Comparative Proteomics Analysis of the Rice Roots Colonized by

Article pubs.acs.org/jpr

Comparative Proteomics Analysis of the Rice Roots Colonized by Herbaspirillum seropedicae Strain SmR1 Reveals Induction of the Methionine Recycling in the Plant Host Dayane Alberton, Marcelo Müller-Santos, Liziane Cristina Campos Brusamarello-Santos, Glaucio Valdameri, Fabio Aparecido Cordeiro, Marshall Geoffrey Yates, Fabio de Oliveira Pedrosa, and Emanuel Maltempi de Souza* Department of Biochemistry and Molecular Biology, Federal University of Paraná, Rua Francisco H. dos Santos s/n Centro Politécnico, Curitiba, Paraná 81531-990, Brazil S Supporting Information *

ABSTRACT: Although the use of plant growth-promoting bacteria in agriculture is a reality, the molecular basis of plant−bacterial interaction is still poorly understood. We used a proteomic approach to study the mechanisms of interaction of Herbaspirillum seropedicae SmR1 with rice. Root proteins of rice seedlings inoculated or noninoculated with H. seropedicae were separated by 2-D electrophoresis. Differentially expressed proteins were identified by MALDITOF/TOF and MASCOT program. Among the identified proteins of H. seropedicae, the dinitrogenase reductase NifH and glutamine synthetase GlnA, which participate in nitrogen fixation and ammonium assimilation, respectively, were the most abundant. The rice proteins up-regulated included the S-adenosylmethionine synthetase, methylthioribose kinase, and acireductone dioxygenase 1, all of which are involved in the methionine recycling. S-Adenosylmethionine synthetase catalyzes the synthesis of S-adenosylmethionine, an intermediate used in transmethylation reactions and in ethylene, polyamine, and phytosiderophore biosynthesis. RT-qPCR analysis also confirmed that the methionine recycling and phytosiderophore biosynthesis genes were up-regulated, while ACC oxidase mRNA level was down-regulated in rice roots colonized by bacteria. In agreement with these results, ethylene production was reduced approximately three-fold in rice roots colonized by H. seropedicae. The results suggest that H. seropedicae stimulates methionine recycling and phytosiderophore synthesis and diminishes ethylene synthesis in rice roots. KEYWORDS: Herbaspirillum seropedicae SmR1, rice roots, proteome, methionine recycling, ethylene

1. INTRODUCTION The diazotroph−grass association is usually beneficial to both partners. Diazotrophs generally do not cause damage to the host organism and promote plant growth by the production and secretion of growth regulators, by antagonistic activity to plant pathogens, and by supplying biologically fixed nitrogen.1 The production of phytohormones, such as auxin and gibberellins, can be stimulated by the presence of diazotrophic endophytes or even produced by them.2,3 However, the intensity of the stimulus for the plant growth, including nitrogen fixation and the transference of the fixed nitrogen to the plant, depends on an efficient plant−bacterial interaction,4,5 which, in turn, depends on the genotype of the plant and the bacterial strain.6,7 Herbaspirillum seropedicae is a diazotrophic endophyte known to associate with many economically important plants, such as rice, sorghum, wheat, and sugar cane.8 Inoculation of H. seropedicae Z67 in different varieties of rice demonstrated that this bacterium enhanced the rice dry weight and carbon and nitrogen content at 28 days after inoculation.9 In micropropagated sugar cane inoculated with H. seropedicae and H. rubrisubalbicans, the total dried material of the plant was © 2013 American Chemical Society

1.3 times higher when compared with noninoculated sugar cane.10 These results suggest that H. seropedicae has a potential for application as a plant inoculant to reduce the use of nitrogen fertilizers, avoiding high emissions of nitrogen oxides and diminishing soil acidification and water eutrophication.11 Proteomic analyses of plant−pathogen associations have received significant attention,12,13 in addition to the symbiotic interactions between microbes and plants.14 However, few proteomic studies of endophyte−plant interactions, especially with grasses, have been conducted. The comparative proteome of Sinorhizobium meliloti 1021 and rice plants showed that this rhizobial symbiont stimulates the expression of plant proteins in the leaf sheath and leaf involved in photosynthesis, while the proteins expressed in roots were exclusively related to defense mechanisms.15 In one of the few studies of the interaction between endophytes and grasses, the distinct colonization characteristics of two sister lineages of Special Issue: Agricultural and Environmental Proteomics Received: May 4, 2013 Published: August 26, 2013 4757

dx.doi.org/10.1021/pr400425f | J. Proteome Res. 2013, 12, 4757−4768

Journal of Proteome Research

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Oryza sativa (IR 36, IR42) by the diazotroph Azoarcus sp. strain BH72 was reflected in differences of their root proteomes.16 The interaction between Azoarcus sp. strain BH72 and IR 42 was less compatible, showing an intense defense response of the plant through the expression of seven proteins involved in defense and stress responses. The more compatible colonization of Azoarcus sp. with IR 36 induced only the expression of two isoforms of a defense protein (SalT). Kandasamy and coworkers17 showed that Pseudomonas fluorescens KH-1 promoted rice plant growth and yield by up-regulating the expression of proteins from the primary metabolism as well as those related to stress and defense systems of the rice leaf sheath tissues. A quantitative mass-spectrometry-based proteomic analysis showed that the endophytic interaction between Gluconacetobacter diazotrophicus with two distinct genotypes of sugar cane (Sp770−1143 and Chunee) elicited different responses. SP70−1143 expressed proteins involved in stress resistance, cellular adaptation, and signaling systems that enabled bacterial root colonization. Chunee elicited robust defense mechanisms against G. diazotrophicus cells, avoiding a successful interaction.18 In this work, we used proteomics analysis and RT-qPCR to study the molecular mechanisms of the plant-bacterial interaction between H. seropedicae SmR1 and roots of Oryza sativa ssp. japonica cv. Nipponbare. The results suggest that H. seropedicae SmR1 activated the methionine recycling, reduced the ethylene biosynthesis in rice roots, and stimulated the phytosiderophores biosynthesis.

2 days to determine the number of endophytic bacteria. The colonies were counted in each dilution from different tissues, and bacterial populations were expressed as CFU/g of fresh roots or aerial part. As negative controls, the roots and aerial parts from the non-inoculated seedlings were rinsed with sterile water, macerated in saline, and plated on Luria agar and NFbHP-malate medium containing 20 mM NH4Cl and incubated at 30 °C. 2.3. Dry Weight of Roots and Aerial Parts

The rice aerial parts and roots of the control and treated plants were separated and dried at 50 °C to constant weight. 2.4. Ethylene Measurement

Ethylene production was determined after 7 days of growth of rice plants in tubes. Tubes containing rice plants subjected to both inoculation with H. seropedicae and control conditions were capped with SubaSeal rubber septa for 24 h, and ethylene production was determined by gas chromatography (Varian Star 3400 CX equipped with a Porapak N column and FID detector). The column and detector temperatures were maintained at 120 and 200 °C, respectively. Ethylene production was expressed in pmol/g of plant dry weight. The quantity of ethylene was calculated using as external standard an authentic ethylene standard (100 ppm) in gaseous nitrogen. Negative controls were carried out in sealed tubes containing propylene beads and Hoagland medium and analyzed under the same conditions. Ten samples of each condition were analyzed with three repetitions.

2. MATERIALS AND METHODS

2.5. Protein Extraction from Rice Roots

2.1. Growth Condition

For gnotobiotic cultures of rice with H. seropedicae, seeds from Oryza sativa ssp. japonica cv. Nipponbare were dehulled and surface-sterilized with 70% ethanol for 5 min, followed by 30 min incubation in 6% (v/v) sodium hypochlorite containing 0.1% Triton X-100. After rinsing 20 times in sterile water, the seeds were incubated overnight in 0.025% (v/v) Vitavax Fungicide solution at 30 °C. Surface-sterilized seeds were germinated in 1% (m/v) sterile agar-water in the dark for 3 days at 30 °C. Three axenic seedlings were transferred aseptically to a sterile glass tube containing propylene beads and 25 mL of carbon- and nitrogenfree Hoagland nutrient solution19 with the following composition: 1 mM KH2PO4, 1 mM K2HPO4, 2 mM MgSO4.7H2O, 2 mM CaCl2.2H2O, 1 mL/L micronutrient solution (H3BO3 2.86 g·L−1, MnCl2·4H2O 1.81 g·L−1, ZnSO4·7H2O 0.22 g·L−1, CuSO4·5H2O 0.08 g·L−1, Na2MoO4·2H2O 0.02 g·L−1), and 1 mL·L−1 Fe-EDTA solution (Na2H2EDTA.2H2O 13.4 g·L−1 and FeCl3.6H2O 6 g·L−1), pH 6.5−7.0. Plants were cultivated at 26 °C under 14 h of light/10 h of darkness for 7 days. H. seropedicae was cultivated in NFbHP malate medium20 containing 5 mM glutamate at 30 °C in a rotary shaker (120 rpm) to OD600nm = 1 (108 cells/mL). For inoculation with H. seropedicae, axenic seedlings were incubated for 30 min in the bacterial suspension (108 cells/mL). The seedlings were then washed with sterile water and transferred to glass tubes containing 25 mL of carbon- and nitrogen-free Hoagland solution for 7 days.

The protein extraction procedure was based on the phenol extraction method.21,22 The rice roots from gnotobiotic seedlings and the seedlings colonized by H. seropedicae were excised and washed with sterile water. The roots were frozen in liquid nitrogen and ground to obtain a fine powder. The powder samples (250 mg) were homogenized in 0.8 mL of precooled extraction buffer (50 mM Tris-HCl pH 8.5, 5 mM EDTA, 100 mM KCl, 30% (m/v) sucrose; 1% w/v DTT, 1 mM PMSF) and sonicated on an ice bath. After sonication, an equal volume of ice-cold Tris-HCl pH 8.0-saturated phenol was added. The mixture was vortexed for 10 min and centrifuged at 16 000g and 4 °C for 20 min. The phenol phase was collected and transferred to another tube. The aqueous phase was extracted again with phenol, which was added to the first phase. The total phenol phase was mixed again with the precooled extraction buffer. The mixture was centrifuged (16 000g, 4 °C, 20 min), and the phenolic phase was recovered in new tubes. Proteins were precipitated with three volume of 100 mM ammonium acetate in methanol overnight at −20 °C. After centrifugation, the pellets were rinsed three times with ice-cold methanol and three times with ice-cold acetone. The pellets were dried at room temperature to remove the acetone and dissolved in isoelectrofocusing buffer (8 M urea, 2 M thiourea, 4% (m/v) CHAPS, 2% (m/v) Triton X-100, 100 mM DTT; 1% (v/v) IPG buffer pH 4−7) by gentle shaking at room temperature for 1 h.

2.2. Herbaspirillum seropedicae SmR1 Cells Count

2.6. Two-Dimensional Polyacrylamide Gel Electrophoresis

After 7 days of growth, the rice plants were carefully removed from the glass tubes. The roots and aerial parts of inoculated plants were separated and submitted to superficial disinfection for 1 min in 70% ethanol, followed by 1 min in 1% chloramine T (Sigma) and rinsed three times with sterile water. Surfacesterilized samples of roots and aerial parts were macerated in sterile saline solution, diluted, plated on NFbHP-malate solid medium containing 20 mM NH4Cl, and incubated at 30 °C for

For isoelectrofocusing, 500 μg of protein were loaded onto a 13 cm, pH 4−7 linear IPG strip (GE Healthcare). The IPG strips were rehydrated for 16 h at 50 V. The isoelectrofocusing run was performed using the following conditions: 500, 800, 11 300, and 13 000 Vh, accumulating ∼26.4 kVh. After IEF, the strips were equilibrated with 6 M urea, 2% (m/v) SDS, 50 mM Tris−HCl (pH 8.8), 30% (m/v) glycerol, and 65 mM dithiothreitol for 30 min. In the second equilibration 4758



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2.10. Quantification of mRNA Levels by Reverse Transcription Quantitative PCR

step (30 min), 216 mM iodoacetamine was used instead of DTT. The second dimension was achieved by 11.5% (acrylamide/bisacrylamide 29:1) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a Ruby SE 600 vertical electrophoresis system (GE Healthcare) at a constant current of 45 mA per gel. Gels were stained according to the Blue Silver protocol.23

Reverse transcription quantitative PCR (RT-qPCR) was carried out using the SYBR Green PCR Master Mix and cDNA as template in a StepOnePlus Real-Time PCR Detection System (both from Applied Biosystems) according to manufacturer’s instructions. Sets of specific primers were designed using the genome sequence of Oryza sativa ssp. japonica cv. (Sequences are in Table S1 in the Supporting Information.) A dissociation cycle was performed after each run to check for nonspecific amplification or contamination. RT-qPCR reactions using the purified RNA without treatment with reverse transcriptase were used as control, and all samples were made in triplicate. Calibration curves for all primer sets were linear over four orders of magnitude (R2 = 0.98 to 0.99) and efficiencies were 90% or higher. The mRNA expression levels were normalized using expression levels of actin 1, tubulin beta-2 chain (beta-2 tubulin),25 and a hypothetical protein (protein kinase)26 using geNorm 3.4 software.27 Relative expression levels were estimated as described.28

2.7. Gel Image Analysis

The 2-DE gels were scanned with a LabScan scanner (GE Healthcare) at a 300 dpi resolution. Gel images were analyzed using ImageMaster Platinum 6.0 (GE Healthcare). The spots were quantified by their relative volume (vol %), defined as the ratio of the individual volume of each spot to the sum of all spots in the gel. The 1.5-fold change in average spot volume was used to define the protein spots chosen for further spectrometric analysis. For each condition (inoculated and noninoculated), three independent biological experiments (biological replicates) were performed, and four technical replicates were obtained for each experiment. 2.8. In-Gel Digestion, MALDI-TOF/TOF MS Analysis, and Database Searches

3. RESULTS AND DISCUSSION

The spots were excised manually from the gels and destained twice for 30 min in acetonitrile 50% (v/v) with 25 mM ammonium bicarbonate, pH 8. The gel plugs were immersed in acetonitrile for 5 min, the acetonitrile was removed, and the gel was dried under a stream of air. In-gel digestion was performed using 20 ng/μL of sequencing-grade trypsin (Promega, Madison, WI) in acetonitrile 10% (v/v) and 40 mM ammonium bicarbonate in a final volume of 10 μL.24 After overnight incubation at 37 °C, aliquots of each digested sample were mixed with a saturated matrix solution of α-cyano-4hydroxycinnamic acid (in 50% acetonitrile, 0.1% (v/v) TFA), spotted onto the MALDI target and allowed to dry. Mass spectra were acquired using a MALDI-TOF/TOF Autoflex II (Bruker Daltonics). MS analyses were performed in a positive ion reflection mode using an accelerating voltage of 20 kV. MS/MS analyses were performed in a positive ion LIFT reflection mode. Peak lists were created using FLEX ANALYSIS 3.0 software (Bruker Daltonics). Trypsin autolysis signals (842.5 and 2211.1 m/z) were used as internal standards when present. Database searching was performed using MASCOT 2.2. Mass lists were searched against the databases of H. seropedicae SmR1 (accession number CP002039.1) and Oryza sativa predicted proteins. Carbamidomethylation of cysteines was set as fixed and oxidation of methionine was set as variable modifications. For peptide mass fingerprint (PMF) search, the maximum error allowed was 200 ppm. For MS/MS ion search, the error was set as 0.6 Da. Only peptides and proteins with scores higher than the Mascot threshold scores (p < 0.05) were considered as positive identification.

3.1. Colonization of Oryza sativa ssp. japonica cv. Nipponbare Plants by H. seropedicae SmR1

H. seropedicae SmR1 was detected internally in the radicular system and in aerial parts of Nipponbare rice plants 7 days after inoculation. The number of H. seropedicae SmR1 in the surface sterilized roots of the rice plants was 105 CFU/g of moist root, confirming that the bacteria colonized the plants internally. The total number of H. seropedicae SmR1 in the roots was ∼109 UFC/g of moist root. In contrast, no bacterial colony was observed in noninoculated plants before or after surface sterilization. 3.2. Protein Identification by MALDI-TOF/TOF MS

Proteomic analysis of rice roots inoculated and noninoculated with H. seropedicae SmR1 was performed on 12 gels of each condition (three independent protein extractions and four gels of each extraction). A total of 566 ± 49 and 548 ± 53 protein spots were detected in the inoculated and noninoculated gels, respectively (Figure 1). Image analyses detected 39 spots differentially expressed in colonized roots, where 32 were more abundant and 7 were less abundant. Among the former 32 spots, 26 were identified, 9 of which corresponded to proteins from rice and 17 from H. seropedicae SmR1 (Tables 1 and 2). Among the seven less abundant spots in colonized rice roots, only two were identified. The mean and standard deviation of the relative volumes of the identified spots are presented in Tables S2 and S3 in the Supporting Information.

2.9. RNA Isolation and First-Strand cDNA Synthesis

3.3. Proteins of Oryza sativa ssp. japonica cv. Nipponbare Roots Down-Regulated by Colonization with Herbaspirillum seropedicae SmR1

Total RNA was isolated from 100 mg rice roots harvested 7 days after inoculation using TRI Reagent (Sigma, St. Louis, MO). Genomic DNA was removed with DNase I (Ambion) before the reverse transcriptase reaction. The integrity and quality of the total RNA was confirmed by spectrophotometric analysis and electrophoresis. cDNA was produced from 2 μg DNase I-treated RNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instruction, and the resulting cDNA was stored at −20 °C.

Spots 913 and 2856 correspond to putative L-ascorbate peroxidases (APX) from Oryza sativa Japonica group (Figure 1). These spots volumes were reduced 1.6- and 2.2-fold in colonized roots, respectively (Table 1). APX belongs to one of the most important class of antioxidant enzymes in plants.29 APX detoxifies hydrogen peroxide using ascorbate as an electron donor, producing dehydroascorbate and water.30 Thus, expression of APXs can be activated by stress elicitors such as pathogen attack, mechanical pressure, injury, UVB radiation, and reactive oxygen species (ROS).31,32 Mittler and 4759



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Figure 1. Protein profiles of rice roots proteins from plants noninoculated (A) and inoculated with H. seropedicae SmR1 (B). Protein spots of H. seropedicae SmR1 present only in inoculated roots are indicated by a circle (○). Rice protein spots differentially expressed in roots 7 days after inoculation with H. seropedicae are indicated by a square (□). For the first dimension, 500 μg of proteins were loaded on a 13 cm IPG strip with a linear gradient of pH 4−7, and 11.5% SDS-PAGE gels were used for the second dimension. Proteins were visualized by blue silver staining.

coworkers33 showed that during viral-induced programmed cell death in tobacco, the expression of cytosolic APX was posttranscriptionally suppressed, while the level of transcripts encoding cytosolic APX protein was induced. In general, a significant decrease in APX levels may lead to a reduction in the capability of cells to detoxify H2O2. RT-qPCR confirmed downregulation of APX913, while APX2856 mRNA levels were not regulated, suggesting post-transcriptional regulation. We cannot explain why H. seropedicae SmR1 down-regulated APX expression in rice roots.

exposed to auxin or cytokinin plant hormones.36 Moreover, tau GSTs are also known to be induced by infection or by treatments that elicit plant defense reactions as well as by osmotic stress and extreme temperatures.37 It is possible that up-regulation of tau GST 4 is due to secretion of indoleacetic acid by H. seropedicae.2 Two spots (542 and 545), 1.8 and 1.9-fold more abundant in colonized roots, were identified as S-adenosylmethionine synthetase (SAMS) and S-adenosylmethionine synthetase 2 (SAMS2), respectively (Table 1). The two proteins share an identity of 93%. These enzymes are involved in the methylthioadenosine (MTA) or Yang cycle38 for methionine recycling and catalyze S-adenosylmethionine (AdoMet) synthesis from methionine and ATP.39 In plants, over 90% of AdoMet is used as a methyl donor in transmethylation reactions and also as a substrate for ethylene, polyamine and phytosiderophore synthesis,40,41 producing MTA as byproduct, which can be recycled to methionine.42,43 SAMS was also up-regulated in shoots of rice colonized by Sinorhizobium meliloti.15 Under stress conditions, SAMS increases plant lignification, providing methyl groups for polymerization of lignin monomers.15,44,45 Two other protein spots corresponding to enzymes of the methionine recycling were found up-regulated in rice roots colonized by H. seropedicae SmR1: the MTK (spot 551), with 6.6-fold increase compared with control plants, and the ARD1 (spot 903), expressed only in colonized roots (Table 1). MTK catalyzes the second step of the methionine recycling, the phosphorylation of methylthioribose to methylthioribosephosphate. ARD1 catalyzes the reaction of acireductone with oxygen to produce the immediate precursor of methionine, 2-keto-4-methylthiobutyrate (KMTB), and formate. In rice, ARD1 gene transcription has been shown to be induced by low levels of ethylene.46 In bacteria, ARD is a unique enzyme with two different enzymatic activities, depending on the metal ion bound as cofactor.47 Fe-ARD acts in the methionine recycling to produce KMTB and the byproduct formate, whereas Ni-ARD catalyzes an off-pathway reaction that produces methylthiopropionic acid, formate, and carbon monoxide. Sauter and coworkers46 showed that apo-ARD1 of deepwater rice displayed a much higher affinity toward Fe2+, thus favoring its participation in the methionine recycling. Overall, the results show that in rice

3.4. Proteins of Oryza sativa ssp. japonica cv. Nipponbare Roots up-Regulated by Colonization with Herbaspirillum seropedicae SmR1

The rice root proteins up-regulated by colonization with H. seropedicae SmR1 were 1-aminocyclopropane-1-carboxylic acid synthetase (ACS), tau class GST protein 4 family, salt stressinduced protein (SalT), adenine phosphoribosyltransferase (APRT), NAD-dependent formate dehydrogenase (FDH), two isoforms of S-adenosylmethionine synthetase (SAMS), methylthioribose kinase (MTK), and acireductone dioxygenase 1 (ARD1) (Table 1). The SalT (spot 810) from rice was overexpressed 1.7 times in colonized roots (Table 1). SalT is a homodimeric cytoplasmic mannose-binding lectin with subunits of 15 kDa.34 The molecular mass observed for SalT was 31 kDa, suggesting that the subunits were tightly bound. This protein can be induced under salt-stress conditions, but other stress elicitors can also induce its expression.35 Michè and coworkers16 showed that SalT was up-regulated in jasmonic acid treated rice roots and in Oryza sativa roots colonized by Azoarcus sp. BH72, but in the former, SalT expression was higher, indicating that the endophyte elicits only a mild stress/ defense response in a compatible interaction. The increase in the SalT expression by H. seropedicae colonization revealed that the bacterium, similarly to Azoarcus sp. BH72, probably elicited rice defense response. Spot 848 was identified as a tau class GST 4 protein. This protein was induced 1.6-fold in colonized plants compared with the control (Table 1). Several tau GSTs are known to be strongly induced during cell division or when plants are 4760


adenine phosphoribosyltransferase

acireductone dioxygenase1

873

903

4761

Os03t0285700-01 Os07t0694700-01

Os03t0161800-01

Os12t0589100-01

Os01t0348900-01 Os10t0528300-01

Os06t0486800-01

32 77 75 58 66 66 82 72 118 68 66 121 48 105 48 78 44 92 75 70

3 21 27 3 5 5 22 3 34 3 35 33 3 35 3 30 6 7 34 21 5/11 4/5

5/7

7/14

3/3 8/15

12/31

6/11

10/35 7/20

Mowse scorea SC (%)b NMP/NSPc exp h

27/5.42 27/5.21

24/5.08

26/9.26

15/5.19 26/4.97

41/6.87

39/5.68

27/5.55 −1.6h 27/5.15 −2.2h

24/5.04 present

27/5.83 +1.7h

31/5.04 +1.7 28/4.86 +1.6h

43/6.65 present

48/5.85 +1.9h

MS /0.6 MS/100 MS/160 MS2/0.6 MS2/0.6 MS2 MS/100 MS2/0.6 MS/100 MS2/0.6 MS/100 MS/100 MS2/0.6 MS/100 MS2/0.6 MS/150 MS2/0.6 MS2/0.6 MS/100 MS/100

2

fold changed MSe/MEf

49/6.18 47/6.37 +6.6 48.9/6.63 46/6.01 present 43/5.83 48/5.98 +1.8h

theor

fragment (ion score)g

1465.6310 YCLEGSGYFDVR (44) 1815.7920 NFFEEHLHTDEEIR (92)

1121.8060 DTIELFVER (48)

1193.6480 FVPAWVATFR (48)

1689.9440 LKPFNCNLLYHDR (68)

1453.5280 FVIGGPHGDAGLTGR (72)

1454.0270 FVIGGPHGDAGLTGR (58) 2418.1410 VLVNIEQQSPDIAQGVHGHFTK (66) VLVNIEQQSPDIAQGVHGHFT (66)

1840.7940 GLCPDHVPEVYHFDR (32)

m/z peak

Mowse score represents the score obtained in the matching with NCBInr database by MASCOT software. bSequence coverage. cNumber matched peaks/number searched peaks. dChange in abundance of protein spots compared to the control analyzed by IMAGE MASTER PLATINUM. Positive numbers mean up-regulated and negative numbers mean down-regulated. eProteins were identified by MS or MS2 mode. fMaximum error (ME) allowed for peptide mass fingerprint (PMF) was 200 ppm and 0.6 Da for MS2. gPeptide fragment identified by MS2 mode using ion score algorithm. Values with higher scores than ion score cutoff are accepted (p < 0.05). hp < 0.05 represents the significance of up- or down-regulation of spots according to the t test through analysis of variance.

a

ascorbate peroxidase ascorbate peroxidase

salt stress-induced protein Tau class GST protein 4

810 848

913 2856

NAD-dependent formate dehydrogenase

622

Os01t0323600-01

S-adenosylmethionine synthetase 2

545

accession n° RAPDB

methylthioribose kinase Os04t0669800-01 1-aminocyclopropane-1-carboxylic acid synthase Os09t0453800-01 Os05t0135700-02 S-adenosylmethionine synthetase

protein name

551 562 542

spot ID

Mr (kDa)/pI

Table 1. Rice Root Proteins Differentially Expressed in Plants Inoculated with Herbaspirillum seropedicae SmR1

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Figure 2. Expression of rice genes by reverse-transcription quantitative-PCR. Significant differences (*p < 0.05, ** p < 0.01, and *** p < 0.001) were determined using one-tailed t test (GraphPad Prism 5). The mRNA levels of genes coding the rice root proteins were differentially expressed, and additional selected genes were normalized using expression levels of three housekeeping genes (actin 1, tubulin beta-2 chain, and protein kinase). Three independent RNA samples were extracted from 100 mg of rice root inoculated or noninoculated with H. seropedicae SmR1, and each sample was analyzed in triplicate. The number is parentheses refers to the spot number in the 2-D gels. Data represent three independent experiments, and each sample was run triplicate.

3.5. Reverse Transcription Quantitative-PCR Analysis and Ethylene Production

roots colonized by H. seropedicae SmR1, the expression of proteins from the methionine recycling is up-regulated, indicating the occurrence of methionine recycling. Spot 562, detected only in the colonized roots, was identified as ACS (Table 1). ACS catalyzes the conversion of AdoMet to 1-aminocyclopropane-1-carboxylate (ACC), which is converted to ethylene by ACC oxidase (ACO).48 Thus, increase in ACS may lead to increased ethylene production, which is in agreement with the observation that colonization by endophytic bacteria may trigger defense reactions in the plant, such as ethylene biosynthesis.48 The enzyme formate dehydrogenase enzyme (FDH) (spot 622) was also up-regulated in the roots of rice colonized by H. seropedicae SmR1 (Table 1). This enzyme catalyzes oxidation of formate to carbon dioxide with the coupled reduction of NAD+ to NADH.49 Formate is a byproduct of acireductone dioxygenase. The detection of FDH in the colonized rice roots is possibly due to the production of formate by ARD1 activity in the roots. Spot 873 was identified as the adenine phosphoribosyltransferase (APRT), whose level was 1.7 times higher in colonized roots than in the control. However, the theoretical and experimental isoeletric points were discrepant (Table 1) with a shift to the acidic pH. Allen and coworkers50 predicted several phosphorylation sites in the APRT sequence, and thus the APRT identified was probably phosphorylated. APRT converts adenine to AMP in a one-step reaction that is thought to be the main salvage pathway for adenine in higher plants.51 Adenine is produced in the methionine recycling from the conversion of MTA to MTR by the MTA nucleosidase. In summary, up-regulation of APRT is in agreement with the previous finding in our comparative proteomic study, indicating that inoculation with H. seropeciae SmR1 up-regulates expression of enzymes of the methionine recycling in rice roots. Figure 5 summarizes these results.

Because of limitations of 2-DE, namely, the possibility of colocalization of more than one protein in a spot, compromising quantification52 reverse transcription quantitativePCR (RT-qPCR) was used to check the regulation of the methionine recycling genes in rice roots upon inoculation with H. seropedicae SmR1 by determining the relative levels of mRNA. Figure 2 shows that the genes coding for MTK, SAMS, SAMS2, ARD1, and FDH were induced upon inoculation with H. seropedicae SmR1, confirming activation of the methionine recycling in colonized rice roots. To support this observation, we also evaluated the expression levels of three other key genes involved in the methionine recycling: MTA nucleosidase, methylthioriburose-1-phosphate isomerase (MTR isomerase), and dehydrogenase−enolase−phosphatase. All three genes had expression levels increased by eight-fold in inoculated plants (Figure 2), further supporting activation of the methionine recycling in colonized roots. Figure 5 summarizes these results. Adomet produced from methionine recycling may be used for ethylene, phytosideropheres, and polyamines synthesis.40,41 To infer to which pathway Adomet is being directed, we evaluated mRNA level of several genes involved in those pathways. ACC synthase (ACS) up-regulated in colonized roots, catalyzes the conversion of AdoMet to ACC, which can follow to any of the above pathways. mRNA levels of ACC oxidase (ACO) was decreased two-fold in colonized roots compared with control (Figure 2), suggesting lower ethylene synthesis. Quantification of ethylene using gas chromatography showed that rice plants colonized by H. seropeciae SmR1 produced ∼824 pmol of ethylene/g of plant dry weight by 24 h, while in noninoculated plants, it reached 300 pmol/g of plant dry weight in the same time (Figure 3). Therefore, the observed 4762



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dehydrogenase. H. seropedicae SmR1 can use ethanol as a carbon and energy source via alcohol dehydrogenase and aldehyde dehydrogenase to yield acetyl-CoA which is fed into the TCA cycle.53 The expression of the alcohol dehydrogenase suggests secretion of ethanol by the rice roots. This was probably due to the hydroponic conditions used in the work, which may cause reduced gas exchange between the plant tissue and the atmosphere, leading to hypoxic conditions in the roots and activation of fermentation pathways to produce the ethanol.56 Another enzyme from H. seropedicae SmR1 expressed in colonized rice roots was the dinitrogenase reductase (spot 760), confirming previous results that H. seropedicae SmR1 sustains nitrogen fixation during association with graminaceous plants.57 H. seropedicae SmR1 also expressed large amounts of glutamine synthetase (GS). This protein was detected in spots 413 and 419. The two isoforms of GS have 59KDa with pI of the 5.68 and 5.62, respectively. Comparing the relative percentage of spot volumes, spot 413 (78.8%) was more abundant than 419 (21.2%). The GS of H. seropedicae SmR1 is reversibly inactivated by adenylylation when ammonium is abundant. In the 2-D proteome reference map of H. seropedicae SmR1 grown under aerobic and high-NH4+ concentration (20 mM), the two forms of GS were also detected, but the most abundant (57%) was the more acidic form that corresponds to the inactive adenylated form.58 In our study, confirmation of adenylylation was not possible due to the absence of the tryptic peptide DLYHLPPEEDK in the acquired spectrum that contains the site of adenylylation, the conserved Y400 residue. Nevertheless, the predominance of the less acidic spot, probably corresponding to the unmodified active form of GS, in H. seropedicae SmR1 colonized rice roots indicates that the bacterial cells are under nitrogen limitation and is in agreement with the expression of dinitrogenase reductase by the bacteria. Spot 643, also detected in colonized rice roots, corresponds to an acetyl-ornitihine aminotransferase (ACOAT) from H. seropedicae SmR1. This enzyme participates in arginine metabolism and catalyzes the reversible conversion of N-acetylglutamate-5-semialdehyde and glutamate to N2-acetylornithine and 2-oxoglutarate.59 The HadL HAD superfamily haloalkanoic acid dehalogenase (spot 881) from H. seropedicae belongs to a large, ubiquitous enzyme superfamily dominated by ATPases (20%) and phosphatases (79%).60 This enzyme could be involved in the halide detoxification reactions because rice paddies are significant sources of CH3Cl, CH3Br, and CH3I.61 Saini and coworkers62 showed that halomethanes are synthesized by higher plants via a methyltransferase. Leaf extracts of Brassiea oleracea catalyze the 5-adenosyl-L-methionine-dependent methylation of halides (Cl¯, Br¯, I¯) to halomethanes.62 The formation of halomethanes provides a mechanism for the elimination of halides, which are known to be phytotoxic. Possibly, H. seropedicae expresses HAD to allow the use of the root-secreted methyl halides. Other proteins from H. seropedicae SmR1 were also identified: Pnp polyribonucleotide nucleotidyltransferase, DnaK, GroEL1 (HSP60), Tig peptidyl-prolyl cis/trans isomerase (trigger factor), UspA, universal stress protein, and the elongation factor EF-Tu. The spots 418 and 422 correspond the GroEL1 (HSP60) isoforms with pIs of 5.02 and 5.08, respectively. In the H. seropedicae 2-D reference map cultivated under high ammonium conditions, the Pnp polyribonucleotide nucleotidyltransferase, DnaK, GroEL1 (HSP60), Tig peptidylprolyl cis/trans isomerase (trigger factor), UspA universal stress protein, and the elongation factor EF-Tu were identified as the

Figure 3. Ethylene production by rice plants colonized and noncolonized with Herbaspirillum seropedicae SmR1. Ethylene production by rice plants 7 days after inoculation was determined by gas chromatography and is expressed in pmol of ethylene produced per 24 h and gram of dry weight of rice plants. The values represent the average and the bars, the standard deviation of ten replicates with three measurements for each replicate.

decrease in mRNA levels of ACO (Figure 2) is in agreement with the lower production of ethylene (Figure 5) in rice roots colonized by H. seropedicae SmR1. In addition, a gene coding for a probable ACC deaminase is present in the H. seropedicae genome, and ACC deaminase is known to compete with ACO for ACC.53 This pathway can further contribute to modulate the levels of ethylene in plants colonized with H. seropedicae SmR1, thus decreasing the stress response promoted by ethylene and allowing appropriate plant growth under stress conditions (Figure 5). However, expression of ACC deaminase from H. seropedicae SmR1 was not tested, and further experiments are necessary to demonstrate its importance to modulate rice ethylene production. Mugineic acids (MAs) are phytosiderophores exudated by graminaceous plants. It was shown that in iron-deficient rice roots, MA production increases and leads to activation of the methionine recycling because the primary precursor of MA biosynthesis is methionine (Figure 5).54 Therefore, we evaluated the expression levels of genes coding for key enzymes of phytosideropheres synthesis pathway. As shown in Figure 2, the mRNA relative level of nicotianamine synthase 1 (NAS1) was 100 times higher in inoculated plants. In addition, an increase of approximately 10-fold in mRNA levels of nicotianamine aminotransferase (NAAT) and deoxymugineic acid synthase (DMAS) genes was observed. Taken together, the data suggest that nicotianamine and phytosideropheres synthesis is induced upon inoculation of rice with H. seropedicae SmR1 (Figure 5). APRT is also involved in the synthesis of MA family phytosiderophores in graminaceous plants.44,55 Inoculation of rice roots with H. seropedicae SmR1 also increased slightly (∼1.4 fold) APRT mRNA level (Figure 2), while the proteome data showed a more significant increase (1.7 fold) in the abundance of the spot corresponding to an acidic form of this protein (Table 1), suggesting post-transcriptional regulation of APRT. The mRNA levels of S-adenosylmethionine decarboxylase (SAMDC), spermine synthase 1 (SPDS1), and spermidine synthase 2 (SPDS2) genes were not regulated in rice inoculated with H. seropedicae SmR1 (Figure 2), suggesting that polyamines biosynthesis is not affected. 3.6. Identified Proteins from Herbaspirillum seropedicae SmR1

The proteins identified from H. seropedicae SmR1 are in Table 2 and Figure 4. Spot 1558 corresponds to a bacterial alcohol 4763


4764

Hsero_2583

Tig peptidyl-prolyl cis/trans isomerase

EF-Tu elongation factor protein

Qor NADPH:quinone oxidoreductase Qor NADPH:quinone oxidoreductase ArgM acetylornithine aminotransferase malate dehydrogenase dinitrogenase reductase UspA universal stress HadL HAD superfamily haloalkanoic acid dehalogenase

AdhA alcohol dehydrogenase NADH:flavin oxidoreductase/NADH oxidase family protein

436

590

629 632 643 729 760 813 881

1558 2395

Hsero_0964 Hsero_3111

Hsero_3499 Hsero_3499 Hsero_3327 Hsero_2976 Hsero_2853 Hsero_2488 Hsero_1129

Hsero_0097

26 54 39 134 62 37 32 53 177 26 51 35 121 56 165 156 79 44 93 74 99 25 81 87

Mowse score 2 12 2 28 10 3 2 10 32 3 15 2 36 4 48 44 22 4 29 24 44 5 16 22

SC (%)

b

6/7 6/9

9/31 5/9 8/18

11/13 9/10 10/38

13/28

7/27

6/20 17/27

13/25 6/11

8/19

NMP/NSP

c

36.5/6.49 39.5/6.25

36/5.68 36/5.68 44/6.06 35/6.25 32/4.94 29/5.21 25/5.96

43/5.48

50/5.18

52/5.48 58/5.09

52/5.48 58/5.05

76/5.35 69/4.97

theor

35/6.8 36/6.59

42/5.91 42/5.76 42/6.36 36/6.73 34/5.09 30/5.37 27/6.35

44/5.62

57/5.20

59/5.62 59/5.08

59/5.68 60/5.02

83/5.46 75/4.99

exp

Mr(Kda)/pI MS /0.6 MS/100 MS2/0.6 MS/100 MS/100 MS2/0.6 MS2/0.6 MS/160 MS/100 MS2/0.6 MS/100 MS2/0.6 MS/100 MS2/0.6 MS/100 MS/100 MS/100 MS2/0.6 MS/100 MS/100 MS/120 MS2/0.6 MS/100 MS/100

2

MSd/MEe

1493.7300

1662.8270

1667.8950

1292.3930

1593.8510

1593.8760 1451.7490

1580.8340

1793.8890

m/z peak

FITFDCYGTLTR (25)

IVQGLEIDEFSQER (44)

LLDQGQAGDNVGVLLR (56)

AEAFPFVLGEGR (35)

AAVEEGVVPGGGVALLR (26)

AAVEEGVVPGGGVALLR (37) YVAAGFNPTDLKR (32)

ALGEFNLEGIPPAPR (39)

ALIAALPAADEFSYSVR (26)

fragment (ion score)f

Mowse score represents the score obtained in the matching with NCBInr database by MASCOT software. bSequence coverage. cNumber matched peaks/number searched peaks. dProteins were identified by MS or MS2 mode. eMaximum error (ME) allowed for peptide mass fingerprint (PMF) was 200 ppm and 0.6 Da for MS2. fPeptide fragment identified by MS2 mode using ion score algorithm. Values with higher scores than ion score cutoff are accepted (p < 0.05).

a


glutamine synthetase chaperonin GroEL (HSP60 family)

419 422


glutamine synthetase chaperonin GroEL (HSP60 family)

413 418


accession no.

polyribonucleotide nucleotidyltransferase DnaK molecular chaperone

protein name

290 307

spot ID

a

Table 2. Proteins of Herbaspirillum seropedicae SmR1 Present in Inoculated Rice Roots and Identified by Peptide Mass Fingerprint (PMF)

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Figure 4. Enlarged area showing the detected proteins of Herbaspirillum seropedicae SmR1. RR and SmR1 indicate rice roots noninoculated and inoculated with H. seropedicae, respectively.

Figure 5. H. seropedicae SmR1 induces the methionine recycling and mugineic acid (MA) biosynthesis and decreased ethylene production in the rice roots. The steps marked with a triangle represent the up-regulated proteins identified in the proteomic studies or up-expressed genes quantified by RT-qPCR analysis in rice roots inoculated with H. seropedicae SmR1, while those marked with an X are either down-regulated or non-regulated. Abbreviations: AdoMet, S-adenosylmethionine; ACC, 1-aminocyclopropane-1-carboxylic acid; ACS, 1-aminocyclopropane-1-carboxylic acid synthase; ACO, 1-aminocyclopropane-1-carboxylic acid oxidase; MTA, 5′-methylthioadenosine; MTR, 5′-methylthioribose; MTK, methylthioribose kinase; MTR-1-P, 5′-methylthioribose-1-phosphate; KMTB, 2-keto-4-methylthiobutyrate; ARD1, acireductone dioxygenase 1; SAMS, Sadenosylmethionine synthetase; APRT, adenine phosphoribosyltransferase; FDH, NAD-dependent formate dehydrogenase; NAS, nicotianamine synthase; NAAT, nicotianamine amino transferase; DMA, deoxymugineic acid; SAMDC, S-adenosylmethionine decarboxylase, SPS; spermine synthase; SPDS2, spermidine synthase 2. In the possible presence of ACC deaminase produced by H. seropedicae, plant ACC is sequestered by bacterial cells and cleaved into ammonia (NH3) and α-ketobutyrate.

most abundant proteins.58 However, under stress condition, such as those during the colonization process, the proteins

GroEL and DnaK could be overexpressed. In the Gluconacetobacter diazotrophicus and sugar cane plantlet proteomes, the 4765



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bacterial chaperones GroEL and DnaK were up-regulated.63 Another bacterial abundant protein, EF-Tu, besides participating in the translational mechanism, can induce innate immunity in plants during plant−bacterial interaction, as observed for Arabidopsis thaliana.64 Dos Santos and coworkers63 showed that isoforms of EF-Tu factor in G. diazotrophicus cocultivated with sugar cane plantlets were repressed, indicating that these genes could be turned off to facilitate bacterial access. The UspA universal stress protein (spot 813) was also identified in the 2-D gel. This protein was one of the most abundant in the H. seropedicae SmR1 reference map.58 It is an important component of the defense system against general stress conditions, such as oxidative stress.65 The Qor NADPH:quinone oxidoreductase was identified in two different spots (629 and 632) in the 2-D gel. In the H. seropedicae SmR1 proteome reference map, five isoforms of this protein were identified.58

accumulate larger amounts of iron and have higher nutritional value than noninoculated plants. Endophytic H. seropedicae SmR1 expressed proteins involved in nitrogen fixation and assimilation in rice roots as well as haloalkanoic acid dehalogenase and alcohol dehydrogenase, which are probably involved in metabolizing methyl halides and ethanol secreted by the roots. The results suggest an interplay between the two partners in the interaction to maximize mutual benefits. Here we show for the first time that rice roots inoculated with H. seropedicae SmR1 induce key genes responsible for phytosiderophore synthesis. Using an proteomics approach and subsequent RT-qPCR, we show that the expression of plant genes involved in methionine recycling are modulated by H. seropedicae SmR1, opening the possibility for the use of plant associative rhizobacteria to promote not only plant growth but also increase in Fe content via induction of MA biosynthesis.

■

4. CONCLUSIONS Our data show that inoculation of rice with H. seropedicae SmR1 lead to up-regulation of plant proteins involved in methionine recycling, whose function is to metabolize the MTA produced from conversion of the AdoMet to ACC and to recycle methionine. ACC is the substrate of ACO for the production of ethylene (Figure 5). Adomet is also a precursor of nicotianamine leading to the synthesis of MA family of phytosideropheres. RT-qPCR showed that genes coding for the MA biosynthesis pathway are induced in colonized roots. Overall, the results support the conclusion that colonization of rice roots by H. seropedicae SmR1 leads to induction of phytosideropheres biosynthesis genes, which are usually activated under Fe-limiting conditions (Figure 5). It is unlikely that the plants were under iron-limiting conditions because the Hoagland’s nutrient solution contained 22 μM of FeCl3, and no sign of iron deficiency, such as chlorosis, was observed. The purpose of phytosiderophore pathway up-regulation in the interaction H. seropeciae−rice is not known yet. It has been reported that secretion of siderophore by plant-growth-promoting rhizobacteria (PGPR) may improve the capacity of the host plant to absorb Fe in deficient soils by taking up Fe−siderophore complex.66 Our results show that stimulation of Fe acquisition by PGPG may be also due to direct modulation of the plant uptake system. Wu and coworkers67 observed an increase in ethylene production in rice under Fe-limiting conditions, in addition to up-regulation of ACS and ACO and MA biosynthesis genes, leading to the suggestion that expression of NAS1 and NAS2 is ethylene-dependent. Contrary to these observations, ACO expression is repressed in H. seropedicae SmR1-colonized roots (and a lower amount of ethylene is produced), while NAS1 and phytosiderophore biosynthesis is induced. Therefore, upregulation of MA biosynthesis enzymes in colonized roots is subject to a hitherto unknown signaling pathway triggered by the endophytic bacteria. It may be possible that increased utilization of Adomet for production of MAs also contributes to lower ethylene production. Iron deficiency is one of the most prevalent nutrient deficiencies in the world. Recently, transgenic iron-biofortified rice has been proposed as a solution for human micronutrient deficiency. Transgenic rice overexpressing nicotianamine synthase gene presents a three-fold increase in Fe content of seeds.68 Anemic mice fed with such transgenic seeds recovered from Fe deficiency, in contrast with when the regular grains were used as control.68 Because inoculation with H. seropedicae SmR1 caused a 100-fold increase in NAS, it is possible that inoculated plant may naturally

ASSOCIATED CONTENT

S Supporting Information *

Nucleotide sequences of primers used for reverse-transcription quantitative PCR (RT-qPCR), mean and standard deviation of relative volume of spots differentially expressed in rice roots colonized by H. seropedicae SmR1, and mean and standard deviation of relative volume of spots from proteins of H. seropedicae SmR1, and statistical justification for using 1.5fold change in spot volumes as a measure of significance. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +55 (41) 33611667. Fax: +55 (41) 32622042. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Roseli Prado, Valter Baura, and Marilza Doroti Lamour for technical assistance. We also thank the National Institute of Science and Technology of Nitrogen Fixation/ CNPq/MCT and Fundaçaõ Araucária for financial support. G.V. thanks PNPD/CAPES for postdoctoral scholarship.

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REFERENCES

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