Comparative Proteomic and Physiological Analyses Reveal the

Article pubs.acs.org/jpr

Comparative Proteomic and Physiological Analyses Reveal the Protective Effect of Exogenous Polyamines in the Bermudagrass (Cynodon dactylon) Response to Salt and Drought Stresses Haitao Shi,† Tiantian Ye,†,‡ and Zhulong Chan†,* †

Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, Hubei, China ‡ University of Chinese Academy of Sciences, Beijing 100039, China S Supporting Information *

ABSTRACT: Polyamines conferred enhanced abiotic stress tolerance in multiple plant species. However, the effect of polyamines on abiotic stress and physiological change in bermudagrass, the most widely used warm-season turfgrasses, are unknown. In this study, pretreatment of exogenous polyamine conferred increased salt and drought tolerances in bermudagrass. Comparative proteomic analysis was performed to further investigate polyamines mediated responses, and 36 commonly regulated proteins by at least two types of polyamines in bermudagrass were successfully identified, including 12 proteins with increased level, 20 proteins with decreased level and other 4 specifically expressed proteins. Among them, proteins involved in electron transport and energy pathways were largely enriched, and nucleoside diphosphate kinase (NDPK) and three antioxidant enzymes were extensively regulated by polyamines. Dissection of reactive oxygen species (ROS) levels indicated that polyamine-derived H2O2 production might play dual roles under abiotic stress conditions. Moreover, accumulation of osmolytes was also observed after application of exogenous polyamines, which is consistent with proteomics results that several proteins involved in carbon fixation pathway were mediated commonly by polyamines pretreatment. Taken together, we proposed that polyamines could activate multiple pathways that enhance bermudagrass adaption to salt and drought stresses. These findings might be applicable for genetically engineering of grasses and crops to improve stress tolerance. KEYWORDS: bermudagrass (Cynodon dactylon), polyamine, putrescine, spermidine, spermine, proteomic, salt, drought

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INTRODUCTION Salinity and drought are two major environmental stresses that seriously affect crop growth and agriculture production worldwide.1−6 Under salt and drought stress conditions, plants have developed many molecular and biochemical adaptive mechanisms to advent stress triggered damage, such as the induction of some important messengers, including abscisic acid, hydrogen peroxide (H2O2), nitric oxide, and polyamines, and the activation of these messengers involving stress sensing and signal transduction.1−4,7 Bermudagrass (Cynodon dactylon) is a warm-season turfgrass and is widely used on home lawns, golf courses, and sport fields.1 Bermudagrass is adapted to cultivation in a range of climate conditions, but some adverse abiotic stress, such as salinity and drought, drastically affect the growth of bermudagrass. Polyamines [mainly putrescine (Put), spermidine (Spd), and spermine (Spm)] have been widely dissected for their protective role in almost all abiotic stresses, including salt, drought, low and high temperatures, ozone, flooding, darkinduced senescence, heavy metals (Cu, Fe, and Ni), acid stress and osmotic and oxidative stresses.7−11 Although many recent researchers investigated the in vivo role of polyamines in abiotic © XXXX American Chemical Society

stress using transgenic overexpression plants or loss-of-function mutants, the classical and popular approach is an exogenous polyamine application experiment.12−33 Application of exogenous polyamines, at least one type among Put, Spd, and Spm, conferred enhanced tolerance to various abiotic stress in Arabidopsis,24 rice,21−23 tobacco,17,20 cucumber,15,16,19,27,30 Atropa belladonna,12 barley,31 Brassica napus,25,28 welsh onion,33 oat,13 Nymphoides peltatum,29 Raphanus sativus,14 and Citrus reticulate.26 All these observations indicated that polyamines might be ideal targets of agricultural biotechnology for crop improvement under variable climate conditions.5 In recent years, the proteomic technique has become an effective approach to identify a wide range of differential expressed proteins and to investigate the molecular mechanism underlying crop responses to environmental stresses.34,35 Zhao et al.6 have identified 54 proteins associated with water-deficit tolerance in bermudagrass leaves using two-dimensional Special Issue: Agricultural and Environmental Proteomics Received: May 22, 2013

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dx.doi.org/10.1021/pr400479k | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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Figure 1. Flow chart of the experiment design in this study. The bermudagrass seeds of Yukon were sown in the plastic container filled with soil in the growth chamber for 21 days. 21-day-old bermudagrass seedlings were irrigated with water, water with 5 mM Put, 5 mM Spd, and 5 mM Spm for 7 days, respectively. After this treatment, the 28-day-old bermudagrass plants with different treatments were used for the next assays of proteomic analysis, physiological parameters, and abiotic stress tolerance at designed times.

27 ± 2 °C, with 65−70% relative humidity, and 16 h light and 8 h dark cycles. Nutrient solutions were irrigated twice every week.

electrophoresis (2-DE) and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOFMS). Functional analysis of differentially displayed proteins between two bermudagrass genotypes differing in drought tolerance indicated that photosynthesis and antioxidant defense mechanisms might play essential roles in bermudagrass adaptation to water-deficit stress.6 Through the proteomic technique, Komatsu’s group has clarified the organ-specific proteomic changes in the roots, root tips, leaves, hypocotyls, and seedlings of flooded soybean plants.34−41 Additionally, they found that soybeans response to flooding stress might be regulated by both modulation of protein expression and phosphorylation state in a specific organ, especially the energy-demanding and production-related metabolic pathways.34−41 All these observations indicated that comparative proteomic analysis was an effective approach to dissect the complex mechanism underlying crop physiology. In this study, to get new insight into the mechanism of polyamines mediated abiotic stress responses, exogenous polyamines (including Put, Spd, and Spm) were applied on bermudagrass plants to dissect the in vivo role of polyamines in plant physiological response to salt and drought stresses. Additionally, proteomic analysis via 2-DE and MALDI-TOFMS was performed to identify differentially displayed proteins affected by Put, Spd, and Spm. The effect of three types of polyamines (Put, Spd, and Spm) on bermudagrass protein level changes was also compared. The results of physiological assays and comparative proteomic analyses might provide some insights to understand the physiological and molecular mechanisms of polyamines in plant response to abiotic stress in bermudagrass.

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Experimental Design of Polyamines Pretreatment, and Salt and Drought Stress Treatments

To test the effect of exogenous polyamines on plant physiological response and abiotic stress tolerance, 21-day-old bermudagrass seedlings were irrigated with water, or 5 mM Put, 5 mM Spd, 5 mM Spm solutions for 7 days, respectively. After polyamine pretreatment, all control and polyamine pretreated 28-day-old plants were subjected to the following salt and drought treatments. For salt stress treatment, 28-day-old bermudagrass plants in soil were irrigated with NaCl solutions for 24 days. And the NaCl concentration was increased stepwise by 50 mM every 2 days to 300 mM. For drought stress treatment, 28-day-old plants in soil with different treatments were subjected to drought conditions by withholding water for 21 days and then rewatered for 3 days. The survival rate of the above stressed plants was recorded at 24 days after stress treatments. The leaf samples from polyamine pretreated 28day-old plants were collected for identification of differentially displayed proteins using the proteomics approach. At indicated time points after salt or drought treatment (referred to as control-, salt-, or drought-0 d, 7 d, 14 d, and 21 d), bermudagrass materials were collected for physiological parameters assay (Figure 1). Assay of Electrolyte Leakage

Electrolyte leakage (EL) was determined according to Shi et al.1 Briefly, about 0.2 g of plant leaves from at least 15 independent lines were incubated in 20 mL of deionized water on a gyratory shaker at room temperature, and then the initial conductivity (ELi) was measured using the conductivity meter (Leici-DDS-307A, Shanghai, China) after 8 h of shaking. The full conductivity of the killed mixtures (ELmax) was determined after boiling at 121 °C for 20 min. Then the relative electrolyte leakage EL (%) was calculated as (ELi /ELmax) × 100.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The bermudagrass seeds of Yukon [kindly provided by American Seed Research of Oregon Company (http://www. sroseed.com/)] were stratificated in deionized water at 4 °C for 3 days in darkness, and then sown in the plastic container filled with soil in the growth chamber. The growth chamber was controlled at an irradiance of about 150 μmol quanta m−2 s−1,

Trypan Blue Staining

Trypan blue staining was carried out on bermudagrass leaves in lactophenol trypan blue solution [0.025% (w/v) trypan blue, B



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data. The minimum score of 43 and the minimum sequence coverage of 6% in MOWSE analysis were used to keep the confidence of the identification results, and the best matched protein with high confidence score was selected for further analysis.

25% (v/v) lactic acid, 25% (v/v) glycerine, 25% (v/v) phenol, and 25% (v/v) deionized water] for 2 min. Then the stained plants were transferred to 70% (v/v) ethanol to remove the chlorophyll and photographed. Determination of Water Loss Rate and Leaf Water Content

Cluster Analysis and Metabolic Pathway Analyses

For the water loss rate assay, the detached leaves from at least 30 independent lines with different treatments in the growth chamber were quantified every 1 h intervals for up to 9 h. Then the leaf water loss rate was expressed as percent change in the rate of leaf fresh weight (FW) at designated time intervals. For the leaf water content (LWC), the leaf samples from at least 30 independent lines with different treatments were harvested at different time points (7, 14, and 21 days). The FW was quantified immediately after harvest, and the dry weight (DW) was quantified after 16 h of 80 °C treatment, and then the LWC (%) was measured as (FW-DW)/FW × 100.

The fold change of differentially expressed proteins was imported for cluster analysis, and hierarchical cluster analysis was performed using an uncentered matrix and complete linkage method with the CLUSTER program (http://bonsai. ims.u-tokyo.ac.jp/∼mdehoon/software/cluster/).49 Resulting tree figures were exhibited using the software package and Java Treeview (http://jtreeview.sourceforge.net/) as Shi et al.1 described. The differentially displayed proteins were loaded and annotated in the Classification SuperViewer Tool (http://bar. utoronto.ca/ntools/cgibin/ntools_classification_superviewer. cgi),50 and functional categories of every protein were assigned using MapMan (http://mapman.mpimp-golm.mpg.de/ general/ora/ora.html).51 The pathway graph of carbon fixation in photosynthetic organisms and in glycolysis/gluconeogenesis was obtained using KEGG (http://www.genome.jp/kegg-bin/ search_pathway).

Protein Extraction and 2-DE

Total protein extraction was determined using the phenol method according to a previously described protocol,43−45 and the protein supernatant was quantified through the Bradford method.46 Briefly, 1 g of plant leaves was ground in liquid nitrogen, homogenized extensively in 4 mL of precooled homogenization buffer [20 mM Tris-HCl (pH 7.5), 1.05 M sucrose, 10 mM EGTA, 1 mM DTT, 1 mM PMSF, and 1% (v/ v) Triton X-100], and then centrifuged at 10000g for 30 min at 4 °C. One volume of Tris-HCl buffered phenol (pH 7.8) was mixed with the supernatant thoroughly, and the mixture was centrifuged at 10000g for 30 min at 4 °C. The top phenol phase was mixed with five volumes of ice-cold saturated ammonium acetate in methanol overnight at −20 °C. Then the pellet was centrifugated, dried, and dissolved in the lysis buffer [7 M urea, 2 M mithiourea, 4% (w/v) of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), 65 mM DTT, and 0.2% (w/v) of carrier ampholyte (pH3.5−10)]. The 2-DE was performed as previously described47 with minor modification. Briefly, 1 mg of total proteins was applied onto an immobilized pH gradient (IPG) strip (17 cm, pH 4−7, Bio-Rad, USA) and rehydrated extensively overnight at room temperature; then the strips were transferred to isoelectric focus (IEF) in the Protein IEF system (Bio-Rad, USA). After IEF, the strips were initially incubated in the first equilibration buffer [6 M urea, 0.375 M Tris-HCl pH 8.8, 2% (w/v) of SDS, 20% (v/v) of glycerol, and 2% (w/v) of DTT] and the second equilibration buffer [6 M urea, 0.375 M Tris-HCl pH 8.8, 2% (w/v) of SDS, 20% (v/v) of glycerol, and 2.5% (w/v) of iodoacetamide] for 15 min, respectively. Then the equilibrated strips were used for sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) as previously described.43,47,48

Determination of Malondialdehyde (MDA) Content

The MDA content in plant samples was extracted using thiobarbituric acid (TBA) regent and boiled at 100 °C for 20 min. After centrifugation, the MDA of supernatant was quantified by subtracting the nonspecific absorbance at 600 and 450 nm from the absorption at 532 nm. Determination of ROS Accumulation and Antioxidant Enzyme Activities

For protein extraction, about 0.2 g of plant leaves from at least 15 independent lines was homogenized in 1 mL of 50 mM sodium phosphate buffer (pH 7.8) and centrifuged at 15294g for 15 min at 4 °C. Then the supernatant was used for the determination of H2O2, O2•− content, and antioxidant enzyme activities, and the protein concentration of plant protein extraction was quantified using the Bradford method.46 For H2O2 content assay, the above plant extraction and 0.1% (w/v) titanium sulfate regent [in 20% (v/v) H2SO4] were mixed at 1/1 (v/v) to precipitate the peroxide−titanium complex. The absorbance of solution was quantified at 410 nm. The H2O2 content was expressed as micromolar [(mg protein)−1]. For the O2•− content assay, a plant O2•− ELISA Kit was used. The kit used purified O2•− to coat microtiter plate wells and make solid-phase antibody; then the added O2•− combined with horseradish peroxidase (HRP) labeled O2•− antibody and became antibody−antigen−enzyme−antibody complex. After washing completely, a substrate solution of 3,3′,5,5′-tetramethylbenzidine (TMB) was added and became blue because of the catalysis of HRP enzyme, and the reaction was terminated by the addition of a sulphuric acid solution. The absorbance of the yellow solution was quantified at 405 nm. The O2•− content was expressed as nanograms [(mg protein)−1]. The superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), and peroxidase (POD, EC 1.11.1.7) activities were determined using a Total SOD Assay Kit (S0102, Beyotime, China), a CAT Assay Kit (S0051, Beyotime), and a Plant POD Assay Kit (A084-3, Nanjing Jiancheng Bioengineering Institute, China), respectively,

Gel Image Analysis and Protein Spot Identification by MALDI-TOF-MS

2-D gels were stained in Coomassie brilliant blue (CBB) R250 staining buffer for 4 h and distained overnight. After scanned using an EPSON PERFECTION V700 PHOTO scanner (Epson), the protein spot images of 2-D gel were analyzed with PDQuest 2-DE Analysis Software (BIO-RAD, USA). The differentially displayed protein spots (fold change ≥2) were excised from the 2-DE gel and used for trypsin digestion and MALDI-TOF-MS analysis with AXIMA-CFR plus (Shimadzu Biotech, Kyoto, Japan) as reported by Li et al.47 MASCOT software (Mascot Wizard 1.2.0, Matrix Science Ltd., http://www.matrixscience.com) was used to analysis the MS C



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Figure 2. Exogenous polyamines improve salt and drought stress tolerances in bermudagrass. (A and B) Photographs showing plant growth (A) and trypan blue staining (B) of 4-week-old plants with different treatments (control, 5 mM Put, 5 mM Spd, and 5 mM Spm, respectively) under control conditions, 300 mM NaCl and rewatered conditions at designated days. Bars = 100 μm in the pictures of trypan blue staining. (C) EL of 4-week-old plants with different treatments (control, 5 mM Put, 5 mM Spd, and 5 mM Spm, respectively) under control conditions, 300 mM NaCl, and drought conditions at designated time intervals. (D) Survival rate of bermudagarss plants after 24 days of control, 300 mM NaCl, and drought treatments. (E−H) Plant shoot length (E), shoot FW (F), root length (G), and root FW (H) under control conditions, 300 mM NaCl, and drought conditions at designated time intervals. The results shown are the means ± SE (n = 4 for C and D, and n = 12 for E−H). Asterisk symbols indicate significant differences in comparison to wild type without polyamine treatment (p < 0.05).

according to Shi et al.1 All these relative enzyme activities were expressed as fold change.

absorbance of 480 and 620 nm, respectively. Both of sucrose and soluble total sugar contents were expressed as mg [(g DW)−1].

Measurements of Proline Content, Sucrose and Soluble Total Sugars

Statistical Analysis

Proline content was determined according to Shi et al.,1−3 and the proline level of sample was expressed as μg [(g FW)−1]. The soluble sugars of plant samples were extracted using the anthrone method as previous described.1 The concentration of sucrose and soluble total sugars were examined at the

All experiments in this study were repeated at least three times in independent experiments. In these results, the Student’s ttest was used to analyze the significant difference, and asterisk symbols indicate the significant difference of p < 0.05. D



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Figure 3. Comparison of proteome patterns of bermudagrass leaves in response to polyamines (Put, Spd, and Spm) treatments. Proteins were separated in the first dimension on the IPG strip (pH 4−7), and in the second dimension on 12.5% SDS-PAGE. The coregulated proteins spots by at least two types of polyamines were marked with arrows as shown.

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RESULTS

plants showed significantly lower water loss rate than that of nontreated plants from 3 to 9 h after leaf detachment (SI Figure S1A). Under well-watered (control) conditions, all the polyamines pretreated and nontreated plants maintained the LWC at about 80% (SI Figure S2B). Under drought stress (water-deficit) conditions, the LWC showed a gradual decrease in all plants; however, the polyamines (Put, Spd, and Spm) pretreated plants showed relatively slower water loss, especially at 14 and 21 days after drought treatment (SI Figure S2B).

Exogenous Polyamines Improved Salt and Drought Stress Tolerances in Bermudagrass

After 7 days pretreatment with polyamines, no significant differences were observed between nontreated plants and pretreated plants (28-day-old). Growth of Put, Spd, and Spm pretreated bermudagrass plants was generally equivalent to that of nontreated plants under well-watered conditions for the following 21 days. After salt and water-deficit treatments, growth of both polyamines pretreated and nontreated plants was inhibited, but polyamines (Put, Spd, and Spm) pretreated plants had greener leaf tissues than that of nontreated bermudagrass plants (Figure 2A). Consistently, polyamines (Put, Spd, and Spm) pretreated plants showed significantly lower trypan blue staining, lower EL, and higher survival rate than those of nontreated bermudagrass plants (Figure 2D). Additionally, polyamines pretreated plants exhibited healthy growth in relative to nontreated plants, with longer shoot and root length and higher shoot and root FW (Figure 2E−H). These results indicated that exogenous polyamines application could improve salt and drought stress tolerances in bermudagrass. Moreover, leaf water loss in vitro and LWC in vivo of polyamines pretreated plants were assayed in this study. As shown in Supporting Information (SI) Figure S1, leaf water loss increased gradually in a time dependent manner after leaf detachment, but polyamines (Put, Spd, and Spm) pretreated

Proteins Induced by Put, Spd, and Spm Pretreatment in Bermudagrass

Based on 2-DE and MALDI-TOF-MS, comparative analysis of samples from the nontreated and Put, Spd, Spm treated bermudagrass leaves showed that some proteins were differentially displayed (Figure 3 and SI Table S1). MALDI-TOFMS was successfully performed to identify 36 proteins induced by Put, Spd, and Spm in bermudagrass, including 32 coregulated proteins and 4 specifically regulated proteins by different polyamines (Figures 35 and SI Figure S2A-D and Table S2). Among 36 proteins, expression levels of 12 proteins were increased by at least two types of polyamines (Put, Spd, and Spm), while those of 20 proteins were decreased, and the other 4 proteins exhibited different expression patterns by three polyamines (Figure 3 and Table 1). Cluster analysis and Venn diagram analysis showed that the expression levels of 9 proteins (U1, U2, U3, U5, U8, U9, U10, U11, and U12) were increased in common by three types of polyamines, and 9 proteins (D1, E



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14 days. After salt and drought treatments for 7 and 14 days, MDA, H2O2, and O2•− contents in polyamines (Put, Spd, and Spm) pretreated plants were significantly lower than those in untreated bermudagrass plants (Figure 6A−C). Consistent with the 2-DE results in Figure 4, the activities of SOD and POD were significantly decreased after 7 days of polyamine pretreatments (Figure 6D−F). Under well-watered (control) conditions for 7 and 14 days, the activities of three major antioxidant enzymes (SOD, CAT, and POD) in polyamines pretreated and nontreated bermudagrass plants showed no significant differences (Figure 6D−F). After salt and drought stress treatments, polyamines (Put, Spd, and Spm) pretreated plants exhibited significantly higher activities of three antioxidant enzymes than those of nontreated plants mainly at 14 days after treatments (Figure 6D−F). These observations indicated pretreatment with exogenous polyamines might activate antioxidant enzyme activities, which further resulted in relatively lower ROS accumulation in bermudagrass. Effect of Exogenous Polyamines on Accumulation of Osmolytes during Salt and Drought Stresses

After polyamine pretreatment for 7 days (control-0 d), contents of sucrose and soluble total sugars were significantly higher than those of nontreated plants (Figure 7B, C). Under wellwatered (control) conditions for 7 and 14 days, no significant differences were observed for the accumulation of osmolytes including proline, sucrose, and soluble total sugars between polyamines pretreated and nontreated plants (Figure 7). After salt and drought stress treatments for 7 and 14 days, endogenous proline, sucrose, and soluble total sugar contents were obviously increased in both polyamines pretreated and nontreated bermudagrass plants (Figure 7). However, contents of proline, sucrose, and soluble total sugar in Put, Spd, or Spm pretreated plants were significantly higher than those of nontreated plants (Figure 7). Together, exogenous polyamine application might positively modulate the accumulations of proline and sugars under salt and drought stress conditions, which in turn increased plant stress tolerance.

Figure 4. Cluster analysis and comparative distribution of coregulated proteins by polyamines treatments. (A) Hierarchical cluster analysis of fold change of coregulated proteins by polyamines treatments. Resulting tree figure was displayed using the software package and Java Treeview. (B) Venn diagram showing the number and spot number of proteins that overlapped among three types of polyamines treatments.

D2, D4, D6, D7, D8, D10, D15, and D17) were decreased by three types of polyamines, indicating these common changed proteins might play important roles in polyamine-mediated stress responses (Figure 4). Metabolic pathway analysis showed that electron transport and energy pathways were largely enriched by polyamine treatment, including nine proteins (U1, U5, U6, U8, U9, U10, D12, F3, and F4) involved in light reaction of photosystem and nine proteins (U3, U4, D4, D5, D6, D14, D15, D17, and D18) involved in the Calvin cycle of the photosystem (Table 2 and SI Table 1). Additionally, three antioxidant enzymes (D1, 2-Cys POD; D13, ascorbate peroxidase (APX); F2, Cu/Zn SOD) and six stress-related proteins (U12, D6, D7, D10, D11, D13, and F1) were also significantly coregulated by three polyamines, indicating ROS metabolism and the underlying antioxidant enzymes might be affected by polyamines (Figure 5). Moreover, expression levels of four stress responsive proteins (U12, D6, D7, D10, and D11) were also commonly decreased by polyamines, while F1 was differentially regulated by three types of polyamines (Figure 5).

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DISCUSSION In this study, the protective role of polyamines in bermudagrass response to salt and drought stresses was assigned through exogenous applications of three types of common polyamines (Put, Spd, and Spm), as confirmed by the assay of EL, survival rate, cell damage, shoot and root growth, and leaf water status (Figure 2). The enhanced abiotic stress tolerance in bermudagrass was consistent with the protective effect of polyamines using the exogenous application approach or genetic the engineering technique by manipulation of polyamine biosynthetic and metabolic pathways in other plant species, including in Arabidopsis,24 rice,21−23 tobacco,17,20 and cucumber.15,16,19,27,30 All these studies provided promising applications that polyamines could function as ideal targets for future crop improvement in variable climates.5,52 However, the underlying mechanism of polyamines-mediated plant stress responses need to be further dissected, especially in bermudagrass. In this study, comparative proteomic and physiological analyses were performed to reveal the protective mechanism of exogenous polyamines in response to salt and drought stresses. Through 2-DE and MALDI-TOF-MS, we successfully identified 12 commonly increased proteins by at least two types of polyamines, 20 commonly decreased proteins and 4 differ-

Effect of Exogenous Polyamines on ROS Accumulation and Antioxidant Enzyme Activities in Bermudagrass

Since three antioxidant enzymes were coregulated by polyamines treatment based on proteomic analysis, the ROS accumulation and underlying antioxidant enzyme activities were further assayed. As the major indicator for stress triggered oxidative damage and ROS level, MDA, H2O2, and O2•− contents were determined during salt and drought stress treatments. As shown in Figure 6A−C, polyamine pretreatments significantly increased H2O2 and O2•− contents, but not MDA, in burmudagrass prior to any further treatments, but the differences disappeared under well-watered conditions for 7 and F



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Figure 5. Analysis of nine coregulated proteins by polyamines treatments involved in stress and redox regulation. (A) Magnified views of nine coregulated proteins by polyamines treatments involved in stress and redox regulation. (B) Relative protein levels of nine coregulated proteins by polyamines treatments involved in stress and redox regulation. The results shown are the means ± SE of three independent experiments. Asterisk symbols indicate significant differences in comparison to wild type without polyamine treatment (p < 0.05).

(germin-like protein), D19 (ubiquitin-conjugating enzyme 2), and F1 (heat shock protein 70) indicated the involvement of exogenous polyamines in cell organization, protein degradation, and stress response. Based on proteomic analysis, three antioxidant enzymes [D1, 2-Cys POD; D13, APX; F2, Cu/Zn SOD] were regulated commonly by polyamines, indicating ROS metabolism, and underlying antioxidant enzymes might be affected after polyamines pretreatments (Figure 5). As reported previously,36,53 the increased level of 2-Cys POD might be the result of a delay in protein degradation. To dissect the involvement of these antioxidant enzymes in polyaminemediated abiotic stress responses, the ROS accumulation and underlying antioxidant enzyme activities were further assayed. It is known that MDA, H2O2, and O2•− contents are major indicators for stress triggered oxidative damage and ROS accumulation.1−4,54 The antioxidant system including multiple antioxidant enzymes can inhibit the accumulation of ROS, thus alleviating ROS-induced oxidative damage in plants under stress conditions.1−4,36 In this study, polyamine pretreatments significantly increased H2O2 and O2•− contents and lower activities of antioxidant enzymes than those of untreated bermudagrass plants (Figure 6), which was consistent with the 2-D results in Figure 5. After salt and drought stress treatments, polyamines (Put, Spd, and Spm) pretreated plants exhibited

entially regulated proteins by polyamines (Figure 3, Table 1, and SI Table 1). Among these proteins, expression levels of 9 proteins (U1, U2, U3, U5, U8, U9, U10, U11, and U12) were increased and those of 9 proteins (D1, D2, D4, D6, D7, D8, D10, D15, and D17) were decreased by all three types of polyamines, respectively (Figure 4). Further metabolic pathway analysis showed that electron transport and energy pathways were largely enriched by the polyamine effect, including 9 proteins (U1, U5, U6, U8, U9, U10, D12, F3, and F4) involved in light reaction of photosystem and 9 proteins (U3, U4, D4, D5, D6, D14, D15, D17, and D18) involved in the Calvin cycle of the photosystem (Table 2 and SI Table 1). Zhao et al.6 have found that differentially displayed proteins in photosynthesis and antioxidant defense pathways might play essential roles in bermudagrass adaptation to water-deficit stress. Consistently, polyamines-mediated proteins in this study mainly functioned in photosynthesis electron transport and energy pathways (18 proteins) and the antioxidant enzyme defense pathway (3 proteins). As an important enzyme for synthesis of Sadenosylmethionine involved in polyamine metabolism,7,10,11 U7 (S-adenosylmethionine synthetase) activity was increased by Spd and Spm, indicating the effect of exogenous Spd and Spm on changes of polyamine metabolism and other related metabolites. The regulation of D3 (β-actin), D7 (heat shock cognate protein 70), D10 (cold shock domain protein), D11 G



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Table 1. List of Commonly Changed Bermudagrass by at Least Two Types of Polyamines (Put, Spd, and Spm) Treatmentsa no. U1 U2 U3 U4 U5 U6 U7 U8 U9 U10 U11 U12 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14 D15 D16 D17 D18 D19 D20 F1 F2 F3 F4

theor

exp

Sc

cov

Mr/pi

Mr/pi

Put

Spd

Spm

84

20%

35.1/6.26

36.6/5.10

2.07

3.16

2.82

69

29%

23.4/4.96

20.2/5.26

5.05

3.62

2.42

71

7%

33.7/7.64

30.0/5.46

2.92

3.42

2.26

55

27%

5.1/5.06

10.6/5.75

2.43

2.97

0.88

80

19%

20.7/6.41

12.0/6.22

13.99

24.97

40.51

52

94%

3.8/9.99

39.7/6.45

2.78

1.48

2.49

68

19%

43.6/5.57

53.7/6.23

1.43

4.31

7.56

172

34%

49.9/6.23

61.1/6.28

2.32

2.95

3.09

243

82%

9.4/6.51

9.6/6.71

7.68

6.71

6.42

cytochrome b6-f complex iron−sulfur subunit, chloroplastic [Oryza sativa subsp. Japonica] fructose-bisphosphate aldolase, cytoplasmic isozyme [Zea mays] nucleoside diphosphate kinase 1 [Mesembryanthemum crystallinum] 2-Cys peroxiredoxin BAS1, chloroplastic [Oryza sativa subsp. Japonica] adenylate kinase, putative [Ricinus communis]

114

34%

24.2/8.54

15.7/6.80

2.34

3.41

2.69

120

28%

39.0/7.52

45.7/6.82

2.88

2.31

2.46

83

10%

16.3/6.3

12.6/6.60

2.45

6.15

5.56

121

27%

28.3/5.67

20.8/4.73

0.48

0.44

0.43

43

6%

29.4/7.71

29.4/5.35

0.50

0.46

0.46

b-actin [Zoysia japonica] ribulose bisphosphate carboxylase/oxygenase activase, chloroplastic [Oryza sativa subsp. Japonica] ribulose bisphosphate carboxylase/oxygenase activase, chloroplastic [Malus domestica] rubisco large subunit-binding protein subunit β, chloroplastic [Secale cereale] heat shock cognate 70 kDa protein [Petunia hybrida] pyruvate orthophosphate dikinase, partial [Eleusine indica] pyruvate, phosphate dikinase, chloroplastic [Flaveria pringlei] predicted protein, cold shock domain protein [Micromonas sp. RCC299] germin-like protein 8−14 [Oryza sativa subsp. Japonica] ATP synthase CF1 epsilon subunit, partial [Eleusine coracana] ascorbate peroxidase [Eleusine coracana] fructose-bisphosphate aldolase, chloroplastic [Oryza sativa subsp. Japonica] ribulose bisphosphate carboxylase/oxygenase activase, chloroplastic [Phaseolus aureus] hypothetical protein, glyoxalase I homologue [Sporobolus stapfianus] ribulose bisphosphatecarboxylase/oxygenase activase, chloroplastic [Solanum pennellii] glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic [Chlamydomonas reinhardtii] ubiquitin-conjugating enzyme E2 2 [Triticum aestivum] triosephosphate isomerase,cytosolic [Lactuca sativa] stromal 70 kDa heat shock-related protein, chloroplastic [Pisum sativum] superoxide dismutase [Cu−Zn], chloroplastic [Oryza sativa subsp. Japonica] ATP synthase CF1 alphasubunit [Eleusine coracana]

96 105

32% 14%

41.9/5.23 51.8/5.43

51.6/5.41 54.8/5.35

0.49 0.40

0.78 0.47

0.52 0.37

133

17%

48.2/8.2

50.8/5.33

0.33

0.41

0.89

138

15%

53.7/4.88

71.0/5.28

0.41

0.47

0.24

156 182

29% 39%

71.6/5.11 75.3/4.90

82.5/5.27 94.3/5.31

0.42 0.43

0.49 0.47

0.40 0.46

78

14%

105.3/5.94

94.7/5.39

0.46

0.73

0.31

80

10%

32.7/9.26

14.0/5.60

0.42

0.02

0.04

60 60

10% 50%

22.0/6.01 15.2/5.03

18.8/5.56 17.4/5.59

0.43 0.77

0.83 0.28

0.17 0.25

107 75

28% 12%

27.6/5.79 42.2/6.38

30.2/5.78 39.8/5.76

0.76 0.80

0.39 0.34

0.19 0.28

134

11%

48.0/7.57

42.7/5.80

0.48

0.49

0.32

52

14%

32.2/5.73

35.4/5.60

1.18

0.36

0.43

82

20%

50.9/8.61

45.9/5.82

0.45

0.45

0.46

78

18%

40.5/9.17

48.9/5.68

0.67

0.41

0.30

30 75 99

11% 38% 16%

17.4/5.67 20.8/5.28 75.6/5.22

14.8/5.67 29.8/5.67 81.6/4.87

1.45 0.43 2.18

0.33 0.83 0.78

0.18 0.17 0.39

stress photosystem, light reaction redox regulation photosystem, Calvin cycle photosystem, Calvin cycle amino acid metabolism photosystem, Calvin cycle photosystem, Calvin cycle protein degradation glycolysis stress

82

19%

21.4/5.79

15.8/5.60

2.08

1.14

0.28

redox regulation

107

27%

55.7/5.72

63.9/5.86

1.13

2.02

0.24

146

13%

55.8/5.87

64.4/5.90

0.29

2.07

0.51

photosystem, light reaction photosystem, light reaction

homo. (species) oxygen-evolving enhancer protein 1, chloroplastic [Fritillaria agrestis] NAD(P)H-quinone oxidoreductase subunit M, chloroplastic [Physcomitrella patens] triosephosphate isomerase, chloroplastic [Fragaria ananas sa] ribulose 1,5-bisphosphate carboxylase oxygenase small subunit protein [Eleusine coracana] cytochrome b6-f complex iron−sulfur subunit [Saccharum hybrid cultivar ROC22] photosystem II subunit H [Bouteloua curtipendula] S-adenosylmethionine synthetase [Cleistogenes songorica] ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit [Harpochloa sp. Hodkinson 579] photosystem I iron−sulfur center [Aegilops tauschii]

ATP synthase subunit α, chloroplastic [Saccharum hybrid]

H

fold change fun. photosystem, light reaction not assigned photosystem, Calvin cycle photosystem, Calvin cycle photosystem, light reaction photosystem, light reaction amino acid metabolism photosystem, light reaction photosystem, light reaction photosystem, light reaction glycolysis nucleotide metabolism redox regulation nucleotide metabolism cell organization photosystem, Calvin cycle photosystem, Calvin cycle photosystem, Calvin cycle protein folding gluconeogenese/ glyoxylate cycle gluconeogenese/ glyoxylate cycle stress



Article

Table 1. continued a

Legend: no., protein spot number; homo., homologous protein; sc., score; cov, sequence coverage; theor, theoretical value; exp, experimental value; Mr, Mr (KD); fun., function bin.

Table 2. Pathway Enrichment Analysis of Commonly Regulated Proteins by Polyamines in Bermudagrass

Figure 6. ROS accumulation and antioxidant enzymes’ activities affected by exogenous polyamine treatment during salt and drought stresses in bermudagrass. (A−F) Quantifications of MDA content (A), H2O2 content (B), O2•− content (C), SOD activity (D), CAT activity (E), and POD activity (F) of bermudagarss plants with different treatments (control, 5 mM putrescine, 5 mM spermidine, and 5 mM spermine, respectively) under control conditions, 300 mM NaCl, and drought conditions at designated time intervals. The relative activities of SOD, CAT, and POD of bermudagrass without treatment under control conditions of 7 days were set as 1. The results shown are the means ± SE of four independent experiments. Asterisk symbols indicate significant differences in comparison to wild type without polyamine treatment (p < 0.05). I



Article

Figure 7. Accumulation of osmolytes affected by exogenous polyamine treatments during salt and drought stresses in bermudagrass. Proline content (A), sucrose content (B) and soluble sugars content (C) of bermudagarss plants with different treatments (control, 5 mM Put, 5 mM Spd and 5 mM Spm, respectively) under control condition, 300 mM NaCl and drought conditions at designated time intervals. The results shown are the means ± SE of 4 independent experiments. Asterisk symbols indicate significant differences in comparison to wild type without polyamine treatment (p < 0.05).

also as a defense response to abiotic stress.11,55 On the other hand, almost all abiotic stresses could induce the burst of ROS,1,2 and both free polyamines and polyamine conjugates served as efficient scavengers of ROS radicals.10 Nucleoside diphosphate kinase (NDPK), a key metabolic enzyme involved in multiple abiotic stress responses,57−59 was commonly increased by Put, Spd, and Spm (Figure 5). Overexpression of AtNDPK2 conferred enhanced tolerance to multiple environmental stress that elicits ROS accumulation through modulating antioxidant enzyme activities.56−58 Thus, the increased NDPK protein levels by polyamines might contribute to enhanced salt and drought tolerances in burmudagrass. Based on our results, polyamines might modulate stress triggered ROS homeostasis and oxidative damage (MDA) by activating some antioxidant enzyme activities including POD, CAT, and SOD, and polyamine-derived H2O2 production might play dual roles under control and abiotic stress conditions.

significantly higher activities of these antioxidant enzymes and lower concentrations of H2O2 and O2•− contents than those of untreated bermudagrass plants at some time points (7 or 14 days or both 7 and 14 days of abiotic stress treatments). As reviewed by Moschou et al.55 and Tavladorake et al.,11 polyamine catabolism in plants is highly connected with the production of H2O2, because H2O2 is the product in many reactions of the polyamine metabolic pathway. As an important second messenger in the signal transduction network, H2O2 could activate downstream ion channels (Ca2+, Na+/K+, H+ATPase, etc.) and modulate stress-responsive gene expression and stomatal closure.11,54,55 Wu et al.56 found that polyamine catabolism derived H2O2 production partly contributed to the activation of the Ca2+-permeable channel. Thus, exogenous polyamines induced H2O2 production (Figure 6B) might be helpful to keep the water status under water-deficit conditions (Figure 2D, E). Additionally, polyamine catabolism derived H2O2 production has been shown to induce plant cell death J



Article

Figure 8. Commonly regulated proteins by polyamines treatment involved in carbon fixation in photosynthetic organisms. (A) Seven commonly regulated proteins by polyamines treatment involved in carbon fixation in photosynthetic organisms. (B) Relative protein levels of seven commonly regulated proteins by polyamines treatments involved in the carbon fixation pathway. The results shown are the means ± SE of three independent experiments. Asterisk symbols indicate significant differences in comparison to wild type without polyamine treatment (p < 0.05).

metabolized to Put and proline through ODC and omithineδ-aminotransferase (OAT), respectively.3,4,11 Because the polyamine and proline biosynthesis pathways shared some common substrates, exogenous application of polyamines might shift more substrates for proline biosynthesis, especially under stress conditions. Two identified proteins (U7, Sadenosylmethionine synthetase; D16, glyoxalase I homologue) involved in amino acid metabolism might also partly contribute to the regulation of proline concentration. Sugars, widely

Proline and soluble sugars are widely known as compatible osmolytes which are important to maintain turgor and stabilize cell molecular structure under various stress conditions.1,2 As shown in Figure 7, exogenous polyamines pretreated burmudagrass plants accumulated more proline and soluble sugars under salt and drought stresses than untreated control, thus balancing osmotic pressure under stress conditions. In the polyamine metabolic pathway, omithine is synthesized from arginine by arginase; then the omithine can be further K



Article

Figure 9. Possible mechanism depicting polyamines application involved in abiotic stress responses of bermudagrass. Exogenous polyamines could reduce water loss and keep MDA content at a lower level, thus to alleviate stress-triggered water-deficit, plant growth inhibition, and damage of cell membrane. Additionally, polyamines increased the protein level of NDPK, and activated antioxidant enzymes activities and osmolytes (proline and soluble sugars), which could alleviate osmotic pressure and other cell damages that were induced by salt and drought stresses. Polyamines might also regulate unknown physiological changes and modulate global proteomic responses including some proteins identified in this study, in turn resulting in other unknown adaptive responses.

known as the major products of photosynthesis, are essential for regulation of several metabolic pathways that enable them to be self-sustainable with basic nutrients and stress responses.60−62 It is well established that insulin synthesis and secretion require glycolysis, and the intermediates of anaerobic glycolysis between phosphoenolpyruvate and fructose 1,6-diphosphate are essential for cell glucose sensing in the pancreatic islets.61−63 In the central metabolism in photosynthetic cells, carbon is converted into starch and sucrose in the plastid and cytosol, respectively, and the corresponding metabolism is partitioned into different pathways by multiple isozymes.62 Interestingly, some of these proteins were identified in this study that were commonly affected by polyamines, including nine proteins [U3 (triosephosphate isomerase, chloroplastic), U4, D4, D5, D6, D14 (fructose-bisphosphate aldolase, choroplastic), D15, D17, and D18 (glyceraldehyde-3phosphate dehydrogenase A, chloroplastic)] involved in the Calvin cycle of the photosystem, U11 (cytoplasmic fructosebisphosphate aldolase) and D20 (cytosolic triosephosphate isomerase) involved in glycolysis, and D8 (pyruvate orthophosphate dikinase) and D9 (pyruvate, phosphate dikinase) involved in the gluconeogenese/glyoxylate cycle (Table 1 and Figure 8). These commonly regulated proteins, especially the seven proteins involved in carbon fixation (Figure 8), might lead to the changes of sugar content, indicating the polyaminesmediated Calvin cycle, glycolysis, and the gluconeogenese/ glyoxylate cycle might play important roles in bermudagrass responses to salt and drought stresses. Additionally, three proteins associated with ATP synthase (D12, F3, and F4) were also identified as polyamine affected proteins, and this might because that ATP to be the major product during the Calvin cycle and glycolysis metabolism.

Collective observations supported the putative roles of polyamines in abiotic stress responses in bermudagrass (Figure 9). Exogenous polyamines could reduce water loss and keep MDA content at a lower level, thus to alleviate stress-triggered water-deficit, plant growth inhibition, and damage of cell membrane. Additionally, polyamines increased the protein level of NDPK, activated antioxidant enzymes activities, and osmolytes (proline and soluble sugars), which could alleviate osmotic pressure and other cell damages induced by salt and drought stresses. Polyamines might also regulate unknown physiological changes and modulate global proteomic responses including some proteins identified in this study, in turn resulting in other unknown adaptive responses. Taken together, this is the first study to assign the protective role of polyamines in bermudagrass responses to salt and drought stresses. Comparative proteomic and physiological analyses revealed the putative mechanisms of exogenous polyamines pretreatments induced salt and drought stress responses. Proteins involved in electron transport and energy pathways were largely enriched, and NDPK and three antioxidant enzymes were extensively regulated by polyamines. Dissection of ROS levels indicated that polyamine-derived H2O2 production might play dual roles under abiotic stress conditions. Moreover, accumulation of osmolytes was also observed after application of exogenous polyamines, which is consistent with proteomics results that several proteins involved in the carbon fixation pathway were mediated commonly by polyamines pretreatment. Collective observations might provide new insights to elucidate the polyamine related signaling pathway and downstream targets, and the connections between these pathways and polyamine-mediated abiotic stress. These results will also be further used for improvement of plant L



Article

catabolism: target for antiproliferative therapies in animals and stress tolerance strategies in plants. Amino Acids 2012, 42, 411−426. (12) Ali, R. M. Role of putrescine in salt tolerance of Atropa belladonna plant. Plant Sci. 2000, 152, 173−179. (13) Borrell, A.; Carbonell, L.; Farras, R.; Puig-Parellada, P.; Tiburcio, A. F. Polyamines inhibit lipid peroxidation in senescing oat leaves. Physiol. Plant. 1997, 99, 385−390. (14) Choudhary, S. P.; Oral, H. V.; Bhardwaj, R.; Yu, J. Q.; Tran, L. S. P. Interaction of brassinosteroids polyamines enhances copper stress tolerance in Raphanus sativus. J. Exp. Bot. 2012, 63, 5659−5675. (15) Du, C. X.; Fan, H. F.; Guo, S. R.; Tezuka, T. Applying spermidine for differential responses of antioxidant enzymes in cucumber subjected to short-term salinity. J. Am. Soc. Hortic. Sci. 2010, 135, 18−24. (16) Duan, J.; Li, J.; Guo, S.; Kang, K. Exogenous spermidine affects polyamine metabolism in salinity-stressed Cucumis sativus roots and enhances shortterm salinity tolerance. J. Plant Physiol. 2008, 165, 1620−1635. (17) Hu, X.; Zhang, Y.; Shi, Y.; Zhang, Z.; Zou, Z.; Zhang, H.; Zhao, J. Effect of exogenous spermidine on polyamine content and metabolism in tomato exposed in salinity-alkalinity mixed stress. Plant Physiol. Biochem. 2012, 57, 200−209. (18) Li, B.; He, Li.; Guo, S.; Li, J.; Yang, Y.; Yan, B.; Sun, J.; Li, J. Proteomics reveal cucumber Spd-responses under normal condition and salt stress. Plant Physiol. Biochem. 2013, 67, 7−14. (19) Janicka-Russak, M.; Kabała, K.; Młodzińska, E.; Kłobus, G. The role of polyamines in the regulation of the plasma membrane and the tonoplast proton pumps under salt stress. J. Plant Physiol. 2010, 167, 261−269. (20) Navakouidis, E.; Lütz, C.; Langebartels, C.; Lütz-Meindl, U.; Kotzabasis, K. Ozone impact on the photosynthetic apparatus and the protective role of polyamines. Biochem. Biophys. Acta 2003, 1621, 160−169. (21) Ndayiragije, A.; Lutts, S. Do exogenous polyamines have an impact on the response of a salt-sensitive rice cultivar to NaCl. Plant Growth Regul. 2006a, 48, 51−63. (22) Ndayiragije, A.; Lutts, S. Exogenous putrescine reduces sodium and chloride accumulation in NaCl-treated calli of the salt-sensitive rice cultivar I Kong Pao. Plant Growth Regul. 2006b, 48, 51−63. (23) Quinet, M.; Ndayiragije, A.; Lefèvre, I.; Lambillotte, B.; DupontGillain, C. C.; Lutts, S. Putrescine differently influences the effect of salt stress on polyamine metabolism and ethylene synthesis in rice cultivars differing in salt reisistance. J. Exp. Bot. 2010, 61, 2719−2733. (24) Sagor, G. H. M.; Berberich, T.; Takahashi, Y.; Niitsu, M.; Kusano, T. The polyamine spermine protects Arabidopsis from heat stress-induced damage by increasing expression of heat shock-related genes. Transgenic Res. 2013, 22, 595−605. (25) Shevyakova, N. I.; Il’ina, E. N.; Stetsenko, L. A.; Kuznetsov, V. I. V. Nickel accumulation in rape shoots (Brassica Napus L.) increased by putrescine. Int. J. Phytoremediation 2010, 13, 345−356. (26) Shi, J.; Fu, X. Z.; Peng, T.; Huang, X. S.; Fan, Q. J.; Liu, J. H. Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response. Tree Physiol. 2010, 30, 913−922. (27) Shu, S.; Guo, S. R.; Sun, J.; Yuan, L. Y. Effects of salt stress on the structure and function of the photosynthetic apparatus in Cucumis sativus and its protection by exogenous putrescine. Physiol. Plant. 2012, 146, 285−296. (28) Verma, S.; Mishra, S. N. Putrescine alleviation of growth in salt stressed Brassica juncea by inducing antioxidative defense system. J. Plant Physiol. 2005, 162, 669−677. (29) Wang, X.; Shi, G.; Xu, Q.; Hu, J. Exogenous polyamines enhance copper tolerance of Nymphoides peltatum. J. Plant Physiol. 2010, 164, 1062−1070. (30) Zhang, W.; Jiang, B.; Li, W.; Song, H.; Yu, Y.; Chen, J. Polyamines enhance chilling tolerance of cucumber (Cucumis sativus L.) through modulating antioxidative system. Sci. Hortic. 2009, 122, 200−208.

abiotic stress tolerance through the polyamine pathway, including bermudagrass, the most popularly used warm-season turfgrass.

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ASSOCIATED CONTENT

* Supporting Information S

Figures showing leaf water status affected by polyamines treatments, and analysis of 27 coregulated proteins by polyamines treatments; tables showing fold change of all bermudagrass proteins regulated by polyamines treatments, and a detailed list of bermudagrass proteins that were commonly changed by polyamines treatments. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +86 27 87510823. Fax: +86 27 87510251. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Pingfang Yang, Mr. Ming Li, and Mr. Xiaojian Yin for their help in this research. This research was supported by “the Hundred Talents Program” and the Knowledge Innovative Key Program of the Chinese Academy of Sciences (Grant No. 54Y154761O01076 to Z.C.), and by the National Natural Science Foundation of China (Grant No. 31200194 to H.S.).

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REFERENCES

(1) Shi, H.; Wang, Y.; Chen, Z.; Ye, T.; Chan, Z. Analysis of natural variation in bermudagrass (Cynodon dactylon) reveals physiological responses underlying drought tolerance. PLoS ONE 2012a, 7, e53422. (2) Shi, H. T.; Li, R. J.; Cai, W.; Liu, W.; Wang, C. L.; Lu, Y. T. Increasing nitric oxide content in Arabidopsis thaliana by expressing rat neuronal nitric oxide synthase resulted in enhanced stress tolerance. Plant Cell Physiol. 2012b, 53, 344−357. (3) Shi, H.; Ye, T.; Chen, F.; Cheng, Z.; Wang, Y.; Yang, P.; Zhang, Y.; Chan, Z. Manipulation of arginase expression modulates abiotic stress tolerance in Arabidopsis: effect on arginine metabolism and ROS accumulation. J. Exp. Bot. 2013a, 64, 1367−1379. (4) Shi, H. T.; Chan, Z. L. In vivo role of Arabidopsis arginase in arginine metabolism and abiotic stress response. Plant Signal. Behav. 2013b, 5, e24138. (5) Varshney, R. K.; Bansal, K. C.; Aggarwal, P. K.; Datta, S. K.; Craufurd, P. Q. Agricultural biotechnology for crop improvement in a variable climate: hope or hype. Trends Plant Sci. 2011, 16, 363−371. (6) Zhao, Y.; Du, H.; Wang, Z.; Huang, B. Identification of proteins associated with water-deficit tolerance in C4 perennial grass species, Cynodon dactylon×Cynodon transvaalensis and Cynodon dactylon. Physiol. Plant. 2011, 141, 40−55. (7) Wimalasekera, R.; Tebartz, F.; Scherer, G. F. E. Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses. Plant Sci. 2011, 181, 593−603. (8) Gill, S. S.; Tuteja, N. Polyamines and abiotic stress tolerance in plants. Plant Signal. Behav. 2010, 5, 26−33. (9) Groppa, M. D.; Benavides, M. P. Polyamines and abiotic stress: recent advances. Amino Acids 2008, 34, 35−45. (10) Hussain, S. S.; Ali, M.; Ahmad, M.; Siddique, K. H. M. Polyamines: Natural and engineered abiotic and biotic stress tolerance in plants. Biotechnol. Adv. 2011, 29, 300−311. (11) Tavladoraki, P.; Cona, A.; Federico, R.; Tempera, G.; Viceconte, N.; Saccoccio, S.; Battaglia, V.; Toninello, A.; Agostinelli, E. Polyamine M



Article

(31) Zhao, F.; Song, C. P.; He, J.; Zhu, H. Polyamines improve K+/ Na+ homeostasis in barley seedlings by regulating root ion channel activities. Plant Physiol. 2007, 145, 1061−1072. (32) Zhao, H.; Yang, H. Exogenous polyamines alleviate the lipid peroxidation induced by cadmium chloride stress in Malus hupehensis Rehd. Sci. Hortic. 2008, 116, 442−447. (33) Yiu, J. C.; Juang, L. D.; Fang, D. Y. T.; Liu, C. W.; Wu, S. J. Exogenous putrescine reduces flooding induced oxidative damage by increasing the antioxidant properties of Welsh onion. Sci. Hortic. 2009, 120, 306−314. (34) Komatsu, S.; Hiraga, S.; Yanagawa, Y. Proteomics techniques for the development of flood tolerant crops. J. Proteome Res. 2012, 11, 68− 78. (35) Nanjo, Y.; Skultety, L.; Uvácǩ ová, L.; Klubicová, K.; Hajduch, M.; Komatsu, S. Mass spectrometry-based analysis of proteomic changes in the root tips of flooded soybean seedlings. J. Proteome Res. 2012, 11, 372−385. (36) Kausar, R.; Hossain, Z.; Makino, T.; Komatsu, S. Characterization of ascorbate peroxidase in soybean under flooding and drought stresses. Mol. Bio. Rep. 2012, 39, 10573−10579. (37) Khatoon, A.; Rehman, S.; Hiraga, S.; Makino, T.; Komatsu, S. Organ-specific proteomics analysis for identification of response mechanism in soybean seedings under flooding stress. J. Proteomics 2012a, 75, 5706−5723. (38) Khatoon, A.; Rehman, S.; Oh, M. W.; Woo, S. H.; Komatsu, S. Analysis of response mechanism in soybean under low oxygen and flooding stresses under gel-base proteomics technique. Mol. Bio. Rep. 2012b, 39, 10581−10594. (39) Komatsu, S.; Kobayashi, Y.; Nishizawa, K.; Nanjo, Y.; Furukawa, K. Comparative proteomics analysis of differentially expressed proteins in soybean cell wall during flooding stress. Amino Acids 2010, 39, 1435−1449. (40) Komatsu, S.; Thibaut, D.; Hiraga, S.; Kato, M.; Chiba, M.; Hashiguchi, A.; Tougou, M.; Shimamura, S.; Yasue, H. Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots. Plant Mol. Biol. 2011, 77, 309−322. (41) Salavati, A.; Khatoon, A.; Nanjo, Y.; Komatsu, S. Analysis of proteomic changes in roots of soybean seedling during recovery after flooding. J. Proteomics 2012, 75, 878−893. (42) Komatsu, S.; Nanjo, Y.; Nishimura, M. Proteomics analysis of the flooding tolerance mechanism in mutant soybean. J. Proteomics 2013, 79, 231−250. (43) Chan, Z. Proteomic responses of fruits to environmental stresses. Front. Plant Sci. 2012, 3, 311. (44) Chan, Z.; Wang, Q.; Xu, X.; Meng, X.; Qin, G.; Li, B.; Tian, S. Functions of defense-related proteins and dehydrogenases in resistance response induced by salicylic acid in sweet cherry fruits at different maturity stages. Proteomics 2008, 8, 4791−4807. (45) Saravanan, R. S.; Rose, J. K. C. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 2004, 4, 2522−2532. (46) Bradford, N. M. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (47) Li, M.; Sha, A.; Zhou, X.; Yang, P. Comparative proteomic analyses reveal the changes of metabolic features in soybean (Glycine max) pistils upon pollination. Sex. Plant Reprod. 2012, 25, 281−291. (48) Chan, Z.; Qin, G.; Xu, X.; Li, B.; Tian, S. Proteome approach to characterize proteins induced by antagonist yeast and salicylic acid in peach fruit. J. Proteome Res. 2007, 6, 1677−1688. (49) Aede Hoon, M. J. L.; Imoto, S.; Nolan, J.; Miyano, S. Open Source Clustering Software. Bioinformatics 2004, 20, 1453−1454. (50) Provart, N.; Zhu, T. A Browser-based Functional Classification SuperViewer for Arabidopsis Genomics. Curr. Comput. Mol. Biol. 2003, 2003, 271−272. (51) Thimm, O.; Blasing, O.; Gibon, Y.; Nagel, A.; Meyer, S.; Kruger, P.; Selbig, J.; Muller, L. A.; Rhee, S. Y.; Stitt, M. MAPMAN: a userdriven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J. 2004, 37, 914−939.

(52) Marco, F.; Alcázar, R.; Tiburcio, A. F.; Carrasco, P. Interactions between polyamines and abiotic stress pathway responses unraveled by transcriptome analysis of polyamine overproducer. OMICS 2011, 15, 775−781. (53) Nishizawa, K.; Komatsu, S. Characteristics of soybean 1-Cys peroxiredoxin and its behavior in seedlings under flooding stress. Plant Biotechnol. 2011, 28, 83−88. (54) Shi, H.; Ye, T.; Wang, Y.; Chan, Z. Arabidopsis ALTERED MERISTEM PROGRAM 1 negatively modulates plant responses to abscisic acid and dehydration. Plant Physiol. Biochem. 2013c, 67, 209− 216. (55) Moschou, P. N.; Wu, J.; Tavladoraki, P.; Angelini, R.; Roubelakis-Angelakis, K. A. The polyamines and their catabolic products are significant players in the turnover of nitrogenous molecules in plants. J. Exp. Bot. 2012, 63, 5003−5015. (56) Wu, J.; Shang, Z.; Jiang, X.; Moschou, P. N.; Sun, W.; Roubelakis-Angelakis, K. A.; Zhang, S. Spermidine oxidase-derived H2O2 regulates pollen plasma membrane hyperpolarization-activated Ca2+-permeable channels and pollen tube growth. Plant J. 2010, 63, 1042−1053. (57) Moon, H.; Lee, B.; Choi, G.; Shin, D.; Prasad, D. T.; Lee, O.; Kwak, S. S.; Kim, D. H.; Nam, J.; Bahk, J.; Hong, J. C.; Lee, S. Y.; Cho, M. J.; Lim, C. O.; Yun, D. J. NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 358−363. (58) Verslues, P. E.; Batelli, G.; Grillo, S.; Agius, F.; Kim, Y. S.; Zhu, J.; Agarwal, M.; Katiyar-Agarwal, S.; Zhu, J. K. interaction of SOS2 with nucleoside diphosphate kinase 2 and catalases reveals a point of connection between salt stress and H2O2 signaling in Arabidopsis thaliana. Mol. Cell. Biol. 2007, 27, 7771−7780. (59) Kim, Y. H.; Kim, M. D.; Choi, Y. I.; Park, S. C.; Yun, D. J.; Noh, E. W.; Lee, H. S.; Kwak, S. S. Transgenic poplar expressing Arabidopsis NDPK2 enhances growth as well as oxidative stress tolerance. Plant Biotechnol. J. 2011, 9, 334−347. (60) Gibson, S. I. Control of plant development and gene expression by sugar signaling. Curr. Opin. Plant Biol. 2005, 8, 93−102. (61) Granot, D.; David-Schwartz, R.; Kelly, G. Hexose kinases and their role in sugar-sensing and plant development. Front. Plant Sci. 2013, 4, 44. (62) Tiessen, A.; Padilla-Chacon, D. Subcellular compartmentation of sugar signaling: links among carbon cellular status, route of sucrolysis, sink-source allocation, and metabolic partitioning. Front. Plant Sci. 2013, 3, 306. (63) German, M. S. Glucose sensing in pancreatic islet beta cells: the key role of glucokinase and the glycolytic intermediates. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1781−1785.

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Comparative Proteomic and Physiological Analyses Reveal the

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