Differential proteomic responses to water stress induced by PEG in two creeping bentgrass cultivars differing in stress tolerance

Journal of Plant Physiology 167 (2010) 1477–1485

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Differential proteomic responses to water stress induced by PEG in two creeping bentgrass cultivars differing in stress tolerance Chenping Xu, Bingru Huang ∗ Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, United States

a r t i c l e

i n f o

Article history: Received 17 January 2010 Received in revised form 25 May 2010 Accepted 25 May 2010 Keywords: Water deficit Electrophoresis Grass Proteomics Stress tolerance

a b s t r a c t Protein metabolism and expression play important role in plant adaptation to water stress. The objectives of this study were to examine proteomic responses to water stress induced by polyethylene glycol (PEG) in creeping bentgrass (Agrostis stolonifera L.) leaves and to identify proteins associated with stress tolerance. Plants of two cultivars (‘Penncross’ and ‘Penn-A4’) differing in water stress tolerance were grown in sand irrigated daily with water (control) or PEG solution (osmotic potential of −0.66 MPa) to induce water stress, for 28 d in growth chambers. Shoot extension rate, relative water content and cell membrane stability were measured to compare drought tolerance between the two cultivars. All parameters maintained at a significantly higher level in ‘Penn-A4’ than in ‘Penncross’ under PEG treatment. After 28 d of water stress, proteins were extracted from leaves and separated by difference gel electrophoresis. Among 56 stress-responsive protein spots, 46 were identified using mass spectrometry. Some proteins involved in primary nitrogen and carbon metabolism were down-regulated by PEG-induced water stress in both cultivars. The abundance of antioxidant enzyme proteins (ascorbate peroxidase, catalase and glutathione-S-transferase) increased under water stress, particularly ascorbate peroxidase in ‘Penn-A4’. The abundance levels of actins, UDP-sulfoquinovose synthase and glucan exohydrolase were greater in ‘Penn-A4’ than in ‘Penncross’ under PEG treatment. Our results suggest that proteins involved in membrane synthesis, cell wall loosening, cell turgor maintenance, and antioxidant defense may play roles in perennial grass adaptation to PEG-induced water stress. Published by Elsevier GmbH.

Introduction Water deficit is a significant problem in agricultural production, including perennial grasses. Plant adaption to water stress may be accomplished through changes at the molecular, cellular, and physiological levels. Physiological studies have demonstrated that changes in water relation, nutrient uptake, hormonal metabolism, carbon metabolism, and antioxidant metabolism play important roles in drought tolerance (Bray, 1997). Transcriptomic studies have revealed that the expression of a wide range of genes is regulated in response to water deficit (Kreps et al., 2002; Seki et al., 2002; Shinozaki and Yamaguchi-Shinozaki, 2007). Study in Arabidopsis showed that 1008 mRNAs were up-regulated in response to water deficit (Kreps et al., 2002). Although RNA and DNA microarrays are

Abbreviations: APX, ascorbate peroxidase; DTT, dithiothreitol; EL, electrolyte leakage; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; GST, glutathioneS-transferase; HSPs, heat shock proteins; IEF, isoelectric focusing; OEE, oxygen evolving enhancer; PEG, polyethylene glycol; PSII, photosystem II; RWC, relative water content; SHMT, serine hydroxymethyltransferase; TCA, trichloroacetic acid. ∗ Corresponding author. Tel.: +732 932 9711; fax: +732 932 9441. E-mail address: [email protected] (B. Huang). 0176-1617/$ – see front matter. Published by Elsevier GmbH. doi:10.1016/j.jplph.2010.05.006

powerful tools in the detection of gene expression, limited knowledge of stress-responsive protein expression remains a major gap in understanding biological functions of genes and the linkage between gene expression and physiological functions. Therefore, comprehensive profiling of stress-responsive proteins is important for further understanding the molecular mechanisms controlling plant drought tolerance. Proteomics, the study of global changes in proteins, offers a powerful approach to discovering the genes and pathways that are crucial for stress responsiveness and tolerance. The identification and characterization of stress-responsive proteins and their corresponding genes has proven to be of immense practical values. Recently, proteomic-based technologies have been successfully applied to the systematic study of the proteomic responses in many plant species to a wide range of abiotic stresses, including water stress (Salekdeh et al., 2002; Plomion et al., 2006; Gazanchian et al., 2007; Hajheidari et al., 2007; Ingle et al., 2007; Kottapalli et al., 2009). These studies indicated that water stress altered the abundance of proteins involved in carbohydrate and energy metabolism, cellular detoxification, protein degradation and processing, signal transduction, and cell wall strengthening. Most of the previous work on water stress related proteomics was performed on annual crops (Salekdeh and Komatsu, 2007). However, very limited infor-

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mation is available on proteomic responses of perennial grasses to water stress. Perennial grasses may express stress-responsive proteins associated with long-term adaptation or stress survival, as perennial grasses must endure stress or persist through the stress period, unlike annual crops which produce seeds and may die in the case of severe drought (DaCosta and Huang, 2009). Thus, developing long-term adaptation mechanisms is critical for the survival of perennial grasses in water-limiting environments. A better understanding of proteomic responses to water stress in perennial grass species is vital for the development of breeding or biotechnology strategies to improve plant growth and productivity, and to reduce water use in areas with limited rainfall or irrigation. Investigation of stress-responsive proteins in tolerant cultivar in comparison to sensitive cultivar may identify specific proteins related to stress tolerance in grass. Fu et al. (2007) reported that drought tolerance of creeping bentgrass was improved by overexpression of LEA3 genes encoding dehydrin proteins. The objective of this study was to identify proteins responsive to polyethylene glycol (PEG)induced water stress in perennial grass by comparing proteomic responses to water stress between two cultivars of creeping bentgrass (Agrostis stolonifera), a grass species widely used as a forage and turf in cool-climatic regions. Materials and methods Plant materials and water stress treatments Plants of creeping bentgrass ‘Penncross’ and ‘Penn-A4’ were collected from field plots in the turfgrass research farm at Rutgers University, New Brunswick, NJ. Previous studies reported that ‘Penn-A4’ had superior drought resistance relative to ‘Penncross’ (McCann and Huang, 2008). All plants were propagated vegetatively in plastic pots (15 cm deep and 15 cm in diameter) filled with washed, fine sand. During the plant establishment period, plants were watered daily until water drained from the bottom of the pots and fertilized twice a week with full-strength Hoagland’s nutrient solution (Hoagland and Arnon, 1950). Plants were maintained in a greenhouse for 30 d and then moved to a growth chamber set at 20/15 ◦ C (day/night temperature), 75% relative humidity, 600 ␮mol m−2 s−1 of photosynthetically active radiation, and 12h photoperiod. Plants were allowed to acclimate to the growth chamber conditions for 7 d before treatments were imposed. The plants were irrigated daily with water (control) or with polyethylene glycol (PEG) 8000 solution to induce water stress for 28 d. Water stress was obtained by adding PEG solution with osmotic potential of −0.15 and −0.30 MPa twice a day for 2-d intervals at each concentration, and followed by adding PEG solution with osmotic potential of −0.66 MPa twice a day for 28 d. Each treatment for each cultivar was replicated in four pots. At each time of watering, sufficient water or PEG solution was applied until the sand medium was fully saturated. During the treatment period, plants were fertilized once a week with full-strength Hoagland’s nutrient solution (Hoagland and Arnon, 1950). Physiological measurements Physiological responses to water stress were evaluated by measuring shoot extension rate, leaf electrolyte leakage (EL) and relative water content (RWC) during the treatment period according to the method of Jiang and Huang (2002). For EL analysis, about 0.2 g (fresh weight) of leaves was placed in test tube containing 30 mL of distilled, deionized H2 O. Test tubes were shaken for 17–18 h at 23 ◦ C, and the initial conductance (Ci ) was measured with a conductivity meter (YSI Instrument, Yellow Spring, OH). Leaves then were killed at 120 ◦ C for 30 min, and the con-

ductance of killed tissue (Cmax ) was measured. The relative EL was calculated as 100 × Ci /Cmax . RWC was calculated using the formula: 100[(FW − DW)/(TW − DW)] where FW is fresh weight, TW is turgid weight, and DW is dry weight following oven-drying leaf samples for 72 h at 80 ◦ C. Shoot extension rate (mm d−1 ) was measured as change in shoot height within each week of treatment. Protein extraction and labeling Protein extraction was performed following a protocol using acetone/trichloroacetic acid (TCA) precipitation as described previously (Xu et al., 2008). At 28 d of treatment, leaves were harvested and immediately frozen in liquid nitrogen, and then stored at −80 ◦ C prior to analysis. Three independent samples were harvested from each treatment. About 0.5 g of leaf samples were homogenized and incubated with 10 mL of precipitation solution (10% TCA and 0.07% 2-mercaptoethanol in acetone) for 2 h at −20 ◦ C. The precipitated proteins were pelleted and washed with ice-cold acetone containing 0.07% 2-mercaptoethanol to remove pigments and lipids until the supernatant was colorless. The pellet was vacuum-dried, resuspended in resolubilization solution (30 mM Tris–HCl pH 8.5; 7 M urea; 2 M thiourea; 4% CHAPS) and sonicated to extract proteins. Insoluble tissue was removed by centrifugation at 21,000 g for 15 min. Protein concentration was determined according to Bradford (1976) using a commercial dye reagent (BioRad Laboratories, Hercules, CA) with BSA as a standard. Proteins from plants of the control and PEG treatment were labeled respectively with Cy3 and Cy5 CyDye DIGE Fluor minimal dyes (GE Healthcare, Amersham, UK) as described previously (Xu et al., 2010). The dye: protein labeling ratio is deliberately kept low so that only proteins containing a single dye molecule are visualized on the gel and variants containing more than one dye molecule are avoided (Tonge et al., 2001). Cyanine dyes were reconstituted in 99.8% anhydrous dimethylformamide and added to labeling reactions in a ratio of 100 pmol CyDye: 25 ␮g protein. Protein labeling was achieved by incubation on ice for 30 min in the dark. The reaction was quenched by the addition of 10 mM lysine followed by incubation on ice for 10 min. In addition, a pooled internal standard composed of equal quantity of protein from all the experimental samples was labeled with Cy2. For each gel, 25 ␮g of each sample was mixed with 25 ␮g protein of the pooled internal standard. Two-dimensional electrophoresis The Immobiline DryStrips (13 cm pH 3–10 linear GE Healthcare) were passively rehydrated with the labeled samples for 1 h. The voltage settings for isoelectric focusing (IEF) were 50 V for 12 hrs, 500 V for 1 hr, 1000 V for 1 hr, and 8000 V to a total 80 kVh. Following IEF, the protein in the strips was denatured with equilibration buffer (50 mM Tris–HCl pH 8.8, 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate, 0.002% bromophenol blue, 1% dithiothreitol/DTT) and then incubated with the same buffer containing 2.5% iodoacetamide instead of DTT for 20 min at room temperature. The second dimension electrophoresis was performed on a 12.5% gel using a Hoefer SE 600 Ruby electrophoresis unit (GE Healthcare, Piscataway, NJ). Labeled proteins were visualized using the Typhoon 9410 imager (GE Healthcare, Piscataway, NJ). The Cy2 images were scanned using a 488 nm laser and an emission filter of 520 nm BP (band pass) 40. Cy3 images were scanned using a 532 nm laser and an emission filter of 580 nm BP 30. Cy5 images were scanned using a 633 nm laser and a 670 nm BP 30 emission filter. The narrow BP emission filters ensure that there is negligible cross-talk between fluorescence channels. Images were analyzed using SameSpots software (Nonlinear). To correct the variability due to staining, the spot volumes were normalized as a percentage of the total volume of all spots on the gel. The detailed procedure

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of protein identification using mass spectrometry was described previously (Xu et al., 2010). Briefly, protein identification was performed by searching the combined MS and MS/MS spectra against the green plant NCBI database using a local MASCOT search engine (V.1.9) on a GPS (V. 3.5, ABI) server. Protein contains at least two unique peptides confirmed by MS/MS analysis with confidence interval (C.I.) values no less than 95% was considered being identified. Experimental design and statistical analysis Treatments were arranged in a complete randomize design with four replicates (4 pots for each treatment). For protein abundance analysis, electrophoresis was run in three extraction sample from each replicate (3 sub-samples per replicate). The mean of the three sub-samples was used to represent a single replicate in the analysis of variance. Physiological and protein abundance data were analyzed with analysis of variance and mean separations were performed with the Fisher’s protected least significance difference test at P = 0.05 using a SAS program (SAS 9.1, SAS Institute Inc., Cary, NC).

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tions (Fig. 2B). At 7, 14, 21 and 28 d of PEG treatment, leaf RWC was 83, 81, 71 and 59% in ‘Penn-A4’ and 81, 76, 62 and 49% in ‘Penncross’, respectively. ‘Penn-A4’ had significantly higher leaf RWC than ‘Penncross’ at 21 and 28 d of treatment. For electrolyte leakage (EL), there was no significant difference between the two cultivars under control condition (Fig. 2C). Leaf EL increased rapidly during PEG treatment, but ‘Penn-A4’ had significantly lower EL than ‘Penncross’ at 21 and 28 d of treatment. PEG-induced water stress caused leaf water deficit in both cultivars as indicated by decline in leaf RWC. ‘Penn-A4’ had higher leaf RWC and lower EL than ‘Penncross’ under PEG treatment, confirming that ‘Penn-A4’ had superior drought resistance compared to ‘Penncross’. This is consistent with previous study which found that ‘Penn-A4’ had higher water use efficiency and root elongation and production than ‘Penncross’ under water stress (McCann and Huang, 2008). The present study indicated that the genotypic variation in drought tolerance for creeping bentgrass might be associated with the differential expression of certain stress-responsive proteins.

Effects of PEG-induced water stress and cultivar variation in protein abundance

Results and discussion Physiological responses to PEG-induced water stress Polyethylene glycol (PEG)-induced water stress resulted in shoot growth inhibition in both cultivars, and the growth inhibition was more severe in ‘Penncross’ than in ‘Penn-A4’, as shown in plant height (Figs. 1 and 2A). Under the control condition, no differences were detected in shoot growth rate between the two cultivars (Fig. 2A). Shoot growth was reduced by PEG-induced water stress, as demonstrated by the lower shoot extension rate of both cultivars during the experiment. ‘Penn-A4’ maintained a significantly higher shoot extension rate than ‘Penncross’ at 14 and 21 d of treatment (Fig. 2A). Both cultivars maintained leaf relative water content (RWC) at about 87% under well-watered control condi-

High-resolution difference gel electrophoresis was used to detect stress-responsive proteins in leaves of both cultivars. A representative gel image is shown in Fig. 3. Protein spots that were altered by PEG-induced water stress in either cultivar or differentially accumulated between the two cultivars under water stress were further analyzed. A total of 56 protein spots were altered by water stress (Table 1). In ‘Penn-A4’, the abundance of 14 spots (spots 13, 83, 85, 92, 96, 97, 105, 109, 111, 116, 122, 127, 145, 147) increased and that of 24 spots (spots 4, 9, 19–21, 42–44, 57, 59, 62–64, 71, 72, 75, 79, 90, 101–104, 107, 118) decreased (Fig. 4). In ‘Penncross’, the abundance of 15 spots (spots 13, 83, 85, 92, 97, 105, 111, 113, 116, 127, 133, 143, 145–147) increased while the abundance of 37 spots (spots 1–4, 9, 17, 19–21, 40, 42–44, 46, 57, 59, 62–65, 71, 72, 75, 76, 79–82, 90, 101–104, 107, 118, 139, 140)

Fig. 1. ‘Penn-A4’ plants (A and B) and ‘Penncross’ plants (C and D) after 28 d of treatment under well-watered control conditions (A and C) and PEG treatment (B and D).

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metabolism, energy, protein destination/storage, and cell structure categories (Fig. 5). The selected stress-responsive proteins are discussed below. Differential expression of proteins in photosynthesis between cultivars and affected by water stress Photosynthetic inhibition is one of the primary detrimental effects of water stress (Lawlor and Cornic, 2002; Kottapalli et al., 2009). Water deficit may limit photosynthesis through stomatal limitation and/or metabolic limitation (Flexas et al., 2004, 2006). PEG-induced water stress resulted in decline in the abundance of proteins involved in all three phases of the dark reaction of photosynthesis (carbon fixation, reduction and regeneration), including Rubisco large subunit (spot 17) and Rubisco activase (spot 40) in ‘Penncross’, and Rubisco large subunit (spots 19–21), Rubisco activase (spots 42, 43), chloroplastic aldolase (spots 62, 63) and chloroplastic glyceraldehydes-3-phosphate dehydrogenase (GAPDH; spot 57) in both ‘Penncross’ and ‘Penn-A4’. However, the extent of protein reduction by water stress, as indicated by percent change from the control in Table 1, were lower in ‘Penn-A4’ than in ‘Penncross’. The chloroplast GAPDH is a key enzyme that catalyzes the reduction of 3-phosphoglycerate to triose phosphate, a key step in photosynthesis linking the photochemical events of the thylakoid membranes with the carbon metabolism. Chloroplastic aldolase is an important enzyme in controlling the Ribulose 1,5-bisphosphate regeneration rate in photosynthesis (Iwaki et al.,

Fig. 2. Effects of water stress on shoot extension rate (A), leaf RWC (B) and electrolyte leakage (C) in ‘Penn-A4’ and ‘Penncross’. Each bar is the mean ± SE (n = 4) for each treatment. Values with the same letter were not significantly different at P < 0.05.

decreased under water stress (Fig. 4). The abundance of 11 spots (spots 13, 83, 85, 92, 97, 105, 111, 116, 127, 145, 147) increased under water stress in both cultivars, and that of 24 protein spots (spots 4, 9, 19–21, 42–44, 57, 59, 62–64, 71, 72, 75, 79, 90, 101–104, 107, 118) decreased under water stress in both cultivars (Fig. 4). Forty-six of these differentially accumulated protein spots were identified by mass spectrometry in a previous study with creeping bentgrass (Xu et al., 2010). Stress-responsive protein spots in both cultivars were grouped into different functional categories using the classification described by Bevan et al. (1998). Proteins with increased level of abundance under water stress belong to metabolism, energy, protein destination/storage, and disease/defense categories (Fig. 5). Proteins with decreased level of abundance were grouped into

Fig. 3. Analytical 2D DIGE gel image of ‘Penncross’ (A) and ‘Penn-A4’ (B). Cy5 image (Blue) of leaf samples from PEG treatment, Cy3 image (red) of leaf samples from the control, and Cy2 (yellow) image of leaf samples for pooled internal standards. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Table 1 Effects of PEG-induced water stress on leaf protein abundance in ‘Penn-A4’ and ‘Penncross’. SID

Protein name [accession number]

PEG effecta (% change in protein abundance from the control)

Differences between cultivars under PEG treatmentb (% difference in protein abundance for ‘Penn-A4’ from for ‘Penncross’)

Penn-A4

Penncross

Category 01: Metabolism 1 Glycine hydroxymethyltransferase (SHMT) [gi|31126793] 2 SHMT [gi|31126793] 3 Glycine decarboxylase P subunit [gi|710308] 4 Alanine aminotransferase [gi|16604499] 9 Methionine synthase [gi|50897038] 13 Beta-d-glucan exohydrolase [gi|20259685]

ns ns ns ↓ 20.44%* ↓ 33.29%* ↑ 35.66%*

↓ 28.45%** ↓ 31.92%*** ↓ 37.72%** ↓ 35.59%** ↓ 29.98%* ↑ 30.34%*

ns ns ns ns ns ↑ 43.84%**

Category 2: Energy 17 RuBisCO large subunit [gi|61378666] 19 RuBisCO large subunit [gi|61378680] 20 RuBisCO large subunit [gi|32966580] 21 RuBisCO large subunit [gi|4376205] 40 RuBisCO activase [gi|3914605] 42 RuBisCO activase [gi|12620881] 43 RuBisCO activase [gi|32481063] 44 Phosphoribulokinase/SBPase [gi|125578/gi|1173347] 46 Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) – cytosol [gi|120680] 57 GAPDH – chloroplast [gi|115458768] 59 Phosphoglycerate kinase [gi|129915] 62 Chloroplastic aldolase [gi|218155] 63 Chloroplastic aldolase [gi|218155] 64 Fructose-biphosphate (FBP) aldolase [gi|8272480] 65 FBP aldolase [gi|8272480] 71 Malate dehydrogenase [gi|48375044] 72 Class III Alcohol dehydrogenase [gi|1675394] 75 ATPase, beta subunit [gi|11583] 76 ATPase, beta subunit [gi|11583] 79 Enolase [gi|90110845] 80 Oxygen-evolving complex protein 1 [gi|739292] 81 Oxygen-evolving complex protein 1 [gi|739292] 82 Oxygen-evolving complex protein 1 [gi|739292] 90 Light-harvesting complex I; LHC I [gi|544700] 74 Cytochrome f [gi|57900550] 83 Oxygen-evolving enhancer (OEE) [gi|131394] 85 OEE2 [gi|131394] 92 Cytochrome b6-f complex iron-sulfur subunit [gi|68566191] 96 Carbonic anhydrase [gi|729003] 97 Carbonic anhydrase [gi|729003]

ns ↓ 28.95%* ↓ 44.00%** ↓ 44.96%* ns ↓ 23.62%* ↓ 27.04%** ↓ 20.56%* ns ↓ 44.05%** ↓ 37.97%** ↓ 16.85%* ↓ 33.31%** ↓ 26.08%** ns ↓ 23.01%* ↓ 10.94%* ↓ 39.27%*** ns ↓ 34.68%** ns ns ns ↓ 15.56%* ns ↑ 60.70%*** ↑ 119.21%* ↑ 37.64%** ↑ 67.90%** ↑ 91.36%**

↓ 61.61%** ↓ 53.11%* ↓ 57.30%** ↓ 46.26%* ↓ 54.38%* ↓ 54.93%** ↓ 53.48%** ↓ 51.13%** ↓ 19.11%* ↓ 55.39%** ↓ 56.57%** ↓ 46.92%* ↓ 44.31%** ↓ 34.74%** ↓ 35.35%** ↓ 35.17%** ↓ 19.31%* ↓ 66.32%** ↓ 58.21%** ↓ 61.94%** ↓ 41.56%* ↓ 48.63%* ↓ 53.09%* ↓ 24.73%* ↑ 21.27%** ↑ 49.77%** ↑ 76.36%** ↑ 89.95%*** ns ↑ 95.88%*

ns ns ns ns ns ↑ 23.08%* ns ns ns ns ↑ 61.12%* ns ns ns ns ns ns ↑ 43.30%* ns ↑ 35.23%* ns ns ↑ 35.60%* ns ns ↓ 10.90%* ↓ 75.29%* ↓ 29.03%*** ↓ 21.29%* ns

Category 6: Protein destination/storage 101 Heat shock protein 70 [gi|1143427] 102 70 kDa heat shock cognate protein 1 [gi|45331281] 103 60 kDa chaperonin subunit beta [gi|2493650] 104 FtsH-like protein Pftf precursor [gi|52075838] 105 Cyclophilin A-2 [gi|13925734]

↓ 16.25%** ↓ 24.12%** ↓ 19.35%** ↓ 21.49%* ↑ 153.50%***

↓ 45.26%** ↓ 59.20%*** ↓ 54.68%*** ↓ 56.85%*** ↑ 225.61%***

ns ↑ 40.96%* ↑ 51.71%** ns ns

Category 9: Cell structure 107 Actin [gi|9965319]

↓ 24.48%**

↓ 50.61%**

↑ 44.26%*

Category 11: Disease/defence 109 Catalase-1 [gi|2493543] 111 Catalase-1 [gi|2493543] 113 Ascorbate peroxidase (APX) [gi|15080682] 116 Glutathione-S-transferase [gi|6683765]

↑ 23.59%* ↑ 17.23%* ns ↑ 84.52%*

ns ↑ 14.20%* ↑ 142.48%** ↑ 73.57%**

ns ns ns ↓ 21.66%**

Category 12: Unclear classification 118 139 140 122 127 133 143 145 146 147

↓ 19.73%** ns ns ↑ 32.08%* ↑ 23.83%* ns ns ↑ 23.52%** ns ↑ 94.54%**

↓ 46.69%** ↓ 35.14%* ↓ 27.95%* ns ↑ 118.79%*** ↑ 111.40%*** ↑ 104.21%** ↑ 58.02%* ↑ 122.60%** ↑ 225.25%**

ns ns ns ns ↓ 16.70%** ↓ 25.11%* ns ↓ 26.43%* ns ns

ns: not significant. a Percent changes in protein abundance (↓ for decrease or ↑ for increase) in PEG treatment relative to the control treatment. b Percent changes in protein abundance (↓ for higher or ↑ for lower) in ‘Penn-A4’ relative to ‘Penncross’ under PEG treatment. * P ≤ 0.05. ** P ≤ 0.01. *** P ≤ 0.001

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carbon assimilation capacity.Oxygen evolving enhancer proteins (OEEs) consist of three subunits, OEE 1, OEE 2 and OEE 3. These are nuclear-encoded chloroplast proteins, and peripherally bound to photosystem II (PSII) on the lumenal side of the thylakoid membrane. In the present study, the abundance of OEE 2 (spots 83, 85) increased under water stress in both cultivars, and ‘Penncross’ had significantly higher OEE 2 than ‘Penn-A4’. Previous studies in other plant species also reported that the expression level of OEE was increased by drought stress (Gazanchian et al., 2007) and salinity stress (Murota et al., 1994; Abbasi and Komatsu, 2004; Xu et al., 2010). It is known that OEE 2 and OEE 3 can be easily removed from PSII complex under osmotic stress (Murota et al., 1994). The enhancement of abundance of OEE2 under water stress might be due to an acceleration of the dissociation of these proteins from the PSII complex, suggesting drought-induced damages in PSII system. Protein changes associated with respiration metabolism under water stress Fig. 4. Venn diagram illustrating the expression patterns of stress-responsive proteins in leaves of creeping bentgrass.

1991). Rubisco and Rubisco activase control carbon fixation. Downregulation of Rubisco activase protein and reduction in its enzyme activity has also been reported in other plant species (Costa et al., 1998; Lawlor and Cornic, 2002; Ingle et al., 2007; Kottapalli et al. 2009). The reduction of Rubisco large subunit (spots 19–21), Rubisco activase (spots 42, 43), chloroplastic aldolase (spots 62, 63) and chloroplastic GAPDH (spot 57) indicate that water stress may impose metabolic limitations for photosynthesis in creeping bentgrass through the abundance reduction of proteins catalyzing carbon fixation, reduction, and regeneration processes. Therefore, the maintenance of higher level of proteins involved all three phases of carbon assimilation in ‘Penn-A4’, in contrast to more severe down-regulation of these proteins in ‘Penncross’, may contribute to its superior stress resistance associated with greater

In both cultivars, PEG-induced water stress resulted in the reduction in the intensity of protein spots identified as phosphoglycerate kinase (spot 59), fructose-bisphosphate aldolase (spots 64, 65), malate dehydrogenase (spot 71), and enolase (spot 79) (Table 1). Phosphoglycerate kinase is a transferase enzyme which transfers a phosphate group from 1,3-biphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate. Malate dehydrogenase catalyzes the interconversion of malate and oxaloacetate. Enolase, also known as phosphopyruvate dehydratase, is a metalloenzyme responsible for the catalysis of 2-phosphoglycerate to phosphoenolpyruvate. All of these enzymes catalyze reactions in glycolysis or the citric acid cycle in respiration metabolism. Plomion et al. (2006) also reported that the abundance of proteins for respiration was reduced under drought stress. The decline in protein levels involved in respiration suggests that respiratory activity may be down-regulated for conservation of carbon in relation to reduced photosynthesis under drought stress (Huang and Fu, 2000).

Fig. 5. Functional classification of stress-responsive protein spots in each cultivar.

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The intensity of spot 46 was decreased only in ‘Penncross’ by 19.11% (Table 1). Spot 46 was identified as cytosolic GAPDH. The cytosolic GAPDH is involved in breaking down glucose and an important pathway for energy and carbon molecule supply. The abundance reduction of cytosolic GAPDH in ‘Penncross’ indicated glycolytic activities may be inhibited, which limits carbon supply for respiratory energy production in drought-sensitive plants. The lack of stress effects on cytosolic GAPDH in ‘Penn-A4’ suggests that tolerant cultivars could be better able to maintain glycolytic activity for continuing energy production through respiration. Water stress-induced decline in proteins involved in amino acid metabolism Water stress reduced the abundance of alanine aminotransferase (spot 4) and methionine synthase (spot 9) in ‘Penn-A4’ by 20.44% and 33.29%, and by 35.59% and 29.98% in ‘Penncross’, respectively (Table 1). The abundance of serine hydroxymethyltransferase (SHMT; spots 1, 2) and P protein of glycine decarboxylase (spot 3) was also decreased in ‘Penncross’. All these four proteins are involved in amino acid metabolism. The glycine decarboxylase catalyzes the degradation of glycine (Bourguignon et al., 1993). Methionine synthase catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in the formation of methionine, from which ethylene and polyamines are produced. SHMT catalyzes the interconversion of serine and glycine and is a key enzyme in the biosynthesis of purines, lipids, hormones and other compounds (Kopriva and Bauwe, 1995). Alanine aminotransferase is an enzyme involved in photorespiration catalyzing the interconversion of alanine and glycine. The abundance reduction of these proteins indicates that physiological damages in creeping bentgrass leaves resulted from water stress could be related to the inhibition of amino acid metabolism, and particularly the synthesis of glycine, methionine, alanine, and serine. However, none of these proteins was previously reported to be responsive to water stress in trees or other annual plants (Costa et al., 1998; Ndimba et al., 2005; Hajheidari et al., 2005, 2007; Ingle et al., 2007; Plomion et al., 2006; Xiao et al., 2009; Salekdeh et al., 2002; Kottapalli et al., 2009). This difference might indicate perennial grasses express different stress responses and defense mechanisms against long-term water stress than annual plants. Differential expression of proteins involved in cell wall loosening and cell growth ␤-d-glucan exohydrolase hydrolyzes ␤-glucan which can be found in plant cell walls. Cell wall proteins play important roles in various processes, including cell elongation (Zhu et al., 2006). Glucan exohydrolase can digest glucan in cell walls and might be involved in cell wall loosening and cell growth (Hrmova et al., 1996). Water stress resulted in an increase of the intensity of ␤-d-glucan exohydrolase (spot 13) by 30.34 and 35.66% in ‘Penncross’ and ‘Penn-A4’, respectively. ‘Penn-A4’ had higher intensity of this spot than ‘Penncross’. The higher accumulation of glucan exohydrolase in ‘Penn-A4’ may be associated with its maintenance of better leaf growth than for ‘Penncross’ under water stress (Fig. 1). The accumulation of actin was reduced by water stress in both cultivars. Similar results were reported in other plant species (Costa et al., 1998). Actin is a protein produced during cell division and elongation and is the principal component of microfilaments of the cytoskeleton. Actin participates in many important cellular functions, including cell division and cytokinesis, vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. Other stud-

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ies reported that tubulin, another cytoskelenton-related protein, was suppressed by osmotic stress in Arabidopsis (Creelman and Mullet, 1991; Bajaj et al., 1999; Plomion et al., 2006). The decrease in expression of actin may result from the decreased utilization of actin associated with growth inhibition and an autoregulatory feed-back system which down-regulates actin gene expression (Creelman and Mullet, 1991). In the present study, ‘Penn-A4’ had 44.26% higher actin level than ‘Penncross’ under water stress. Since one of the fundamental cellular adaptive strategies to osmotic changes involves cell size adjustments, these results suggest that the response of cytoskeleton-related proteins to osmotic stress may be associated with cellular adaptive strategies to osmotic changes involving cell size adjustments for cell turgor maintenance and cellular hydration. The higher actin level in ‘Penn-A4’ compared to ‘Penncross’ might be related to the maintenance of higher RWC under water stress. Increased abundance of proteins involved in antioxidant metabolism Drought stress often causes accumulation of reactive oxygen species such as singlet oxygen, superoxide radical, hydroxyl radical and hydrogen peroxide in plant cells (Apel and Hirt, 2004). Antioxidant enzymes, such as ascorbate peroxidase (APX), catalase, and glutathione-S-transferase (GST) may be activated to scavenge these toxic compounds during early phase of plant adaptation to drought stress (Zhang and Kirkham, 1994). In both Xerophyta viscose and Amaranthus hypochondriacus, the abundance of APX decreased under drought stress (Ingle et al., 2007; Huerta-Ocampo et al., 2009). However, APX was found to be differentially regulated during acclimation to decreasing water availability in Populus cathayana leaves (Xiao et al., 2009). GSTs are abundant proteins and have functions in the conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles. The increased expression of GSTs has been identified in several proteomics or transcription analyses of plants that were exposed to different stresses (Sappl et al., 2004; Smith et al., 2004; Roth et al., 2006; Gazanchian et al., 2007; Yang et al., 2007). Hajheidari et al. (2007) reported that drought stress increased GST in tolerant cultivar of wheat while decreased it in sensitive cultivar. In the present study, the intensity of spot 111 (catalase) and spot 116 (GST) was increased under water stress in both cultivars, and the intensity of spot 109 (catalase) was increased only in ‘Penn-A4’ while that of spot 113 (APX) was increased only in ‘Penncross’ under PEG-induced water stress. The increased accumulation of APX, catalase and GST in this study indicated that water stress-induced oxidative stress in creeping bentgrass and different antioxidant enzymes could be involved for antioxidant defense against the oxidative stress in cultivars differing in stress resistance. Changes in heat shock protein (HSP) and related proteins Heat shock proteins (HSPs) have chaperone functions in preventing aggregation of incorrectly folded proteins, and facilitating correct folding of proteins. Some studies reported increases in HSP abundance under different stresses (Georgopoulos and Welch, 1993; Wang et al., 2004). However, in this study the abundance of some HSPs (spots 101–103) were decreased under water stress in both cultivars, but ‘Penn-A4’ had higher level of 70 kDa heat shock cognate (HSC 70) (spot 102) and 60 kDa chaperonin (spot 103) than ‘Penncross’ by 40.96% and 51.71%, respectively. HSP 70 has essential functions in preventing aggregation and in assisting refolding of non-native proteins (Hartl, 1996; Frydman, 2001; Wang et al., 2004). They are also involved in protein import and translocation processes, and in facilitating the proteolytic degradation of unstable proteins (Hartl, 1996). Some family members of

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HSP 70 are constitutively expressed and are often referred to as HSC 70. These members are often involved in assisting the folding of de novo synthesized polypeptides and the import/translocation of precursor proteins (Zhang and Glaser, 2002; Wang et al., 2004). The reduction in abundance of HSC 70 under water stress suggests a decrease in the transportation of newly synthesized peptides into mitochondria, which could be due partly to decreased de novo protein synthesis under stress conditions (Ndimba et al., 2005; Jiang et al., 2007). FtsH proteins constitute a small family of membrane-bound zinc metalloproteases containing an ATPase domain. In Arabidopsis, two of the nine chloroplast-targeted FtsH proteases have been shown to play a role in the repair of PSII after oxidative damage (Bailey et al., 2002; Sakamoto et al., 2003). Other studies have suggested that the FtsH protease complex may also degrade an unassembled Rieske Fe–S thylakoid protein and D1 proteins of PSII (Adam, 2000; Sinvany-Villalobo et al., 2004). In the present study, the accumulation of FtsH-like protein (spot 104) was reduced by water stress in ‘Penn-A4’ and ‘Penncross’, but the reduction in protein abundance was less severe in the former cultivar (by 21.49%) than the latter (by 56.85%). The decreased abundance of FtsH protease under water stress suggests that drought-sensitive plants may have reduced ability to repair damage to the D1 polypeptide during stress. Cyclophilin catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and accelerates the folding of proteins. Besides its protein folding function, it also plays a role in signal transduction and thus may be crucial for stress responsiveness (Romano et al., 2004). Cyclophilins were induced during water stress in sugarbeet (Beta vulgaris L.) (Hajheidari et al., 2005). Its abundance increased in susceptible genotype and remained unchanged in tolerant genotype of peanut (Arachis hypogaea) (Kottapalli et al., 2009). However, the cyclophilin-like peptidyl-prolyl cis-trans isomerase was reduced during dehydration in the tolerant cultivar of chickpea (Cicer arietinum) (Bhushan et al., 2007). The increased accumulation of cyclophilin under salinity stress was reported in creeping bentgrass (Xu et al., 2010). In this study, the abundance of cyclophilin A-2 (spot 105) increased under water stress in both cultivars, and there was no difference in its abundance between two cultivars. Under water stress, cyclophilin may facilitate correct folding of proteins and avoid aggregation of incorrectly folded proteins. It might also be involved in signal transduction and thus play a crucial role in stress responsiveness (Kottapalli et al., 2009). In conclusion, PEG-induced water stress caused abundance reduction of some proteins involved in amino acid synthesis, photosynthesis and respiration in both creeping bentgrass cultivars, which could be associated with the growth inhibition. The extent of protein reduction by water stress was lower in ‘Penn-A4’ than that in ‘Penncross’, including Rubisco large subunit, Rubisco activase, chloroplastic aldolase and chloroplastic GAPDH, suggesting that stress-tolerant ‘Penn-A4’ was better able to maintain the metabolism of proteins involved in carbon fixation, reduction, and regeneration in photosynthesis. The lack of PEG effects on cytosolic GAPDH in ‘Penn-A4’, relative to severe reduction in ‘Penncross’, suggests that tolerant cultivar could be better able to maintain glycolytic activity for continuing energy production through respiration. Water stress also caused increased accumulation of APX, catalase and GST, indicating that oxidative stress could be induced in creeping bentgrass, and increases in the accumulation of antioxidant proteins could be involved for antioxidant defense against the oxidative stress induced by water stress. It is worth noting that the functions of some of these differentially expressed proteins and direct involvement in stress tolerance are not clearly understood, which warrants further investigation for revealing the underlying molecular and metabolic pathways.

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