Protoplasma DOI 10.1007/s00709-015-0826-1
ORIGINAL ARTICLE
Oxidative and antioxidative responses in the wheat-Azospirillum brasilense interaction Manuel Méndez-Gómez 1 & Elda Castro-Mercado 1 & Gladys Alexandre 2 & Ernesto García-Pineda 1
Received: 14 January 2015 / Accepted: 30 April 2015 # Springer-Verlag Wien 2015
Abstract Azospirillum is a plant growth-promoting rhizobacteria (PGPR) able to enhance the growth of wheat. The aim of this study was to test the effect of Azospirillum brasilense cell wall components on superoxide (O2·−) production in wheat roots and the effect of oxidative stress on A. brasilense viability. We found that inoculation with A. brasilense reduced O2·− levels by approx. 30 % in wheat roots. Inoculation of wheat with papain-treated A. brasilense, a Cys protease, notably increased O2·− production in all root tissues, as was observed by the nitro blue tetrazolium (NBT) reduction. However, a 24-h treatment with rhizobacteria lipopolysaccharides (50 and 100 μg/mL) alone did not affect the pattern of O2·− production. Analysis of the effect of plant cell wall components on A. brasilense oxidative enzyme activity showed no changes in catalase activity but a decrease in superoxide dismutase activity in response to polygalacturonic acid treatment. Furthermore, A. brasilense growth was only affected by high concentrations of H2O2 or paraquat, but not by sodium nitroprusside. Our results suggest that rhizobacterial cell wall components play an important role in controlling plant cell responses and developing tolerance of A. brasilense to oxidative stress produced by the plant.
Handling Editor: Adrienne R. Hardham * Ernesto García-Pineda
[email protected] 1
Instituto de Investigaciones Químico Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Ciudad Universitaria, Edif. A1’, Morelia, Michoacán CP 58040, Mexico
2
Department of Biology and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996-0840, USA
Keywords Azospirillum . Plant growth-promoting rhizobacteria (PGPR) . Oxidative stress . Superoxide . Superoxide dismutase (SOD) . Catalase (CAT)
Introduction Numerous species of bacteria associated with the plant rhizosphere have been shown to have beneficial effects on plant growth, crop yield, and quality. Such bacteria are collectively called Bplant growth-promoting rhizobacteria^ (PGPR). PGPR promote plant growth indirectly by reducing or eliminating the deleterious effects of pathogenic organisms via induction of host resistance (Van Loon and Glick 2004; Van Loon 2007) and, directly via increased nutrient uptake, atmospheric nitrogen fixation, solubilizing phosphorus and other minerals, producing siderophores that solubilize and sequester iron, and synthesizing phytohormones (e.g., auxins, cytokinins, gibberellins) and enzymes that enhance or modulate plant growth and development (Lucy et al. 2004; Gray and Smith 2005). In this way, bacteria of the genus Azospirillum represent one of the best characterized PGPR. The plant growth-promoting effect of Azospirillum brasilense is mainly attributed to the production of the phytohormone indole-3acetic acid (IAA) (Dobbelaere et al. 1999), known to be the most abundant naturally occurring auxin. Notably, IAA is involved in the coordination of plant growth and development (Abel and Theologis 2010). Because of the agronomical importance of PGPR, many studies in recent years have analyzed the cellular and molecular responses of plants and bacteria after establishing an interaction. These studies are important for understanding the relationship between these two organisms and may serve to increase crop production and quality. Although many biochemical and molecular aspects of the plant-bacteria
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interaction are known, new data confirm the complexity and number of molecular players participating in plant development. Several bacterial genetic determinants have been implicated in the adaptation of Azospirillum to the plant rhizosphere. For example, the adaptation of Azospirillum lipoferum 4B to the rice rhizosphere seems to involve genes related to reactive oxygen species (ROS) detoxification and multidrug efflux, as well as complex regulatory networks (Drogue et al. 2014). Wheat root exudates also induce changes in the A. brasilense exopolysaccharide composition under normal conditions consisting of high amounts of arabinose and xylose, but not when grown under saline stress. Root exudates also induced changes in the lipopolysaccharide (LPS) profile, both under normal and stress conditions (Fischer et al. 2003). The expression of genes implicated in exopolysaccharide production, LPS biosynthesis, and ABC sugar transporters is increased upon wheat root surface colonization by A. brasilense. Together, these data suggest that Azospirillum sp. can respond to chemical conditions in the rhizosphere and respond by modulating cell surface properties and metabolism (Camilios-Neto et al. 2014). The study of molecules present on the cell surface of the rhizobacteria, like proteins, allows knowing the contribution level of these molecules to modulate the interaction with plants. On the other end, different responses of plants to the presence of PGPR in the root system have been described. A previous proteomics study, reported a total of 31 differentially expressed proteins (22 upregulated and nine downregulated) in rice upon interaction with Bacillus cereus. Upregulated proteins included xyloglucan and endotransglycosylase, which are known to be involved in plant growth and development. Moreover, defense proteins were also upregulated, including peroxidases, glutathione S-transferases, and kinases (Wang et al. 2012). Previously, Arabidopsis thaliana was used as a host plant to gain insight into the molecular mechanisms that govern its interaction with wild-type Sp245 A. brasilense or an auxin biosynthesis mutant by microarray analysis. Extensive changes to the root transcriptome were observed upon A. brasilense inoculation. The wild-type bacterial strain induced changes in hormone-, defense-, and cell wall-related genes. In particular, 274 differentially regulated genes were part of the plant’s response to the bacterium independent of auxin production, indicating the extent of the response to this interaction (Spaepen et al. 2014). Among the upregulated genes, there was enrichment in defense-related genes implicated in systemic acquired resistance (Fu and Dong 2013), including the camalexin biosynthesis gene PAD3, at 3 dpi, suggesting the plant immune system initially recognizes PGPR as an invader (Spaepen et al. 2014). Similar responses have been reported for wheat inoculated with A. brasilense, where bacterial colonization caused changes in the expression of 776 wheat ESTs belonging to
various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones, and flavonoids biosynthesis (Camilios-Neto et al. 2014). The molecular basis of the association between PGPR and the roots of the plants they colonize is, however, not yet elucidated. There is growing evidence that ROS play important roles as signaling molecules required for cell expansion during the morphogenesis of organs such as roots and leaves (Carol and Dolan 2006). Evidence suggests that peroxidasecatalyzed production of hydroxyl radicals (·OH) from O2·− and H2O2 within the plant cell wall causes polysaccharide cleavage, which is directly responsible for cell wall loosening and cell expansion in the growing zone of roots (Schopfer et al. 2001; Liszkay et al. 2004; Dunand et al. 2007). Despite increasing evidence supporting the participation of ROS in root growth, its role in plant-rhizobacteria interactions remains to be characterized. Plant growth responses stimulated by A. brasilense appear clearly influenced by reciprocal interactions between ROS, produced by the plant, and auxins, produced by the rhizobacteria. In this sense, reports have shown that IAA reacts with dioxygen (O2) catalyzed by peroxidase (POX) resulting in production of O2·− (Kawano 2003). During IAA-induced ROS-dependent cell elongation, ·OH can easily be produced from H2O2 and in turn cleave polymer in cell walls. The peroxidase, which is involved in this process, is hypothesized to be located in the cell wall (Liszkay et al. 2004). In addition, it has been reported that auxin induces a series of ROS in the root elongation zone (Schopfer 2001). Plant responses to the presence of numerous microorganisms are stimulated by molecular components within the microbial cell wall or cellular membrane. This aspect of the plant-PGPR interaction has been focused mainly on the study of induced systemic resistance. The molecular structures of microorganisms recognized by plant immune system are called microorganism-associated molecular patterns (MAMPs). These molecules are essential polysaccharides and polynucleotides not found in host plants that are able to elicit different responses in plants, such as the production of ROS, production of nitric oxide (NO), alterations in the plant cell wall, induction of antimicrobial compounds, and the synthesis of pathogenesis-related (PR) proteins (Newman et al. 2013) and differ only slightly from microorganism to microorganism. The most important MAMPs are conserved cellsurface structures like flagellin, lipopeptides, peptidoglycans, and LPS, which are uniquely found in bacteria (Nurnberger et al. 2004). In this study, we focused on the cellular and molecular responses of both the plant and the PGPR to one other. Wheat plant ROS production in response to different cellular components of A. brasilense was analyzed. Similarly, A. brasilense enzymatic responses to cell wall components
Oxidative and antioxidative responses in the wheat-A. brasilense
of the root plant and/or their effect in inducing tolerance to oxidative stress in the bacterium were also studied.
Material and methods Unless otherwise indicated, chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). A. brasilense Sp245 was grown in Luria Broth (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 0.186 g/L MgSO4, 0.277 g/ L CaCl2, 15 g/L agar) and stored in nutrient broth with 15 % glycerol at −80 °C long term. Bacterial cultures were grown overnight at 27 °C, at 100 rpm until reaching the exponential phase. Cultures were washed twice in 0.9 % NaCl with centrifugation (4300×g, 10 min, 4 °C) in an Eppendorf centrifuge, re-suspended in sterile water, and adjusted to a final concentration of 106 colony-forming units (CFU)/mL for use as an inoculum. Triticum aestivum seeds (cv Nana F2007) were kindly provided by Dr. Mario González-Chavira (INIFAP, Celaya, México). Seeds were washed by shaking in 1 % sodium dodecyl sulfate (SDS) for 3 min, surface sterilized for 5 min in a 1 % sodium hypochlorite (NaClO) solution, washed four times with sterile distilled water, and germinated on filter paper sterilized and wetted with sterile distilled water on Petri dishes for 3 days in the dark at 28 °C. After this time, seedlings were aseptically transferred to assay tubes containing 5 mL of liquid Murashige and Skoog (MS) medium (pH 5.7). Inoculation with A. brasilense was done by adding 100 μL of a 106 cells/mL suspension to MS medium. Treatment of A. brasilense with papain A culture of A. brasilense (25 mL) in the exponential phase of growth (16 h of growth) was treated with papain (100 μM). The culture was incubated for 2 h at 37 °C then centrifuged at 3200 rpm in an Eppendorff centrifuge. The pellet was resuspended in LB medium and used to inoculate seedlings after 3 days of germination. Bacteria treated with autoclaved papain were used as a control. Culture filtrate (2 mL) from bacteria in the exponential phase of growth was added to seedlings after 3 days of germination in MS liquid medium. Superoxide production was analyzed 24 h after treatment. 4′,6-Diamidino-2-phenylindole and nitro blue tetrazolium staining 4′,6-Diamidino-2-phenylindole (DAPI) staining of root tips was performed as follows: root seedlings were fixed in a solution of 4 % paraformaldehyde for 24 h at 4 °C, washed three times in water, and mounted on cover slips. Roots were stained with 1 μg/mL DAPI for 30 min and analyzed with an Olympus BX60 fluorescence microscope (Ex/Em: 365/
420–540 nm; Zeiss, Jena, Germany) (Tsukagoshi 2012). To visualize the localization and/or rate of O2·− production, seedlings were stained for 15 min with a solution of 0.1 % NBT in 50 mM sodium phosphate buffer [pH 7.5] (Causin et al. 2012). NBT quantification Quantification of O 2 · − was assayed as described by Arthikala et al. (2014). Briefly, NBT-stained tissue was ground in liquid nitrogen, dissolved in 2 M KOH-DMSO, and then centrifuged for 10 min at 12,000×g. NBT was quantified by measuring the optical density (OD630) of samples compared to a standard. The standard curve was prepared by measuring the OD630 of known concentrations of NBT dissolved in freshly prepared 2 M KOHDMSO (1:1.16v/v). LPS isolation The wet cell pellet was used for isolation of LPS. Bacterial cells were suspended in 15 mL of 10 mM Tris-HCl buffer (pH 8), 2 mM MgCl2, 100 μg DNase I per 1 mL, and 25 pg RNase A per 1 mL of cell solution. Cells were sonicated (Bronwill Scientific Inc., Bronsonik) for two 30-s bursts at a probe intensity of 75. After a 2 h incubation at 37 °C, samples were extracted with chloroform (20 μL/g of cells), vortexed, and incubated at room temperature for 10 min, then centrifuged at 50,000×g for 30 min at 20 °C to remove peptidoglycans. Two volumes of 0.375 M MgCl2 in 95 % ethanol were added and cooled in a −20 °C freezer. After overnight incubation, samples were centrifuged at 12,000×g for 15 min at 0–4 °C. The pellet containing the LPS was then re-suspended in distilled water (Darveau and Hancock 1983). Antioxidant enzyme activity of A. brasilense Bacterial cultures were grown in LB medium with added pectin (1 mg/mL), polygalacturonic acid (1 mg/mL), wheat root exudates (2.4 %v/v), or paraquat (100 μM), at 27 °C and 100 rpm rotation for 18 h (exponential growth phase). The cultures were then washed twice in 0.9 % NaCl by centrifugation (4300×g, 10 min, 4 °C) in an Eppendorf centrifuge, resuspended in sterile water, and assayed for catalase (CAT) and superoxide dismutase (SOD) activity with native polyacrylamide gel electrophoresis (PAGE). To extract the enzymes, cultures (25 mL) were centrifuged (4300×g, 5 min, 4 °C) and then re-suspended in 2 mL of 50 mM KPO4 (pH 7.0). Samples were then sonicated for 15 s seven times, centrifuged for 15 min at 10,000×g, and the cleared supernatant used immediately to assay for SOD activity using the method described by Beauchamp and Fridovich (1971). In brief, supernatants (50 μg/lane) were
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separated by PAGE under non-denaturing (in the absence of SDS) conditions using 12.5 % (w/v) gels at 100 V and 4 °C. Following electrophoresis, the gel was immersed in 2.45 mM NBT for 20 min, followed by a 15-min soak in a solution containing 28 mM tetramethylethylenediamine, 28 μM riboflavin, and 36 mM KPO4 (pH 7.8). SOD activity was detected by illuminating the gel with a bright light, which caused the gel to turn uniformly blue except at positions exhibiting SOD activity. When maximum contrast was achieved, the reaction was terminated by rinsing the gel with water. To assay for CAT, 50 μg of total protein was loaded per lane. Gels were washed in distilled water and then soaked for 45 min in 50 mM phosphate buffer (pH 7.0) containing 50 μg/mL horseradish peroxidase. The gel was then incubated with 5 mM H 2 O 2 for another 10 min, rapidly rinsed in distilled water, and placed in 0.5 mg/mL of 3′3′-diaminobenzidine in 50 mM phosphate buffer (pH 7.0). CAT was characterized as achromatic zones against a uniform brown background (Bestwick et al. 2001). The protein content of extracts was determined using the Bradford (1976) assay with the Bio-Rad dye reagent (Bio-Rad, Hercules, CA, USA) and bovine serum albumin as the standard. Effect of oxidative stress on A. brasilense Cultures of bacteria were grown for 16–18 h at 30 °C in LB. Cells in the exponential phase were harvested by centrifugation at 4000×g for 5 min, washed twice in 0.9 % NaCl, resuspended in sterile water, adjusted to a final concentration of OD600 = 0.5, and used as inoculum. Aliquots of bacteria (100 μL) were transferred to 50 mL of LB media in Erlenmeyer flasks containing different concentrations of H2O2, paraquat, or sodium nitroprusside (SNP). After mixing, cultures were incubated at 27 °C with constant agitation (100 rpm) for 20 h, and growth was assayed spectrophotometrically at OD600. Each experiment was repeated at least three times and results are presented as mean±standard deviation. Statistics Statistical differences between mean values were determined with the Student’s t test. Differences at the level of P
