Frankia strains were tested: Frankia sp. strains M16467 isolated from Myrica pensylvanica (Bloom et al. 1989), ACN14a isolated from A. crispa (Normand and Lalonde 1982) and Ea112 isolated from E. augustifolia (Fernandez et al. 1989). Both M16467 and ACN14a Frankia strains nodulate promiscuous and non-promiscuous species, contrary to Ea112 strain which nodulates only promiscuous species (Huguet et al. 2004). All strains were grown on nitrogen-free BAP medium (BAP-) (Murry et al. 1984) with stirring at 28C. Five-day-old cultures (end of the exponential growth phase) were used as inocula. BAP- medium was inoculated with a normalized cell number equivalent to 4 mg l21 of bacterial proteins. My. gale or Mo. cerifera methanolic extracts were added to 200 ml BAP- medium simultaneously to Frankia inoculation. Three concentrations of phenolics mixture were tested: 1, 10 or 30 mg l21, contained in 50 ml of methanol. Uninduced standards were obtained by adding the same volume of methanol (50 ml). Methanol volume used has no effect on Frankia growth and no differences in Frankia proteins profiles were detected. Frankia cells from 5-day cultures were sedimented by centrifugation at 5000 g for 15 min, washed twice with sterilized ultrapure water and homogenized by repeated forced passages through a 21-g needle with a syringe. Protein extraction Frankia cells treated with or without seed extracts were harvested and centrifuged at 5000 g for 15 min. Cell pellets were washed two times with sterilized ultrapure water and resuspended in 1 ml of water. The suspension was sonicated on ice for 5 1 min (pulse cycles: 5 s run/5 s pause), at 700 W (cumulative value) with a Vibra Cell apparatus (Bioblock Scientist, Illkirch, France). Sonicated cells were centrifuged at 13 000 g for 30 min at 4C. Pellets containing cell debris were frozen at 280C. Protein concentration in the supernatant (the hydrosoluble extract, HY) was measured with the bicinchoninic acid (BCA) protein assay according to the manufacturer’s protocol (Pierce, Rockford, IL). To remove most of the residual hydrosoluble proteins, thawed pellets were washed twice with sterilized ultrapure water and then resuspended in a solubilizing solution consisting of 1% (v/v) sodium dodecylsulfate (SDS) and 10 mM Trisbase (pH 9.5) and boiled for 5 min. Insoluble debris were removed by centrifugation at 4C (13 000 g 10 min). 382
Protein concentration of these solubilized pellet extracts (PE) was determined as described above. Aliquots containing 600 mg of proteins were used for 2DE. To remove salts and insoluble impurities, and to concentrate Frankia protein aliquots, a 2-D Clean Up Kit (Amersham Bioscience, Orsay, France) was used. The pellets were resolubilized in 200 ml of rehydration buffer which contained 8.5 M urea, 4% w/v 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate, 0.25% Nonidet P40, 50 mM dithiothreitol (DTT), 0.2% immobilized pH gradient (IPG) Buffer 4–7 (Amersham Bioscience) and 0.001% Bromophenol Blue. Two-dimensional polyacrylamide gel electrophoresis (2DE) IPG strips (pH 4 to 7 linear; 11 cm, BioRad, Marnes-laCoquette, France) containing 600 mg of HYor PE proteins were passively rehydrated overnight. The first dimension isoelectric focusing was performed with an IEF Protean cell (BioRad) at 20C with the following profile: 50 mA per strip maximum, 10 h at 50 V with a rapid voltage ramping slope, 2 h at 100 V, 2 h at 1000 V and 2.5 h at 8000 V with a linear voltage ramping slope, 8000 V until a total of 45 000 Vh was reached. IPG strips were placed at 280C for at least 2 h. Prior to the second dimension, IPG strips were equilibrated first for 15 min in equilibration buffer (6 M urea, 30% v/v glycerol, 3% w/v SDS, 0.05 M Tris–HCl buffer pH 6.8, 1% w/v DTT) followed by a second 15-min equilibration step in the same solution but with the substitution of 4.5% w/v iodoacetamide for DTT and 0.001% of Bromophenol Blue. SDS–polyacrylamide gel electrophoresis was performed as described by Lae¨mmli (1970) with a 13% w/v SDS-polyacrylamide resolving gels and a 4% w/v polyacrylamide stacking gels. Gels were run at 100 V for 1 h and at 250 V for 4 h. Separated proteins were revealed using silver staining kit (BioRad). Gels analysis – MALDI-TOF mass spectrometry Three replicates have been made for ACN14a and Ea112 strains and five for M16467 strain. Gel images were analyzed using PD QUEST software (BioRad). Differential spots [presence/absence or intensity differences between induced (My. gale or Mo. cerifera extracts) and noninduced cultures] were excised from the gel, reduced and alkylated before digestion with trypsine and analyzed by MALDI-TOF mass spectrometry (Institut de Biologie et Chimie des Proteines, Lyon, France) using a Voyager DEPRO MALDI-TOF mass spectrometer (Applied Biosystems, Courtaboeuf, France). Peptide mass fingerprints were identified using Frankia ACN14a and EAN1pec proteome database (http://www.genoscope.cns.fr/agc/ Physiol. Plant. 130, 2007
mage, FrankiaScope database). The confidence level for the identification results was established by evaluating the probability that a protein identification hit in Frankia sequence database corresponded to the protein being analyzed based on the MOWSE score, the number of peptide mass matches, the percentage of sequence coverage and the match values for the isoelectric point and the molecular weight.
Results Approximately 500 hydrosoluble protein spots per gel were revealed by silver nitrate staining for the ACN14a and M16467 strains, whereas about 200 proteins spots were detected in Ea112 strain (Fig. 1). About 700 proteins were revealed for each strain in the pellet using Tris base– SDS buffer, which also contains hydrophobic proteins. Two-dimensional electrophoretic protein patterns were reproducible except for Ea112 strain that exhibited variations. Low amount of proteins in Ea112 protein gels and variations could be explained by the presence of red pigments in Elaeagnus-infective strains, which could interfere with proteins during focalization and thus influence migration. In all strains, the majority of visible proteins migrated to the acidic portion of the gel (between pH 4 and 6) during the isoelectric focusing. Changes in intensity were detected on a total of 115 spots in the gels obtained from hydrophilic (HY) and pellet extracts (PE) with the three Frankia strains treated by exposure to seed extracts. Fig. 1 shows an example of the effects of 10 mg l21 of My. gale phenolic extracts. The other protein patterns obtained with other concentrations and with the other plant species are not shown. These proteins were analyzed by MALDI-TOF mass spectrometry and 50 were efficiently identified using the ACN14a and EAN1pec Frankia protein data banks. The other half of proteins analyzed could not be identified with a high confidence level. The genome sequences have allowed to identify, with a high degree of confidence, 62% of ACN14a, 53% of M16467 and 37% of Ea112-analyzed spots. These 115 differential spots were common to My. gale and Mo. cerifera inductions. Seven proteins (four for M16467 and three for Ea112 strains) were differentially induced by My. gale and Mo. cerifera extracts. However, these weakly expressed spots were not identifiable by MALDI-TOF mass spectrometry. Different responses were observed according to the phenolic mixture concentrations used: at 30 mg l21, biomass production as evaluated by pellet weight was lowered and many proteins were inhibited, whereas at 1 and 10 mg l21, a global reorganization of protein biosynthesis was observed. Table 1 shows a summary of the MALDI-TOF mass spectrometry results classified Physiol. Plant. 130, 2007
Fig. 1. Two-dimensional gel electrophoresis of the three different Frankia strains used: ACN14a (A), M16467 (M), Ea112 (E) after silver staining. Isoelectric focusing in the first dimension was carried out on a linear pH gradient (4 to 7), 11 cm immobilized pH gradient (IPG) strips loaded with 600 mg of protein. AS, MS, ES correspond to standard cultures (non-induced) and AI, MI, EI to cultures induced with 10 mg l21 of My. gale phenolic extracts. Circles indicate differentially expressed proteins.
according to the cluster of orthologous genes (COG) classification. Among the 50 differentially expressed proteins, 20 were upregulated and 30 were downregulated in the presence of plant phenolic extracts. Among upregulated proteins (Table 1A), the most obvious response observed is the over-expression, at the three concentrations tested, of stress, mainly oxidative stress resistance proteins, several of them being very abundant: a Fe–Zn superoxide dismutase (FeSOD; FRAAL4337) is present as a major spot in all gels. This 383
Table 1. Differentially displayed proteins upregulated (1) (1A) and downregulated (2) (1B) by Myricaceae seed phenolic extracts. Proteins are classified into one or several COG categories. (0) corresponds to proteins with the same expression as the standard (non-induced culture) and (nd) corresponds to uncertain results. Phenolic concentrations ACN14a or EaN1pec matching protein (spot no.)
Frankia strains
1A. Differentially displayed proteins upregulated with phenolic extracts [T] Signal transduction mechanisms Tellurium resistance protein TerE (14a-14b) M16467 Tellurium resistance protein TerE (4a) ACN14a Putative tellurium resistance protein (4b) ACN14a [O] Post-translational modification, protein turnover, chaperones Molecular chaperone HSP18 (small HSP) (16) M16467 [J] Translation, ribosomal structure and biogenesis 50S ribosomal subunit protein L9 M16467 30S ribosomal subunit protein S13 M16467 50S ribosomal subunit protein L6, rplF Ea112 [C] Energy production and conversion Membrane-bound ATP-synthase F1 sector M16467 beta-subunit, atpD Aconitate hydratase M16467 Electron-transfer flavoprotein alpha subunit (alpha ETF) ACN14a Electron-transfer flavoprotein large subunit (ETFLS) Electron-transfer flavoprotein alpha subunit (alpha ETF) M16467 electron-transfer flavoprotein large subunit (ETFLS) (13) [P] Inorganic ion transport and metabolism Putative molybdate-binding protein precursor M16467 Superoxide dismutase (Fe–Zn) (FeSOD I), ACN14a sodF, EC 1.15.1.1 Superoxide dismutase (Fe-Zn) (FeSOD I), M16467 sodF, EC 1.15.1.1 [I] Lipid transport and metabolism Enoyl CoA hydratase M16467 [E] Amino acid transport and metabolism Putative Leu, Ile, Thr, Val-binding protein precursor – ACN14a putative signal peptide (3) [R] General function prediction only Conserve hypothetical protein Enterochelin esterase Ea112 (ferric enterobactin) (27) Putative phospholipid-binding protein (5) ACN14a [S] Function unknown – unclassified Conserve hypothetical protein (6) ACN14a Hypothetical protein (17) M16467
1 mg l21
10 mg l21
30 mg l21
ACN14a gene no (FRAAL)-EaN1pec gene no. (FREAN)
1 1 1
1 1 1
1 1 1
FRAAL5898 FRAAL5898 FRAAL0606
62 72 80
10 7 9
HY HY–PE HY
1
1
1
FRAAL4031
21
3
HY
0 0 1
1 1 nd
0 0 nd
FRAAL6850 FRAAL1107 FRAEA7352
62 47 33
9 9 5
PE PE PE
1
1
nd
FRAAL5931
17
5
HY
0 1
1 1
1 nd
FRAAL2064 FRAAL5877
36 40
7 8
HY PE
0
1
nd
FRAAL5877
49
9
HY
1 1
1 1
1 1
FRAAL3835 FRAAL4337
26 66
5 10
HY HY
1
1
1
FRAAL4337
35
9
HY
1
2
2
FRAAL3799
43
8
HY–PE
1
1
1
FRAAL1482
53
12
HY
0
1
nd
FRAAL4718
19
3
HY–PE
1
1
nd
FRAAL0033
42
4
HY–PE
1 1
1 1
nd nd
FRAAL2823 FRAAL0413
26 56
3 6
HY HY
2
nd
FRAAL1133
35
4
HY
0 2
nd 2
FRAEA6206 FRAAL4690
19 18
3 5
PE PE
2
2
FRAAL5181
24
4
PE
2
2
FRAAL2834
17
3
HY
0 0 2 2
2 2 2 2
FRAAL0954 FRAAL5843 FRAAL6655 FRAEA1478
22 26 19 20
4 6 3 3
HY HY HY HY
1B. Differentially displayed proteins, downregulated by seed phenolic extracts [O] Post-translational modification, protein turnover, chaperones groS chaperone HSP10 (GroES), part of GroE Ea112 2 chaperone system (25–11) RuvB Holliday Junction helicase b-subunit Ea112 2 Carbamoyl transferase of the NodU/CmcH family M16467 2 [D] Replication, recombination and repair DNA-repair protein, recN M16467 2 [C] Energy production and conversion N5,N10 methylenetetrahydromethanopterin M16467 2 reductase-related protein Oxydoreductase M16467 0 Beta-isopromylmalate dehydrogenase (EC 1.1.1.85), leuB M16467 0 FbaA – fructose bisphosphate aldolase (EC 4.1.2.13) (1) ACN14a 2 Polyphosphate glucokinase (EC: 2,7,1,63), ppgK (22) Ea112 2
384
Sequence coverage (%)
No of matching peptides
Extract
Physiol. Plant. 130, 2007
Table 1. Continued [K] Transcription RNA polymerase alpha subunit rpoA M16467 [J] Translation, ribosomal structure and biogenesis Putative lysyl tRNA synthetase like protein (21) Ea112 Putative lysyl tRNA synthetase-like protein Ea112 [I] Lipid transport and metabolism Short chain 3 hydroxyacyl CoA dehydrogenase enoyl M16467 CoA hydratase (9) Enoyl CoA hydratase (7) M16467 Carbamoyl transferase of the NodU/CmcH family M16467 1 acyl-sn-glycerol-3-phosphate O-acyltransferase Ea112 (EC 2.3.1.51) (20) [P] Inorganic ion transport and metabolism Putative ABC type Fe31 siderophore transport system M16467 Putative thiosulfate sulfurtransferase ACN14a (Rhodanese-like protein) (2) High affinity phosphate transport protein M16467 (ABC superfamily), pstS [H] Coenzyme transport and metabolism Putative pantoate beta-alanine ligase Ea112 (panthotenate synthetase) (EC 6.3.2.1) Dethiobiotin synthetase (Cobyric acid synthase), bioD M16467 [G] Carbohydrate transport and metabolism Putative GDP D mannose dehydratase M16467 Polyphosphate glucokinase (EC: 2,7,1,63), ppgK (22) Ea112 [F] Nucleotide transport and metabolism Phophoribosylaminoimidazole-succinocarboxamide M16467 synthase (EC 6.3.2.6) purC (SAICAR synthetase) Dethiobiotin synthetase (Cobyric acid synthase), bioD M16467 [Q] Secondary metabolites biosynthesis, transport and catabolism Conserve hypothetical protein putative signal peptide M16467 (Isochorismatase family) (10) Conserved hypothetical protein (23) Ea112 [M] Cell wall/membrane/envelope biogenesis Putative GDP D mannose dehydratase M16467 [V] Defense mechanism DNA-repair protein, recN M16467 [L] Replication, recombination and repair DNA-repair protein, recN M16467 [R] General function prediction only Oxydoreductase M16467 O-methyltransferase M16467 Putative aminoglucoside phosphotransferase M16467 Aldo keto reductase (NADP1) Ea112 Conserved hypothetical protein (24) Ea112 Unclassified Hypothetical protein (8) M16467 Hypothetical protein M16467
2
nd
0
FRAAL1110
42
14
PE
2 2
2 0
2 nd
FRAAL1791 FRAEA1466
30 29
4 3
HY HY
0
2
2
FRAAL5547
43
6
HY–PE
1 2 2
2 2 2
2 2 2
FRAAL3799 FRAAL4690 FRAAL4897
40 18 23
6 5 3
HY–PE PE HY
2 2
2 2
nd 2
FRAAL4168 FRAAL0943
24 22
4 4
HY HY
2
0
nd
FRAAL6535
20
4
HY
2
2
2
FRAAL3483
30
5
HY
0
2
2
FRAAL2887
21
6
HY
0 2
2 2
2 2
FRAAL5662 FRAEA1478
30 20
7 3
HY HY
2
2
nd
FRAAL6667
28
8
HY
0
2
2
FRAAL2887
21
6
HY
2
2
2
FRAAL2926
19
3
HY
2
2
2
FRAAL5401
21
4
HY
0
2
2
FRAAL5662
30
7
HY
2
2
2
FRAAL5181
24
4
PE
2
2
2
FRAAL5181
24
4
PE
0 2 0 2 2
0 2 2 2 2
2 nd 2 2 2
FRAAL0954 FRAAL6092 FRAAL4065 FRAAL2956 FREAN8328
22 62 25 20 27
4 10 6 3 3
HY HY–PE HY HY HY
0 2
2 2
2 nd
FRAAL0413 FRAAL0413
48 55
6 8
HY PE
protein, together with two tellurium resistance proteins, Ter (FRAAL5898 and FRAAL0606), are upregulated in Frankia strains, ACN14a and M16467 exposed to seed extracts. Moreover, a heat shock protein (HSP), HSP18 (FRAAL4031), was also upregulated in M16467 strain. Phenolic mixture of 1 and 10 mg l21 induced the Physiol. Plant. 130, 2007
upregulation of proteins associated with translational activity, primary metabolism and energy production and conversion. The upregulation of three ribosomal protein subunits, L9, S13 and L6, was observed in M16467 and Ea112 strains (FRAAL6850, FRAAL1107 and FRAEA7352, respectively, corresponding genes in 385
ACN14a and EAN1pec strains). Other proteins are involved in Krebs cycle and energy production such as an aconitate hydratase (FRAAL2064), which catalyses the formation of isocitrate from citrate during the second step of the citric acid cycle and three respiratory chain proteins (a membrane-bound ATP-synthase F1, FRAAL5931 and two electron-transfer flavoprotein FRAAL5877). An enoyl coenzyme A (CoA) hydratase (FRAAL3799) involved in lipid metabolism was induced by 1 mg l21 of plant extracts. A putative Leu, Ile, Thr, Val-binding-protein precursor (FRAAL1482) and a putative molybdate-binding-protein precursor (FRAAL 3835) were also induced by plant material. Other upregulated proteins have only a general prediction or are not classified with the COG classification: two hypothetical proteins, a phospholipidbinding protein (FRAAL0033) and a conserved hypothetical enterochelin esterase (FRAAL4718) involved in iron metabolism. Plant extracts also inhibited the expression of 30 proteins (Table 1B). Two chaperones: a GroES chaperone HSP10 (FRAAL1133), a RuvB Holliday Junction helicase b-subunit (FRAEA6206) are downregulated in Ea112 in the presence of 1 mg l21 of seed extract. A M16467 DNA-repair protein RecN (FRAAL5181) was affected by the three concentrations tested. DNA transcription and translation seems to be affected by phenolic mixtures because in M16467 and Ea112 strains, the a-subunit of RNA polymerase rpoA (FRAAL1110) and two lysyl tRNA synthetases (FRAAL1791 and FRAEA1466) were downregulated. Five proteins were associated with energy production and conversion (FRAAL2834, FRAAL0954, FRAAL5843, FRAAL6655 and FRAEA1478), among which two enzymes are involved in glycolysis or gluconeogenesis pathways [a fructose bisphosphate aldolase (FRAAL6655) and a polyphosphate glucokinase (FRAEA 1478)]. Lipid transport and metabolism were also affected: two enoyl CoA hydratase, FRAAL5547 and FRAAL3799 involved in fatty acid biosynthesis were repressed by the higher phenolics concentrations 10 and 30 mg l21, an O-acyltransferase involved in glycerolipid biosynthesis (FRAAL4897) and a carbamoyl transferase of the NodU/ CmcH family (FRAAL4690). Downregulations were also associated with coenzyme metabolism [pantothenate synthase (FRAAL3483) and cobyric acid synthase (FRAAL2887)], carbohydrate transport and metabolism (FRAAL5662 and FRAEA1478), purine metabolism [5#phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR) Synthetase, FRAAL6667]. Three transport proteins, two ATP-binding-ABC tranport system (FRAAL4168 and FRAAL6535) and a sulfurtransferase (FRAAL0943) are also downregulated. Iron metabolism and transport seems to be affected by plant extracts: a homolog to an isochorismatase family protein (FRAAL2926) and a puta386
tive ABC type Fe31 siderophore transport system (FRAAL4168) are downregulated with the three phenolic concentrations tested in the M16467 protein pattern, whereas a conserved hypothetical enterochelin esterase (FRAAL4718) is upregulated in Ea112.
Discussion This work was aimed at studying the molecular mechanisms that control the first steps of actinorhizal symbiosis. We sought to identify modulated proteins of Frankia grown in the presence and in the absence of different concentrations of Myricaceae plant extracts. Identification with high confidence levels was obtained for less than half of the analyzed spots. In spite of the availability of the three Frankia genomes, the absence of genome sequences for the strains M16467 and Ea112, have restricted MALDI-TOF mass spectrometry analysis. Indeed, because of their phylogenetic distance from the Frankia sequenced strains, many spots correspond to proteins not yet described in databases. Normand et al. (2007) observed that only 50% of proteins have homologs (identity > 30%) in the three sequenced genomes. This proportion could be approximately the same for other non-sequenced strains. Concerning the sequenced strain ACN14a, 62% of analyzed spots were identified instead of the 92% expected (Alloisio et al. 2007). The silver staining used, because of its higher sensitivity compared to colloidal blue, is known for its lower MALDI-TOF compatibility. Whatever the concentration used, Myricaceae extracts affected deeply Frankia cells, inducing a global reorganization of protein biosynthesis. A majority of downregulations was observed (two-thirds of analyzed proteins). A similar response was described in Sinorhizobium meliloti, with the upregulation of 130 proteins and the downregulation of at least 350 proteins in the symbiotic state (bacteroid) compared to in vitro culture (Natera et al. 2000). Exposure to Myricaceae seed extracts can represent an environmental stress for Frankia. The adaptation to this stress, particularly to oxidative stress requires energy, and re-orientation of bacterial metabolism that could lead to the downregulation of many different metabolic pathways such as glycolysis and gluconeogenesis, transcription, fatty acid biosynthesis, coenzyme transport and metabolism and carbohydrate metabolism. Frankia could also acquire nutriments through degradation of compounds found in seed extracts. Indeed, the methanolic extract used, mainly composed of phenolic compounds, can also contain other methanol-soluble or amphiphilic molecules. Among upregulated proteins, oxidative stress proteins have been identified as major spots in the gels. FeSOD Physiol. Plant. 130, 2007
is considered as one of the key enzymes in the oxidative defense system of aerobic organisms involved in superoxide anion (O2 2 ) removal. Ter is a protein associated with resistance to tellurium salts, in which mechanism of resistance has not been determined (Silver and Phung 1996). Homologs to FeSOD and tellurium resistance proteins were yet found upregulated in Frankia induced by A. glutinosa root exudates (Hammad et al. 2001) and in the extracellular compartment of Streptomyces coelicolor induced by Lemna minor extract (Langlois et al. 2003), suggesting an adaptation against extracellular host defenses. The need for a bacterial response to the oxidative stress during plant-microbe mutualistic interactions is supported by the fact that an FeSOD mutation affects nodulation in S. meliloti (Ampe et al. 2003, Santos et al. 2000, 2001) and the ability to colonize the rhizosphere in Pseudomonas putida (Kim et al. 2000). SOD and catalases have also been implicated in the survival of some pathogenic bacteria such as Mycobacterium, Xanthomonas and Agrobacterium during infections (Xu et al. 2001). Common mechanisms could take place in the development of symbiosis and pathogenic interactions, which may reflect general bacterial adaptation against host plant defense. This oxidative stress response could be caused either by plant flavonoids or by reactive oxygen species. The methanolic seed extracts used contain mainly a mixture of flavonoids. Among them several are known for their antioxidant activity. However, several recent studies (Cao et al. 1997, Ohshima et al. 1998, Yoshino et al. 1999) revealed that the capacity of flavonoids to act as antioxidants depend upon their molecular structure: number and position of hydroxyl groups, presence or not of metal cofactors such as copper or iron. As a consequence, some flavonoids such as quercetin have been reported to be mutagenic or carcinogenic, effects attributable to their prooxidant activity. The probable presence of prooxidant flavonoids in the complex phenolic mixture may induce the oxidative stress response observed. Moreover, the upregulation of enzymes from Frankia respiratory chain (a membrane-bound ATP-synthase F1, FRAAL5931 and two electron-transfer-flavoprotein FRAAL5877) can also generate reactive oxygen species. Similar early upregulation of SOD and respiratory chain have been observed during interaction between the mycorrhizal fungus Gigaspora margarita and legumes host root exudates (Lanfranco et al. 2005). Other types of stress besides the oxidative stress appear to play a role in host–symbiont interactions. Different chaperones were found to be either upregulated or downregulated, according to the strains. A heat shock protein, HSP18, was upregulated in M16467 strain. HSP18 upregulation has been also observed in ACN14a Physiol. Plant. 130, 2007
strain induced with Alnus root exudates (Hammad et al. 2001). The role of HSP18 in thermotolerance was reported in Streptomyces albus (Servant and Mazodier 1995) and it is hypothesized that it may act as a chaperone in protein folding and unfolding events (Jakob and Buchner 1994). On the contrary, in Ea112 strain, a GroES chaperone (HSP10) and a RuvB Holliday Junction helicase (also involved in DNA replication) were downregulated. Nevertheless, Ea112 pigments could interact with proteins during isofocalization modifying their migration. Thus, downregulated proteins may be present elsewhere on the gel. Several proteins known to be involved in the symbiotic process were identified. The downregulation of a carbamoyl transferase with a significant similarity to NodU of Rhizobium and to CmcH of Nocardia lactamdurans was detected. NodU seems to carry out the carbamoylation of Rhizobium Nod factors but until now, its role in the synthesis of Rhizobium lipochitooligosaccharides remains unclear. The expression of nodU, nodC and nodS genes were shown to be inducible by the isoflavone daidzein in Bradyrhizobium japonicum. (Gottfert et al. 1990). CmcH is involved in Cehamycin (an antibiotic) biosynthesis (Coque et al. 1995). In Frankia ACN14a genome, this carbamoyl transferase belongs to an operon that could be involved in biosynthesis of b-lactam metabolites. The enhancement of antibiotic production was reported for several actinobacteria under environmental stress conditions or nutritional deficiencies (Ghorbel et al. 2006). The possibility to detect other possible Nod proteins would be limited by two-dimensional technique. Indeed, only rhizobial NodE and NodB were identified with proteomic approach (Guerreiro et al. 1997), most of the other Nod proteins being not detectable because of their low concentration (about 10212 M). Moreover, there is presently no evidence for the existence of a comparable Nod-factor-based induction mechanism in actinorhizal symbiosis. Although homologs to Rhizobium nod genes such as nodC, nodB and nodD-like genes were detected in Frankia genomes (Normand et al. 2007), they present low Blast scores and are not clustered in a symbiotic island, as it is the case in Rhizobium. Availability and transport of iron and molybdate were affected by seeds extracts. These two metals are essential for nitrogenase structure and functioning (electron transport, energy production, hydrogenase uptake, leghemoglobine, MoFe cofactors, etc.). More generally, molybdate is also necessary for nitrate reductase, xanthine oxidase and enzyme cofactors. Several differential proteins were found including an upregulated molybdate-binding-protein precursor. For iron, seed extract effects seem to be strain dependant. In M16467 387
strain, a homolog to an isochorismatase family protein, involved in the biosynthesis of a siderophore homologous to enterobactin, was repressed, whereas in Ea112 strain, the biosynthesis of an enterochelin esterase homologous protein, involved in the same enterobactin metabolism pathway, was upregulated. Phenolic compounds such as flavonoids are known to chelate iron (Deng et al. 1997, Morel et al. 1993), consequently their presence in seed extracts could modify iron availability in the culture medium and thus siderophore biosynthesis. Two ABC transporters, a Fe31 siderophore transport system and a phosphate transport system were downregulated. These inhibitions are congruent with the known capacity of flavonoids to inhibit enzymatic activity of ATP-binding proteins (Di Pietro et al. 1975, Thiyagarajah et al. 1991) by binding to the ATP sites and thus ABC transport systems (Conseil et al. 2000). Myricaceae seed extracts repressed a SAICAR synthetase which catalyses the ATP-dependant synthesis of SAICAR during purine biosynthesis. The purine biosynthesis pathway is implicated in Legume infection process as shown by purine auxotrophs (Pur-) of several Rhizobium species, that were found to elicit pseudonodules with abnormal morphological features, thus leading to defective symbiosis (Denarie and Bergeron 1976, Pain 1979). In our study, the inhibition observed could be the presence in the seed extract used of purine precursors able to inhibit the purine biosynthesis pathway. A putative phospholipids-binding protein (FRAAL0033), upregulated by phenolic extracts, also has homology to an inhibitor of the Raf-signalling cascade and several other cell-signalling cascades. In prokaryotes such as Escherichia coli (Serre et al. 2001), raf kinase inhibitor protein homologs have been observed and may be part of a mitogen-activated protein kinase cascade. Alloisio et al. (2007) also found this protein to be upregulated in Frankia alni under nitrogen-fixing conditions and suggested a possible role in phosphorylation and adaptation to symbiosis. Binding to phospholipids may play a role in either stabilization of the membranes or in building the hopanoid-containing cell wall of diazovesicles, which protect nitrogenase against oxygen. The primary significance of this report is to offer the first extensive protein database for understanding how Frankia strains react to the presence of host plant extracts. The differentially expressed proteins are important for adaptation to the host environment, such as protection against host defenses mechanisms and could be involved in symbiosis initiation. Other approaches such as new quantitative proteomic methods together with phytochemical progresses in phenolic purification from actinorhizal plants will provide more insight for elucidating the molecular bases of actinorhizal symbiosis initiation. 388
Acknowledgements – Thanks are expressed to M Becchi and I Zanella-Cleon (UMR 5086 CNRS/UCBL, IBCP, Lyon, France) for MALDI-TOF analysis of the protein spots, to JJ Madjar (IFR41 DTAMB, Lyon, France) for proteomic technical advice, G Comte and C Bertrand (UMR CNRS 5557, Lyon, France) for phytochemistry competences and to M Blanchard (Conservatoire Botanique National Aquitaine/Poitou-Charentes, Jardin Botanique, Bordeaux, France) for seed collection facilities.
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