Biochemical and Proteomic Analyses Reveal that Populus cathayana

Article pubs.acs.org/jpr

Biochemical and Proteomic Analyses Reveal that Populus cathayana Males and Females Have Different Metabolic Activities under Chilling Stress Sheng Zhang,† Lihua Feng,† Hao Jiang,† Wujun Ma,‡ Helena Korpelainen,§ and Chunyang Li*,† †

Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, Chengdu 610041, China ‡ Department of Agriculture and Food Western Australia, and Centre for Comparative Genomics, Murdoch University, South Perth, WA 6150, Australia § Department of Agricultural Sciences, University of Helsinki, P.O. Box 27, FI-00014, Finland S Supporting Information *

ABSTRACT: Male and female poplars (Populus cathayana Rehd.) respond differently to environmental stresses. However, little is known about sex-dependent responses to chilling at the proteome level. To better understand these differences, a comparative proteomics investigation combined with a biochemical approach was used in the current study. Three-month-old poplar cuttings were treated at 25 or 4 °C for 14 days. Results revealed significant sexual differences in nitrogen metabolic enzymes and free amino acid components in response to chilling. The chillingtreated males showed higher activities of nitrate reductase and glutamine synthetase and higher contents of reduced glutathione, serine, arginine, leucine, glycine, proline and methionine than chilling-treated females. A total of 65 chillingresponsive spots were found, of which 48 showed significant sexual differences. These proteins are involved in photosynthesis, carbon and energy metabolism, metabolic processes of proteins, lipid metabolism, vitamin metabolism, stress defense, and gene expression regulation. The study shows that males have more effective metabolic processes and protective systems to chilling than females.

KEYWORDS: plant proteome, chilling, sexual difference, poplar, metabolism

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INTRODUCTION Plants are anchored in soil and are not able to escape unfavorable environmental conditions like animals. To cope with various environmental stresses, plants have evolved a series of adaptive mechanisms during evolution. A low temperature stress is one of the common environmental stresses that plants are constantly facing during their life cycles, especially in the higher altitude mountain areas. In plants adapted to a low temperature environment, the exposure to chilling (0−15 °C) induces gene expression and metabolic changes that allow them to withstand cold stress and increase resistance to further freezing temperatures.1−4 Proteomics research bridges the gap between gene expression and metabolism,5 making it an appropriate solution to increase understanding of plants’ adaptive mechanisms to chilling stress and to develop strategies to improve resistance. © 2012 American Chemical Society

Most chilling related proteomics studies have been performed with herbaceous plants.6−10 Proteomics studies on woody plants exposed to chilling are seldom reported. Recently, several physiological and metabolic studies on poplars in relation to chilling stress have been reported.1,2,11,12 In the proteomics area, Renaut et al.13 performed a study on poplars exposed to 4 °C and found 60 differential protein spots with 26 being successfully identified. The low protein identification rate was due to the fact that the Populus genome had not been sequenced at that time. The complete genome sequence of P. trichocarpa is now available, which facilitates in-depth proteomic studies on this species. Received: July 2, 2012 Published: October 17, 2012 5815

dx.doi.org/10.1021/pr3005953 | J. Proteome Res. 2012, 11, 5815−5826

Journal of Proteome Research

Article

Populus cathayana Rehd. is a dioecious, fast-growing tree species, widely distributed in the northern, central and southwestern regions of China. The altitude of distribution is around 800−3200 m. Our previous studies have shown that P. cathayana males and females demonstrate different adaptability to a series of abiotic stresses at physiological and proteome levels.14−18 Importantly, we found that sexually different responses at the physiological level were significant under chilling: P. cathayana males possessed higher total chlorophyll and soluble sugar contents and higher antioxidant enzyme activities than did females.19 In order to further understand the molecular mechanism of sexual differences in chilling responses, a combination of proteomic and biochemical approaches were utilized in this study. Our objectives are to (1) evaluate the sexdependent changes in the leaf proteome under chilling stress and (2) understand the sex-dependent metabolic responses.

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high performance liquid chromatography with precolum derivatization. Standard amino acids or samples (200 μL) were mixed with derivatizational solution (200 μL norleucine, 100 μL triethylamine and 100 μL phenylisothiocyanate) and kept at room temperature for 1 h. Then, 400 μL of n-hexane was added and the solution was used for the determination of amino acid compounds. Norleucine was used as an internal standard and three replicates were included. The detailed determination was carried out according to the manufacturer’s manual. Activity Assays of Nitrogen Metabolism Related Enzymes

Nitrate reductase (NR) activity was measured in vitro according to Scheible21 with some modification. Briefly, a total of 0.5 g leaf tissue was homogenized with ice-cold extraction buffer [0.1 M Tris-HCl (pH 8.0), 2 mM MgSO4.7H2O, 5 mM EDTA, 2 mM DTT, 1% polyvinylpyrrolidone (PVP), 2 mM monosodium glutamate and 2 mM cysteine]. The homogenate was centrifuged at 12 000× g for 10 min. Then, 0.2 mL of extract was mixed with 1 mL of assay buffer [0.1 M Tris-HCl (pH 8.0), 5 mM KNO3, 0.25 mM NADH] and kept at 25 °C for 30 min. The reaction was stopped by adding 0.5 mL 1% (w/v) hydroxylamine hydrochloride, and the color was developed by adding 0.5 mL 0.2% (w/v) of α-naphthylamine. Tubes were allowed to stand for 20 min at room temperature, and then measurements were conducted at 540 nm using a spectrophotometer. A standard curve in the range of 0−0.2 μg mL−1 NO2− was used. Glutamine synthetase (GS) activity was determined according to Häusler22 with minor modification. Briefly, 0.5 g of leaf tissues was homogenized in ice-cold extraction buffer [0.2 M phosphate buffer (pH 7.2), 0.5 mM EDTA, 50 mM K2SO4]. After centrifugation at 12 000× g for 10 min, 0.5 mL of extract was added into assay mixture [0.6 mL of 0.25 M Tris-HCl (pH 7.0), 0.4 mL of 0.3 M monosodium glutamate, 0.4 mL of 30 mM ATP and 0.2 mL of 0.5 M MgSO4]. Then, 0.2 mL of 0.5 M hydroxylamine hydrochloride was added to start the reaction, which continued for 20 min at 25 °C. The reaction was stopped by adding 1.0 mL of ferric-chloride reagent [20% (w/v) TCA, 3.5% (w/v) FeCl3 and 2% HCl (v/v)]. After centrifugation at 10 000× g for 5 min, the absorbance was measured at 540 nm. A standard curve in the range of 0−5 μM γ-glutamylhydroxamate was used. Glutamate dehydrogenase (GDH) activity was determined according to Loulakakis and Roubelakis-Angelakis23 with some modification. Briefly, 1.0 g of leaf tissues were ground in icecold extraction buffer containing 0.1 M Tris-HCl (pH 7.5), 5 mM MgCl2, 1.0 mM EDTA, 1.0 mM EGTA, 10% (v/v) glycerol and 5 mM DTT. The homogenates were centrifuged at 12 000× g twice for 20 min. The deaminating (NAD-GDH) GDH activities were determined at 340 nm. One unit of GDH activity was defined as the reduction or oxidation of 1 mM of NAD per min at 30 °C.

MATERIALS AND METHODS

Plant Materials and Experimental Design

The experimental material and design are similar as described in a previous report.20 Briefly, the study included 40 males and 40 females, which originated from 20 F1 individuals derived from a controlled intraspecific cross between two P. cathayana genotypes with divergent phenotypes. Cuttings were planted in 10-L plastic pots filled with 8 kg homogenized soil and 8 g slow release fertilizer (13% N, 10% P and 14% K) in a greenhouse. After the plantlets were about 50 cm high, they were moved to a phytotron. Two temperature regimes, 25 and 4 °C, were used with 25 °C being the control and 4 °C the chilling condition. The light intensity was 120 μmol m−2 s−1 (12 h light and 12 h darkness), the relative air humidity was 80% and CO 2 concentration was 400 ± 10 μmol mol−1. Five replicates were used in each treatment with each replicate consisting of four cuttings of each sex. After 14 days of treatment, the fourth and fifth fully expanded leaves were collected for further analyses. Chlorophyll Fluorescence Measurements

Five cuttings (the fourth fully expanded leaves) from each treatment were randomly taken for chlorophyll fluorescence measurements. Leaf samples were placed in dark for 30 min using an aluminum foil cover, and the minimal fluorescence yield (Fo) and the maximal fluorescence yield (Fm) were measured. The leaves were then exposed to sunlight for 15 min, followed by measurements of fluorescence yields, including minimal fluorescence (Fo′) and maximal fluorescence (Fm′), under actinic light with a saturating white light pulse of 8 000 μmol m−2 s−1 applied for 0.8 s. The measurements were carried out in the morning from 08:00 to 09:30. Chlorophyll fluorescence kinetics parameters (Fv/Fm, variable and maximum fluorescence; yield, the effective quantum yield of PSII; qN, nonphotochemical quenching coefficient; and qP, photochemical quenching coefficient) were calculated according to Kooten and Snel20 with a PAM chlorophyll fluorometer (PAM 2100, Walz, Effeltrich, Germany).

Glutathione Content Measurements

Extraction and Determination of Free Amino Acid Compounds

Reduced glutathione (GSH) was assayed according to Guri24 with minor modifications. Briefly, a total of 0.3 g of leaf tissue was homogenized in ice-cold 5% TCA (containing 5 mM EDTA). The homogenate was centrifuged at 10 000× g for 10 min. The reaction mixture contained 0.5 mL of distilled water, 1.0 mL of leaf homogenate, 1.0 mL of 0.2 M potassium phosphate buffer (pH 7.5), and 0.5 mL of the reagent dithiobis2-nitrogenzoic acid (DTNB). GSH was determined at 412 nm

Soluble free amino acids were extracted from 1 g of dried leaf using an ultrasonic extractor. The leaf sample was mixed with 5 mL methanol and 1 mL 0.1 M HCl, and shook for 30 min at 30 °C. After centrifugation, 0.5 mL of supernatant was filtered using a 0.45 μm membrane. The determination of amino acid compounds used Venusil-AA (Angela, USA) inreversed-phase 5816



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°C for 45 min, and dehydrated with 100% ACN. After drying, the gels were incubated in 10−20 mL trypsin solution for 1 h. Then, 15 mL digestion buffer was added (40 mM NH4HCO3, 10% ACN) to cover each gel and incubated overnight at 37 °C. Tryptic digests were extracted using Milli-Q water, followed by double extraction with 50% ACN and 5% TFA for 1 h. The combined extracts were dried in a vacuum concentrator at room temperature. The samples were then subjected to mass spectrometry analysis. Mass spectra were acquired using a Q-TOF mass spectrometer (Micromass, Manchester, UK) fitted with an ESI source (Waters). Tryptic digests were dissolved in 18 μL of 50% ACN. MS/MS was performed in the data-dependent mode, in which the top 10 most abundant ions for each MS scan were selected for MS/MS analysis. Trypsin autolysis products and keratin-derived precursor ions were automatically excluded. The MS/MS data were acquired and processed using MassLynx software (Micromass), and MASCOT (version 2.1, http://www.matrixscience.com/) was used to search the NCBInr (March 2012 release; including 14 683 223 sequences and 5 021 252 203 residues) and SwissProt (March 2012 release; including 530 264 sequences and 187 941 074 residues) databases for protein identification. Database searches were carried out using the following parameters: taxonomy, Viridiplantae (green plant); enzyme, trypsin; peptide mass tolerance, ±1.2 Da; fragment mass tolerance, ±0.6 Da; allowed, one missed cleavage. Fixed modifications of cysteine carbamidomethylation and variable modifications of methionine oxidation were allowed. Because there is no Populus in MASCOT, green plant was used for searching. The data format was selected as micromass peak list, and the instrument was selected as ESI-QUAD-TOF. Proteins with at least two peptides exceeding the score threshold (P < 0.05), which indicates identification at the 95% confidence level for the matched peptides, were identified. For proteins identified by a single peptide and with a score higher than 40 (those with lower ones were discarded), the spectra were manually inspected using the following criteria: (i) the MS/MS spectra must be of good quality with fragment ions clearly above baseline noise; (ii) there should be a continuous stretch of the peptide sequence covered by either the y- or the b-series ions. In the case of peptides that matched multiple members of a protein family in different species, the one with the highest score was reported unless the species was a poplar. Functional categorization of proteins was carried out according to the Gene Ontology (GO) rules using GO three independent sets of ontologies that were used to describe a gene product, including GO Molecular Function, GO Biological Process, and GO Cellular Component. All identified proteins were further checked against the Populus proteome by submitting the peptide sequences to BLASTP (http://www.phytozome.net/) against the Populus proteome database v2.0 (45 033 sequences and 17 235 199 total letters). Those proteins that were identified and positive in the Populus proteome database (identity above 50%) were accepted.

using a spectrophotometer. A standard curve in the range of 0− 100 μM GSH was used. Protein Extraction and 2-DE

Leaf proteins from the fourth and fifth leaves were extracted using a TCA/acetone procedure.17 One gram of frozen leaf material was ground in liquid nitrogen and suspended in 20 mL of prechilled acetone containing 10% (w/v) TCA and 0.07% (w/v) DTT and kept overnight at −20 °C. The suspension was centrifuged at 15 000× g for 10 min at 4 °C and washed twice with prechilled acetone containing 0.07% DTT. The obtained powder was dried using a vacuum pump and resuspended in solubilization buffer [8 M urea, 4% (w/v) CHAPS, 2% (v/v) IPG buffer pH 4−7 (GE Healthcare, UK), 40 mM DTT]. The protein concentration was determined using a 2-D Quant Kit (GE Healthcare; Little Chalfont, UK) with BSA (1 mg−1 mL−1) as a standard. Three hundred micrograms of total protein was loaded onto each IPG-strip. IEF was conducted using the IPGphor II system (GE Healthcare). The linear pH 4−7 IPG strips (GE Healthcare) were rehydrated in 450 mL rehydration protein solution at 20 °C for 12 h (6 h for rehydration and 6 h for focusing at 30 V). IEF was subsequently run for 1 h at 500 V, 1 h at 1000 V, then a gradient was applied from 1000 to 8000 V for 1 h and a final focusing was conducted at 8000 V for a total of 72 000 Vh. After IEF, the IPG strips were equilibrated twice in the equilibration buffer supplemented (i) with 1% DTT for 15 min and then (ii) with 2.5% iodoacetamide for 15 min. The second dimension electrophoresis was carried out on 20 × 18.5 × 0.1 cm, 12.5% (w/v) acrylamide gels run at 2 W per gel for 45 min, followed by 17 W per gel for about 5 h. After SDS-PAGE, the gels were visualized by silver staining without glutaraldehyde. Three biological replicates (three individual cuttings) were used for each treatment and sex, and three gels were processed for each sample. Image Acquisition and Data Analysis

Images of silver-stained gels were acquired by Image Scanner (Amersham Biosciences, Uppsala, Sweden) in a transmission mode. The 2-DE gels were analyzed using the Melanie software version 6.0 (GeneBio, Geneva, Switzerland). After automated detection and matching, manual editing was carried out to correct the mismatched and unmatched spots. Spots that could not be reliably validated as true matches were disregarded. Three well separated gels of each treatment and sex were used to create “replicate groups”. Spots were considered reproducible when they were well resolved in the three biological replicates. For each matched spot, a measurement was carried out for each biological replicate, and normalized volumes were computed using the total spot volume normalization procedure of the Melanie software version 6.0. The normalized volume of each spot was assumed to represent its expression abundance. The Tukey test was performed to compare data from the four treatments. Only the spots that showed the abundance ratio over 1.5 folds and at least one factor (sex, temperature or their interaction) being significant (P < 0.05) based on Tukey test were selected for MS analysis.

Statistical Analysis

In-gel Digestion and ESI-Q-TOF MS/MS

The effects of chilling, sex and their interaction were analyzed by analysis of variance (ANOVA) for randomized completeblock design using SPSS 16.0 (SPSS, Chicago, IL). Before ANOVA, data were checked for the normality and homogeneity of variances. Posthoc comparisons were tested using the Tukey test at a significance level of P < 0.05. Mean values and

In-gel digestion of proteins was carried out using MS-grade Trypsin Gold (Promega, Madison, WI) according to the manufacturer’s instructions in the Sichuan University, China. Briefly, spots cut out of the gel were destained twice with 50 mM NH4HCO3 buffer (pH 8.8, containing 50% ACN) at 37 5817



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Compared to females, chilling-stressed males showed higher NR and GS activities and a GSH content but a lower GDH activity.

standard errors were determined for each variable. We used the hierarchical clustering software PermutMatrix v1.9.3 (http:// www.lirmm.fr/∼caraux/PermutMatrix/)25 to cluster the sexspecific proteins (Fs < 0.05 and Fs×t < 0.05). The Ward’s algorithm was used.

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Sexual Differences in Proteome

One master 2-D gel profile (25 °C of males) is shown in Figure 4, while all other gels are available through Supporting Information 1. Out of more than 700 reliably matched spots on all gels, 65 spots showed the abundance ratio over 1.5 folds and at least one factor (sex, temperature or their interaction) being significant. The coefficients of variation of these different spots in each treatment were 31.45% (control males), 32.84% (4 °C of males), 35.79% (control females) and 36.08% (4 °C of females). Among the 65 different spots, 53 spots displayed a significant temperature effect (T; Pt < 0.05), while sex (S; Ps < 0.05) and sex by temperature interaction (S×T; Ps×t < 0.05) effects accounted for 32 and 31 spots, respectively (Figure 5). Figure 6 shows a close-up view of some differential protein spots. Compared with controls, chilling caused a significant upaccumulation of 27 spots in males and 25 spots in females, and 17 spots were down-accumulated in males and 10 spots in females. Among these changed spots, 5 spots (spots 25, 27, 29, 31 and 56) and 4 spots (spots 7, 24, 27 and 31) were NEW PRESENT to the chilling-stressed males and females, respectively. Spots 17 and 39 were absent in the chillingstressed males, while spots 3 and 17 were absent in the chillingstressed females. Compared to females, 26 spots showed a higher abundance and 18 spots showed a lower abundance in chilling-stressed males than in chilling-stressed females. Interestingly, there were 4 spots (spots 12, 35, 51 and 56) unique to the chilling-stressed males and 4 spots (spots 4, 13, 20 and 24) were detected only in chilling-stressed females (Table 2 and Supporting Information 2).

RESULTS

Sexual Differences in Physiological and Biochemical Changes

In order to estimate the plant’s condition after stress, chlorophyll fluorescence parameters were measured. Results showed that chilling significantly decreased Fv/Fm, yield and qP in both sexes, and chilling-stressed males showed higher Fv/Fm and yield than chilling-stressed females, while there was no significant sexual difference at 25 °C. Additionally, chilling decreased qN in females but not in males (Figure 1).

Figure 1. Chlorophyll fluorescence parameters of P. cathayana males and females at 25 °C (white bars) and 4 °C (gray bars). (A) Fv/Fm, the maximum efficiency of PS II; (B) yield, the effective quantum yield of PS II; (C) qP, photochemical quenching coefficient; (D) qN, nonphotochemical quenching coefficient. Ps, sex effect; Pt, temperature effect; Ps×t, sex and temperature interaction effect. Different letters above bars indicate statistically significant differences between treatments at P < 0.05 (n = 5).

Identification and Functional Classification of Differentially Expressed Proteins

The 65 different expressed spots were successfully identified by ESI-Q-TOF MS/MS (Table 2 and Supporting Information 2 and 3). To obtain functional information for the proteins annotated either as unknown or hypothetical proteins, we searched databases with BLASTP (www.ncbi.nlm.nih.gov/ BLAST/) for their homologues using their protein sequences as queries. Except for spots 63 and 64, four proteins (spots 11, 21, 25 and 52) shared positive identity of more than 70% with their homologues at the amino acid level (Supporting Information 4). Based on the functional features, all identified proteins were classified into six major categories, including photosynthetic proteins, metabolic proteins, stress and defense response proteins, gene expression regulation, basic biosynthetic process and others (Table 2). The largest functional category was metabolism related proteins, including carbon and energy metabolism, lipid metabolism, protein metabolism, vitamin metabolism and secondary metabolism. The numbers of chilling-responsive proteins in various functional categories were different between male and female poplars. Additionally, 48 sex-specific proteins (Ps < 0.05 or Ps×t < 0.05) were subjected to a hierarchical clustering (Figure 7). The clustering analysis gathered proteins into four distinct subtrees according to their abundance changes.

Using the Venusil-AA amino acid system, 17 types of free amino acids were measured but histidine (His), tyrosine (Tyr) and vlaine (Val) were not detected in poplar leaves in this study. The total free amino acid content was higher in females than in males, and chilling caused a significant increase in both sexes (Table 1). Methionine (Met) and isoleucine (Ile) increased significantly but threonin (Thr) decreased significantly in both chilling-stressed sexes. Glycine (Gly) and proline (Pro) increased significantly only in chilling-stressed males and showed higher contents than those in chilling-stressed females. At 25 and 4 °C, males had higher contents of serine (Ser), arginine (Arg) and leucine (Leu) but a lower content of asparagic acid (Asp) than did females (Figure 2). At 25 °C, males showed a higher GS activity but lower NR activity and GSH content than did females, but no significant sexual difference was detected in the GDH activity (Figure 3). Chilling significantly increased the NR activity and GSH content in males but decreased them in females. The GDH activity decreased under chilling in males but not in females. 5818



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Table 1. Contents of Free Amino Acid Components in Male and Female P. cathayana Leaves at 25 and 4 °Ca component (μg mL−1) Asp Glu Ser Gly Arg Thr Ala Pro Met Cys Ile Leu Phe Lys Total

M25 33.56 96.63 8.43 15.85 11.41 50.57 19.70 2.92 15.93 34.77 12.66 32.44 9.89 3.45 348.21

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.81a 2.51a 0.09b 1.11a 0.78b 1.66c 1.20a 0.45a 0.89b 1.27ab 0.84a 1.57b 1.24a 0.25a 6.32a

M4 24.58 99.45 8.17 40.00 12.38 6.36 17.57 7.68 69.09 23.29 45.11 37.19 9.39 2.40 402.66

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

F25 1.41a 4.05a 0.09b 2.57b 0.88b 0.50a 1.16a 0.45b 1.89c 1.59a 2.20b 2.44b 0.94a 0.63a 9.46b

286.40 100.25 2.85.26 15.32 5.31 38.82 17.80 2.50 2.46 40.42 17.53 1.71 10.51 4.81 543.84

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

F4 8.15b 4.56a 0.26a 1.35a 0.45a 2.35b 0.82a 0.26a 0.28a 3.77b 1.04a 0.52a 1.09a 0.78a 7.25c

308.12 109.21 3.21 22.26 6.64 4.78 17.05 3.64 19.79 31.01 52.36 0.64 9.70 3.13 591.54

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

9.51b 1.60a 0.14a 0.74a 0.51a 0.53a 0.57a 0.47a 1.47b 3.09ab 3.36b 0.11a 1.19a 0.39a 15.15d

Ps

Pt

Ps×t

0.000 0.084 0.000 0.001 0.000 0.002 0.247 0.001 0.000 0.035 0.014 0.000 0.687 0.096 0.000

0.343 0.121 0.758 0.000 0.129 0.000 0.175 0.000 0.000 0.004 0.000 0.247 0.577 0.038 0.001

0.043 0.392 0.086 0.001 0.792 0.009 0.495 0.002 0.000 0.705 0.556 0.084 0.892 0.581 0.751

Each value is the mean ± SE (n = 3). M25, 25 °C of males; M4, 4 °C of males; F25, 25 °C of females; F4, 4 °C of females; Ps, sex effect; Pt, temperature effect; Ps×t, sex and temperature interaction effect; Asp, aspartic acid; Glu, glutamic acid; Ser, serine; Gly, glycine; Arg, arginine; Thr, threonine; Ala, alanine; Pro, proline; Met, methionine; Cys, cystine; Ile, isoleucine; Leu, leucine; Phe, phenylalanine; Lys, lysine. a

Figure 2. Components of free amino acids in male and female P. cathayana leaves at 25 and 4 °C. (A) Males at 25 °C; (B) males at 4 °C; (C) females at 25 °C; (D) females at 4 °C.

Sexual Differences in Free Amino Acids and Nitrogen Metabolic Enzymes

Figure 3. Activities of (A) nitrate reductase, (B) glutamine synthetase, and (C) glutamate dehydrogenase and (D) glutathione content in male and female P. cathayana leaves at 25 °C (white bars) and 4 °C (gray bars). Ps, sex effect; Pt, temperature effect; Ps×t, sex and temperature interaction effect. Different letters above bars indicate statistically significant differences between treatments at P < 0.05 (n = 5).

In plants, chilling induces a series of physiological and biochemical changes, and these changes are clone- and sexdependend.12,26−28 A previous study has shown that chilling causes different physiological responses in P. cathayana males and females.19 Similarly as in that study, we investigated here all free amino acids and nitrogen metabolic enzymes. Apart from Pro, we found that Asp, Arg, Ser, Gly, Met and Leu showed significant sexual differences (Figure 2). Especially the Asp content was markedly higher in females than in males, leading to a higher total free amino acid content in females. Interestingly, six amino acids (Ser, Gly, Arg, Pro, Met and Leu) showed higher contents in the chilling-stressed males than in the chilling-stressed females, demonstrating that sexual differences in responses to chilling were not only expressed in amino acid contents but also in types. The sexual difference in free amino acids may contribute to different nitrogen metabolism.

NR and GS are key enzymes involved in nitrogen metabolism. It is reported that a low temperature causes an increased NR activity in some plants29−31 but a decrease in other plants.32−34 Such inconsistent results may be due to differences in responses among plant species. In this study, we found that different genders of P. cathayana also exhibited a difference in the NR activity and GSH content: increases in males and decreases in females at 4 °C. NR catalyzes the reduction of nitrate to nitrite and plays a key role in the regulation of nitrate assimilation and production of nitric oxide (NO) in higher plants.15,35 NR and GSH can react nonenzymatically to form S-nitrosoglutatione, which is a longdistance signal molecule that can operate under certain stress conditions.36−38 Although chilling did not cause a statistically significant change in the GS activity, it was always higher in males than in females. Therefore, the higher activities of NR

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DISCUSSION

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carbon and energy metabolisms between the two sexes under chilling stress conditions. Sexual Differences in Proteome

Differently Expressed Photosynthetic Proteins. Chilling significantly decreases the photosynthetic rate of many plants. The disassembly of RuBisCO might be the main cause of photosynthetic rate reduction under chilling conditions.6,43 It is reported that subtilisin-like proteases (subtilases) are not only associated with programmed cell death in plants but they also play an important role in the proteolytic degradation of RuBisCO under biotic and abiotic stresses.44,45 In this study, we found that the RuBisCO large subunit (spot 1) and glycosyltransferase (spot 8) decreased in abundance, while the presence of subtilases (spot 27) was new in chilling-stressed individuals. The glycosyltransferase family protein has an essential function in releasing inhibitory sugar phosphates from the active site of RuBisCO, allowing continuous CO2 fixation.46 Thus, the presence of subtilases may accelerate the degradation of RuBisCO, while the decrease of glycosyltransferase may affect the RuBisCO activity under chilling conditions. More interestingly, chilling-stressed males exhibited a higher abundance of the transient complex of poplar plastocyanin with turnip cytochrome F (Pc-Cyt f, spot 6) than chilling-stressed females. Plastocyanin (Pc) functions as an electron carrier between PS I and the Cyt f complex.47 The down-regulation of Pc-Cyt f indicated that the electron transport between PS I and the Cyt f is affected by chilling and this is more obvious in females than in males. In addition, based on the analysis of chlorophyll fluorescence parameters, we found that chilling causes a disorder in PS II. However, the higher Fv/Fm and yield values in chilling-stressed males are attributable to the occurrence of less photodamage to PS II reaction centers in males than in females at 4 °C. These different responses in PS I and PS II might lead to a difference in photosynthesis between sexes under chilling stress. Differently Expressed Metabolic Proteins. Sexually different chilling-responsive proteins in metabolic process are involved in carbon and energy metabolism, metabolic processes of proteins, and vitamin and secondary metabolism. In the current study, cytoplasmic and cytosolic malate dehydrogenases (MDH, spots 9 and 10) always showed higher abundances in males than in females, although both sexes showed a general trend of decrease after chilling. Previous evidence showed that cytoplasmic and cytosolic MDH plays a central role in plant nutrition and tolerance to cold stress.48,49 The succinate dehydrogenase [ubiquinone] flavoprotein subunit (SDH, spot 14), which is involved in complex II of the mitochondrial electron transport chain and is responsible for transferring electrons from succinate to ubiquinone, significantly increased in its abundance only in chilling-stressed females. Both MDH and SDH are involved in the citric acid cycle. These results demonstrate a significant sexual difference in the citric acid cycle in response to chilling stress. The citric acid cycle is a key component of the metabolic pathway to generate energy through the oxidization of acetate derived from carbohydrates and proteins into carbon dioxide and water. In this study, some proteins related to glycolysis, starch and protein metabolism showed significant sexual differences in responses to chilling. Two proteins related to glycolysis increased in abundance under chilling stress, of which 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAMi, spot 11) increased in males and cytosolic

Figure 4. Reference 2-D map of P. cathayana leaves (25 °C of males). The 65 spots submitted to MS/MS analysis are marked and numbered.

Figure 5. Venn diagram showing the total number of spots displaying at least one significant effect based on the two-way ANOVA. S, sex effect; T, temperature effect; S×T, sex and temperature interaction effect.

Figure 6. Close-up views of differently expressed proteins in the treatments. M25, 25 °C of males; M4, 4 °C of males; F25, 25 °C of females; F4, 4 °C of females.

and GS and the higher content of GSH in chilling-stressed males clearly indicate that P. cathayana males possess a higher ability of nitrogen and ammonia assimilation than do females under chilling. Additionally, we found that the GDH activity decreased only in chilling-stressed males. Although the exact physiological role of GDH in plant metabolism remains speculative,39 the evidence suggest that GDH functions to funnel the carbon skeletons of glutamate into the citric acid cycle for energy production under certain environmental conditions.40−42 Therefore, the different GDH activity may indicate different 5820



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Table 2. List of Identified Proteins

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Table 2. continued

a

Assigned spot numbers as indicated in Figure 4. bDatabase accession numbers according to SwissProt and NCBInr. cTheoretical and observed masses (kDa) and pI of differently expressed proteins. dP values according to ANOVA; Ps, sex effect; Pt temperature effect; Ps×t, sex and temperature interaction effect. eMeans of relative protein abundances.

studies have reported that both PGAMi and PGK show different changes to drought between sexes or among provenances.17,50 Wang et al.51 reported that low temperature

phosphoglycerate kinase (PGK, spot 13) increased in females. Spot 13 is a fragment of PGK, because its observed molecular weight is lower than the theoretical one (Table 2). Previous 5822



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these proteins are partially degraded products of their intact proteins. Most degraded proteins are metabolic and photosynthetic, for example, the RuBisCO large subunit, ATP synthase beta subunit and succinate dehydrogenase. These results are similar as those observed in rice under chilling stress6,52 and poplar under drought and salt stresses.17,18 Additionally, the isoelectric point of some proteins differ from the theoretical value, suggesting that post-transcriptional regulation may happen in these proteins. Chilling induced two glycine-rich RNA-binding proteins (GRPs) in males (spot 24) and females (spot 25). GRPs are involved in post-transcriptional regulation of genes, which have been found to play a role in increasing cold tolerance.53,54 The glycine cleavage H-protein (spot 26) is a component of the glycine cleavage system (GCV) and catalyzes the degradation of glycine. It has been reported that a low temperature results in GCV H-protein down-regulation in cold-sensitive rice but not in cold-tolerant cultivars.55 In this study, no significant temperature effect on the GCV H-protein was observed. However, it always exhibited a higher abundance in males than in females under both control and chilling stress conditions. This result indicates that males may have a higher glycine metabolism rate than females. In addition, chilling induced pyridoxine biosynthesis protein PdxS (spot 29−33) accumulation and higher abundances in chilling-stressed males than in chilling-stressed females. PdxS is a subunit of the pyridoxal 5′-phosphate (PLP) synthase, an important enzyme in de novo biosynthesis of PLP. PLP is an essential cofactor in enzymatic reactions, is involved in numerous cellular processes and plays a role in oxidative stress responses.56The greater accumulation of pyridoxine biosynthesis proteins can enhance PLP production and further result in a higher antioxidant ability in chilling-stressed males. Differently Expressed Stress and Defense Proteins. Plant responses to chilling involve the induction of a number of sex-specific changes in stress response and defense proteins. Two of such proteins increased in abundance (spot 38, universal stress protein and spot 42, phi class glutathione transferase GSTF1) only in chilling-stressed males, while another protein (spot 41, macrophage migration inhibitory factor) increased in both sexes. Comparably as in this work on chilling stress, previous studies have shown that the universal stress protein and GSTF1 increase significantly in abundance in P. cathayana males under drought or salt stress,17,18 suggesting that these two proteins are male-specific responsive proteins and may play similar functions under drought, salt and chilling stresses. Interestingly, most identified stress and defense proteins show a higher abundance in males than in females under chilling stress and also under drought and salt. Although the precise function and regulation mechanisms of these stress response proteins are not well-known, many of them are believed to be crucial components of the plant’s self-defense mechanism. For example, plant GSTF has been implicated in numerous stress responses, including those arising from pathogen attack, oxidative stress, and heavy metal toxicity.57 Similarly as in a previous study on poplars’ responses to chilling,13 we found that there are more abundant heat shock proteins (HSPs) at 4 °C than under optimal growth conditions. Furthermore, we found that the accumulation of HSPs was greater in chilling-stressed males than in chilling-stressed females: a total of eight HSPs (spots 43−49) increased significantly in chilling-stressed males. Except for spot 45, the other identified HSPs are small HSPs. It has been reported that

Figure 7. Hierarchical clustering of 48 identified sex-specific proteins (Ps < 0.05 or Ps×t < 0.05) recorded from two-way ANOVA. The hierarchical cluster analysis was conducted using the software PermutMatrix v1.9. The used data set is with normalized rows (Zscores) and the hierarchical cluster method is Ward’s algorithm. M25, 25 °C of males; M4, 4 °C of males; F25, 25 °C of females; F4, 4 °C of females.

induced a high expression of the PGK gene in Eupatorium adenophorum Spreng. In our study, we observed an upregulation of the PGK fragment only in females but not in males. A granule-bound starch synthase I (spot 12) was found to increase in abundance in males but not in females, which indicated that chilling strengthens starch synthesis ability in males. Several proteins involved in metabolic processes of proteins also showed sexually different responses to chilling. The abundances of FKBP-type peptidyl-prolylcis−trans isomerase (spot 18) and protein disulfide isomerase (spot 19) increased significantly in chilling-stressed males and females, respectively. These two proteins function as protein folding chaperones to ensure a correct protein folding state. The main function of the proteasome is to degrade misfolded or damaged proteins by tagging with a ubiquitin. In this study, we found a probable proteasome inhibitor (spot 20), which inhibits the hydrolysis of protein and peptide substrates by the 20S proteasome, was down-regulated and only detected in females, while a ubiquitin fusion-degradation protein (spot 22) exhibited a higher abundance in females than in males. These results indicate that males and females may have different abilities in degrading unneeded or misfolded proteins in vivo. In general, the apparent molecular weight predicted by SDSPAGE has an error deviation of about ±10% compared with the theoretical value.52 Among the 65 identified proteins, 14 identities were found with observed molecular weights much smaller than the theoretical values (Table 2), suggesting that 5823



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(No. 30930075), and the National Natural Science Foundation of China (No. 31170572).

the accumulation of small HSPs can be triggered by abiotic stresses in plants and is considered as an essential response.58,59 In vivo, small HSPs are believed to confer a protective function by preventing unfolding or disassembly of other proteins.60,61Interestingly, under chilling stress, three out of seven small HSPs (spots 43, 46 and 48) showed a higher abundance in males than in females, similarly as in P. cathayana under drought and salt stresses.17,18 Other Proteins. Two phenylcoumaran benzylic ether reductases (PCBER, spot 34 and 35) showed a higher abundance in chilling-stressed males than in chilling-stressed females. PCBER is an enzyme that catalyzes lignan biosynthesis, which plays important roles in the plant host defense systems and in the regulation of plant growth and wood development.62 Chilling also changed basic biosynthetic processes. For example, a pterin-4-alpha-carbinolamine dehydratase (spot 56), which is involved in the purine biosynthetic process, was only present in chilling-stressed males. The nucleoside diphosphate kinase Group I (spot 57) that catalyzes the exchange of phosphate groups between different nucleoside diphosphates to form ATP was up-regulated only in chillingstressed males.

CONCLUDING REMARKS Our results showed that there are significant sexually different responses to chilling in the free amino acid components, NR activity and proteome dynamics. More than half of the sexspecific proteins are involved in metabolic processes, such as carbon and energy metabolism, metabolic processes of proteins, and lipid, vitamin and secondary metabolism. Especially under chilling, males showed higher abundances of proteins involved in stress defenses, which is a similar result as observed in previous studies on the responses of P. cathayana to drought and salt stresses. It is concluded that males have more effective metabolic activities and protective systems than do females in response to chilling. However, further evidence from metabonomics and other approaches are needed.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Information 1. Repeated 2-D gels of P. cathayana males and females at 25 and 4 °C. Supporting Information 2. Identified proteins as indicated in Figure 3. Supporting Information 3. MS/MS identified and searched proteins. Supporting Information 4. Corresponding homologues of unknown or predicted proteins. Blastp (http://blast.ncbi.nlm. nih.gov/Blast.cgi) was used to search for the homologues of unknown proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

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ABBREVIATIONS

Ala, alanine; Arg, arginine; Asp, aspartic acid; Cys, cystine; Cyt f, cytochrome F; Fv/Fm, variable and maximum fluorescence; GCV, glycine cleavage system; GDH, Glutamate dehydrogenase; Glu, glutamic acid; Gly, glycine; GRP, glycine-rich RNAbinding protein; GS, glutamine synthetase; GSH, reduced glutathione; GSTF, phi class glutathione transferase; HSP, heat shock protein; Ile, isoleucine; Leu, leucine; Lys, lysine; MDH, malate dehydrogenase; Met, methionine; NR, nitrate reductase; Pc, plastocyanin; PCBER, phenylcoumaran benzylic ether reductase; PGAMi, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase; Phe, phenylalanine; PGK, phosphoglycerate kinase; PLP, pyridoxal 5′-phosphate; Pro, proline; qN, nonphotochemical quenching coefficient; qP, photochemical quenching coefficient; RuBisCO, ribulose-1,5-bisphosphate carboxylase oxygenase; SDH, succinate dehydrogenase; Ser, serine; TCA, trichloroacetic acid; Thr, threonine; yield, the effective quantum yield of PS II

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 86-28-85557542. Fax: 86-2885222258. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Key Basic Research Program of China (No. 2012CB416901), the Key Program of the National Natural Science Foundation of China 5824



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Biochemical and Proteomic Analyses Reveal that Populus cathayana

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