Biochimica et Biophysica Acta 1844 (2014) 818–828

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Expression and interaction of small heat shock proteins (sHsps) in rice in response to heat stress Xinhai Chen a,b,1, Shoukai Lin a,c,1, Qiulin Liu a, Jian Huang a, Wenfeng Zhang a, Jun Lin a, Yongfei Wang a, Yuqin Ke a, Huaqin He a,⁎ a b c

College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China Center for Proteomics, State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, PR China Putian University, Fujian 351100, PR China

a r t i c l e

i n f o

Article history: Received 28 January 2014 Accepted 12 February 2014 Available online 22 February 2014 Keywords: Rice (Oryza sativa L.) Heat stress Small heat shock protein Expression Interaction Protein complex

a b s t r a c t The inherent immobility of rice (Oryza sativa L.) limited their abilities to avoid heat stress and required them to contend with heat stress through innate defense abilities in which heat shock proteins played important roles. In this study, Hsp26.7, Hsp23.2, Hsp17.9A, Hsp17.4 and Hsp16.9A were up-regulated in Nipponbare during seedling and anthesis stages in response to heat stress. Subsequently, the expressing levels of these five sHsps in the heattolerant rice cultivar, Co39, were all significantly higher than that in the heat-susceptible rice cultivar, Azucena. This indicated that the expressive level of these five sHsps was positively related to the ability of rice plants to avoid heat stress. Thus, the expression level of these five sHsps can be regarded as bio-markers for screening rice cultivars with different abilities to avoid heat stress. Hsp18.1, Hsp17.9A, Hsp17.7 and Hsp16.9A, in the three rice cultivars under heat stress were found to be involved in one protein complex by Native-PAGE, and the interactions of Hsp18.1 and Hsp 17.7, Hsp18.1 and Hsp 17.9A, and Hsp17.7 and Hsp16.9A were further validated by yeast 2-hybridization. Pull down assay also confirmed the interaction between Hsp17.7 and Hsp16.9A in rice under heat stress. In conclusion, the up-regulation of the 5 sHsps is a key step for rice to tolerate heat stress, after that some sHsps assembled into a large hetero-oligomeric complex. In addition, through protein–protein interaction, Hsp101 regulated thiamine biosynthesis, and Hsp82 homology affected nitrogen metabolism, while Hsp81-1 were involved in the maintenance of sugar or starch synthesis in rice plants under heat stress. These results provide new insight into the regulatory mechanism of sHsps in rice. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Unlike animals, the inherent immobility of rice plants limited their abilities to avoid stress and required them to contend with heat stress through their innate defense abilities [1] in which heat shock proteins (Hsps) might play important roles [2]. Hsps, which possessed molecular chaperone activities, were key factors contributing to cellular homeostasis in cells under both normal and adverse growth conditions [3]. Hsps were highly conserved proteins, suggesting the parallel functions in different organisms [4]. Hsps were divided into highmolecular-mass proteins, comprising Hsp100, Hsp90, Hsp70, Hsp60, and small-molecular-mass proteins consisting of Hsp20 or small heat shock proteins (sHsps) [5]. SHsps were designated as a group of Abbreviations: Hsps, heat shock proteins; sHsps, small heat shock proteins; GS, glutamine synthetase; IEF, iso-electrophoresis focus; β-Me, β-mercaptoethanol; CI, confidence interval; PVP, polyvinylpyrrolidone ⁎ Corresponding author at: College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China. Fax: +86 591 83789352. E-mail addresses: [email protected], [email protected] (H. He). 1 Authors contributed equally to this work.

http://dx.doi.org/10.1016/j.bbapap.2014.02.010 1570-9639/© 2014 Elsevier B.V. All rights reserved.

proteins with a molecular mass of 15 to 42 kDa in both prokaryotic and eukaryotic cells [6]. In mammalian cells, sHsps were known to be involved not only in enhancing cell survival in response to stress but also in the regulation of other cellular function, including apoptosis and differentiation, via their participation in the modulation of cellular redox states [7]. In rice, the expression of some sHsp genes was regulated differentially by abiotic stresses and abscisic acid (ABA), implying that these sHsp genes may play important roles in rice plant development and abiotic stress responses [8]. Based on the comprehensive sequence and expression profile analysis, Ouyang et al. [9] suggested that the expression patterns of OsHsp20 genes differed not only in different developmental stages but also in different variety levels. Sarkar et al. [5] also revealed that 23 sHsp genes were differentially expressed under several stresses and at different stages in the life cycle of rice plant by using microarray analysis and RT-PCR. High temperature increased the expression of sHsps in rice during seedling [10] and caryopsis development [11] and anthesis [12]. However, the investigation of functional characterization of sHsps in plant was limited. Recently, some interesting results on rice sHsp had been reported, which allowed us to partly predict the functions of

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sHsp. Transgenic rice lines with overexpression of the sHsp17.7 gene clearly showed higher drought tolerance compared to untransformed control rice plants during the seeding stage [13]. In addition, overexpression of a rice chloroplast sHsp in Escherichia coli conferred better tolerance not only to heat stress but also to oxidative stress [14]. In maize, mitochondrial sHsps protected Complex I electron transport during NaCl stress [15]. Moreover, the chloroplast sHsp had been found to protect photosynthesis of rice during heat [16], oxidative [17] and heavy metal stress [18]. Taken together, these results indicated that rice sHsps could be induced by abiotic stresses and sHsp improved tolerance to abiotic stresses. However, the relationship between sHsp expression levels and ability of plant adaptation to environmental stresses was poorly understood. After inducing by stresses, sHsp stably binds heat-denatured proteins and then cooperate with Hsp70, an ATP-dependent chaperone, to reactivate a metaprotein [19,20]. Stable binding of several nonnative proteins to Hsp25 creates a reservoir of folding intermediates for reactivation [21]. Thus, it can be inferred that cell protection by Hsps in plants under abiotic stresses was achieved by the binding of sHsps to other proteins. However, there was no direct evidence to prove this inference in rice because limited focus had been given to intensive study of the complex of sHsps in rice in response to heat stress. In addition, two-dimensional Blue Native/SDS-PAGE (BN-PAGE) showed greatly higher resolution and had been generally used for identification of protein complexes [22]. However, compared with BN-PAGE, a potential advantage seemed to be the milder conditions of NativePAGE, which might allow us to determine protein complexes under a condition that was close to the physiological situation of cells since proteins' electrophoretic migration depended perfectly on protein interior charges [23]. Therefore, it was assumed that more tethered rice soluble protein complexes could be identified using Native-PAGE than BN-PAGE. In this study, rice varieties (Oryza sativa L.) with different abilities to tolerate heat stress were employed as materials to analyze the expression levels of sHsps, and their interactions with other proteins in response to heat stress by using a combination of techniques, including two-dimensional gel electrophoresis (2-DE), Native-PAGE, RT-PCR, yeast two-hybridization, pull-down and mass spectrometry. The objectives of this study were to: (i) determine the abundance of the main sHsp in different rice cultivars; and (ii) investigate the Hsp complexes, especially sHsp complexes, in rice in response to heat stress. 2. Materials and methods 2.1. Plant growth and heat-stress treatment In this study, three rice varieties (O. sativa L), Nipponbare, Azucena (Susceptible to heat) and Co39 (Tolerant to heat), with different abilities to tolerate heat stress [24], were used as materials. Rice seedlings were grown in a greenhouse (28 °C ± 2° day/22°C ± 2° night) under natural light conditions (14 h light/10 h darkness period) in nutrient solution after germination at 30 °C for 48 h [25]. For high-temperature treatments, 3-week-old seedlings were exposed to 42 °C (Treatment) or 28 °C (Control) for 12 h and 24 h, respectively. Nipponbare plants during anthesis stage were exposed to 42 °C (Treatment) or 28 °C (Control) for 6 h and 12 h. Leaves were sampled and stored at −80 °C immediately after treatment. 2.2. IEF and SDS-PAGE Total protein for 2-DE experiment was extracted from leaves using the methods of He and Li [26]. Leaf proteins (300 μg) were loaded on prepared iso-electrophoresis focus (IEF) tube gels (17 cm length and 2 mm diameter), which contained 12 M urea, 5.5% (w/v) acrylamide, 3% NP-40 and 7.5% Biolyte (pH 3–10 and 5–8; Bio-Rad), 0.02%w/v ammonium persulfate, and 0.15%v/v N, N, N, N-N-tetraethylethylenediamine. The IEF was performed at 200 V, 300 V, 400 V, 500 V and 600 V for

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30 min, followed by 800 V for 16 h and 1000 V for 4 h. After IEF, the gels were immediately equilibrated for 25 min in equilibration buffer (60 mM Tris–HCl pH 6.8, 2.5% SDS, 9% (v/v) glycerol and 5% (v/v) β-mercaptoethanol (β-Me)). The second-dimension electrophoresis was carried out by using 12.5% resolving gel and 5% stacking gel. The gels were then stained with Coomassie. 2.3. Evaluation of protein expression abundance The images of 2-D gels were analyzed by ProteinMaster 6.0 software (FortuneSun corporation, China). 2-D gels were aligned and matched after spot detection and background subtraction (lowest on boundary mode), and spot volumes were quantitatively determined (total spot volume normalization mode). The 2-DE experiments were performed in triplicate using three separate samples. 2.4. Image analysis and MALDI-TOF/TOF MS analysis In MALDI-TOF/TOF MS analysis, protein spots were digested with sequencing-grade trypsin (Promega, USA) as described previously [27]. The resulting peptides were desalted with C18 ZipTips (Millipore), mixed with 5 mg/ml alpha-cyanocinnamic acid in 70% acetonitrile and 0.1% trifluoraoacetic acid, and spotted onto a MALDI sample plate. Mass spectra were acquired on a MALDI TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems) in both the MS and MS/MS modes. Data were analyzed using MASCOT software (Matrix Science, UK). NCBI or Swiss-Prot was selected as the database. Typical search parameters were set as: mass tolerance, 0.5 Da; missed cleavages, 1; enzyme, trypsin; fixed modifications, carbamido-methylation; variable modification, oxidation (M); taxonomy, O. sativa. For a match to be considered a valid identification, a confidence interval (CI) greater than 95% was required [27]. 2.5. Native-PAGE and SDS-PAGE Total protein extracted from leaves for Native-PAGE were carried out following the protocol described by Kügler et al. [28] and Sun et al. [29] with some modifications. Briefly, rice leaves were ground in liquid nitrogen for at least 30 min. The powder was rapidly transferred to centrifuge tubes and the solubilization buffer (500 mM 6-aminohexanoic acid, 50 mM imidazole/HCl PH7.0, 1 mM EDTA, 0.6% polyvinylpyrrolidone (PVP), 1 mM PMSF, 1 mM phenantroline) was added subsequently [30]. The mixture was incubated on ice for 15 min. After mild lysis, the protein solution was separated by centrifugation for 25 min at 15,000 rpm. The supernatant could then be quickly frozen in liquid nitrogen and stored at −80 °C. Protein concentrations were measured according to Bradford [31]. The protocol for Native-PAGE and SDS-PAGE analysis was performed as described by Pan et al. [32]. Briefly, the first-dimension Native-PAGE gels consisted of an 8% separating gel and a 4% stacking gel. Prior to electrophoresis, triton X-100 was added to a final concentration of 2.5%. The mixtures were allowed to stand for 5 min on ice before the addition of sample buffer (100 mM Tris–HCl pH 6.8, 50% glycerol, 0.2% bromophenol blue). Native-PAGE was carried out at 4 °C at a constant voltage of 80 V in Tris–glycine buffer. The NativeMark™ Unstained Protein Standard (Invitrogen) was used for Native marker. After 1-D Native-PAGE, the gels were stained with imidazole–sodium dodecyl sulfate–zinc reverse staining [33]. The protein lanes were cut out according to the results of staining and then were equilibrated in equilibration buffer (60 mM Tris–HCl pH 6.8, 3% SDS, 10% (v/v) glycerol and 8% (v/v) β-mercaptoethanol) for not less than 35 min. Seconddimension SDS-PAGE was performed at room temperature with 12% resolving gel and 5% stacking gel, and then the gels were stained with Coomassie. The experiments were also performed in triplicate using three separate samples.

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2.6. RT-PCR analysis Total RNA was isolated from 0.5 g of rice leaves by RNeasy Plant kit (Qiagen, USA) as recommended by the manufacturer. Total RNA (1 μg) was used for first-strand cDNA synthesis using M-MLV reverse transcriptase (Invitrogen, USA). The full length rice cDNA clones were used as the template for PCR amplification of sHsp genes. The gene-specific primers used in PCR are listed in Supplementary Table s1, and actin was used to check the RNA and amplification conditions of the RT-PCR reaction internally. The PCR reactions were subjected to 29 cycles consisting of 95 °C, 30s; 55 °C, 30s; and 72 °C, 30s with 3 replicas. 2.7. Yeast two-hybrid assay Specific primers for genes and oligonucleotides with restriction sites were designed and are listed in Supplementary Table s2. The PCR fragments were cloned into pMD18-T vector (TaKaRa) and then used for the transformation of E. coli strain DH5α. The constructs were checked by direct sequencing in BGI-Shenzhen Co. Ltd. The cDNA fragments were recovered from plasmids of the correct sequence and then ligated into plasmid pGADT7 or pGBKT7. Recombinant plasmids were verified by restriction enzyme digestion. Subsequently, the fusion constructs were used for the transformation of yeast strain AH109. Before plated on SD/-Leu-Trp selective media, self-activation of each gene of the binding domain needed to be validated. Result showed that all recombinant plasmids could not grow in SD/-Trp/-Ade and SD/-Trp/-His selective media, and the color of single colony presented the white in SD/-Trp/ X-α-Gal drop-out media, revealed that the self-activation problem of the binding domain did not exist in these sHsps genes (data not shown). Positive colonies from SD/-Leu-Trp plates were chosen and subsequently grown on the stringent SD/-Leu-Trp-His-Ade drop-out media. Positive clones were further confirmed by assaying yeast reporter LacZ via the metabolism of X-gal. In this assay, the interaction of pGBKT7-53 and pGADT7-T was used as the positive control and the interaction of pGBKT7-Lam and pGADT7-T as the negative control [34]. The experiments were also performed in triplicate using three separate samples. 2.8. Pull-down assay A pair of primer for PCR amplification of gene Os01g0136100 (Hsp16.9A) was showed in Supplementary Table s3. The accurate cDNA fragment was cloned into plasmid pET-28a and then expressed in E. coli BL21 strain. Recombinant plasmid was selected and confirmed by restriction enzyme analysis. Then, His-tagged Hsp16.9A was bound to Ni-NTA Super flow resin (Qiagen), and incubated with native protein in samples for overnight on ice with gentle shaking. After washing, according to the manufacturer's instructions, the retained proteins were eluted by boiling for 10 min in 1 × SDS sample buffer (2% SDS, 30 mM Tris pH 6.8, 10% glycerol, and 0.01% bromophenol blue), separated by 12% SDS-PAGE, and then stained with Coomassie blue dye. Special bands were excised and then mixed for subsequent LC MS/MS analysis. 2.9. LC MS/MS analysis Protein band digestion was performed following the protocol described in Image analysis and MALDI-TOF/TOF MS analysis. The eluted peptide solution was dried thoroughly using a vacuum centrifuge and then resuspended with 5% ACN in 0.1% formic acid, separated by nanoLC and analyzed by online electrospray tandem mass spectrometry. The experiments were performed on a LC-20AD system (Shimadzu, Tokyo, Japan) connected to an LTQ Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) equipped with an online nanoelectrospray ion source (Michrom Bioresources, Auburn, CA). The separation of the peptides took place in a 15 cm reverse phase column (100-μm id, MICHROM Bioresources, Inc., Auburn, CA).

The peptide mixtures were injected onto the trap-column with a flow of 60 μl/min and subsequently eluted with a gradient of 5–45% solvent B (95% ACN in 0.1% formic acid) over 90 min, and then injected into the mass-spectrometer at a constant column-tip flow rate of 500 nl/min. Eluted peptides were analyzed by MS and data-dependent MS/MS acquisition, selecting the 8 most abundant precursor ions for MS/MS with a dynamic exclusion duration of 1 min. The mass spectra were searched against the NCBInr database using the Bioworks software (Version 3.3.1; Thermo Electron Corp.) based on the SEQUEST algorithm. The parameters for the SEQUEST search were as follows: enzyme, fullTrypsin; missed cleavages, two; fixed modification, carboxyamidomethylation (C); variable modifications, deamidation (N) and oxidation (M); peptide tolerance, 10 ppm; MS/MS tolerance, 1.0 Da. Positive protein identification was accepted for a peptide with cross-correlation score (Xcorr) of greater than or equal to 2.5 for triply and 1.8 for doubly charged ions, and all with ΔCn ≥ 0.1. Furthermore, database search results were statistically analyzed using PeptideProphet, which effectively computes a probability for generating statistical validation of MS/MS search engines' spectra-to-peptide sequence assignments. A minimum PeptideProphet probability score (P) filter of 0.95 was used to remove low-probability peptides. At least two different peptides were required to assign confident protein. 2.10. Phylogenetic tree and multiple sequence alignment All rice sHsp (23 sHsps) sequences were downloaded from Uniprot (http://www.uniprot.org/). A phylogenetic tree of rice sHsps was constructed using MEGA (version 5.2) [35], while the neighbor-joining method and the bootstrap test were carried out with 1000 iterations. Multiple sequence alignments using the Clustalx 2.1 with the default parameters were performed on the indicated sequences. 2.11. Statistical analysis All data were subjected to analysis of variance (ANOVA) to assess the treatment effects on the fold changes of differentially expressed proteins both in 2-DE and Native-PAGE by using DPS 3.2 software. The means were separated and compared using the least significant difference test at P = 0.05 and 0.01. Novel protein spots that appeared in heat-stress treatments were included in the comparison as increasing spots. 3. Results 3.1. Differentially expressed proteins in rice under heat stress In this study, we carried out 2-DE analysis to identify differentially expressed proteins in rice (O. sativa L. cv. Nipponbare) in response to 12-h and 24-h heat stress compared to no heat stress. Protein profiles, with three replicas, were acquired and visualized with Coomassie staining (Fig. 1a). More than 1000 protein spots could be detected on each of the 2-DE maps within the pI range of 3.5–10 and MW range of 10–90 kDa. Among these proteins, 14 differentially expressed spots (spots 1–14) were found to have a more than 1.5-fold reproducible change in abundance. As shown in Table 1, seven proteins (spots 1–7) were down-regulated by heat stress, while six proteins (spots 8 and 10–14) were up-regulated. Spot 9 protein increased with the addition of heat but further decreased as the time of heat treatment increased. After digestion by trypsin, the differentially expressed proteins were analyzed by MALDI-TOF–TOF mass spectrometry. The differentially expressed protein spots were identified as putative mRNA binding protein (spots 1 and 7), Rubisco large chain (spot 2), uncharacterized proteins (spots 3, 4, 6 and 13), Os01g0791033 protein (spot 5), allene oxide synthase 3 (spot 8), putative tyrosine phosphatase (spot 9) and sHsp (spots 10, 11, 12 and 14) (Table 1), respectively.

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Fig. 1. Expressing profile of differential proteins (a), especially 5 sHsps (b, c) and its mRNA (d), in rice in response to heat stress. (a) Second-dimension gels for the investigation of altered proteins in rice (O. sativa L. cv. Nipponbare) in response to heat stress. (b) Enlarged images of the expression of five sHsps in Nipponbare during seedling and anthesis stages under heat stress. (c) Expressing abundance of the 5 sHsps investigated in (b). In the bar chart for each protein spot, the ordinate value represents the relative abundance of protein expression with three biological replicates (average ± SE). The symbols (**) show that they are significantly different from the controls at 0.01 level of Student's t-test. A, Control; B, rice plant during seedling under 12 h heat stress; C, rice plant during seedling under 24 h heat stress; D, rice plant during anthesis under 6 h heat stress; E, rice plant during anthesis under 12 h heat stress. (d) mRNA abundance of the sHsp (Hsp26.7, Hsp23.2, Hsp17.9A and Hsp16.9A) in Nipponbare during seedling and anthesis under heat stress. 1, Control; 2, rice plant during seedling under 12 h heat stress; 3, rice plant during seedling under 24 h heat stress; 4, control; 5, rice plant during anthesis under 6 h heat stress; 6, rice plant during anthesis under 12 h heat stress.

3.2. Level of expression of sHsps in rice during seedling and anthesis stages under heat stress Out of these differentially expressed proteins, four protein spots (spots 10, 11, 12 and 14), were identified as 26.7 kDa heat shock protein (Hsp26.7), 23.2 kDa heat shock protein (Hsp23.2), 17.9 kDa class I heat shock protein (Hsp17.9A) and 16.9 kDa class I heat shock protein 1 (Hsp16.9A) (Table 1). Spot 13, labeled in Fig. 1, which was identified as an uncharacterized protein (Table 1), was found to be 100% homologous to 17.4 kDa class I heat shock protein in rice (Table 2). Therefore, spot 13 was briefly named as Hsp 17.4. Rice during anthesis was most sensitive to heat stress [36]. In the present study, the expressing levels of the above five sHsps in Nipponbare during anthesis in response to heat stress were also determined (Supplemental Fig. s1). As expected, five sHsps (Hsp26.7, Hsp23.2, Hsp17.9A, Hsp17.4 and Hsp16.9A) were found to be upregulated by heat stress in rice during anthesis (Fig. 1b and 1c).

In addition, RT-PCR assays were performed to determine the mRNA abundance of four sHsps (Hsp26.7, Hsp23.2, Hsp17.9A and Hsp16.9A) in rice during seedling and anthesis stages under heat stresses. As the expressing abundance of these four sHsps, the abundances of their mRNAs were also up-regulated by heat stress (Fig. 1d). 3.3. Expression level of sHsps in different rice varieties under heat stress We examined expression levels of the above five sHsps in different rice varieties with different abilities to tolerate heat stress. Total proteins extracted from the leaves of two rice varieties with different abilities to avoid heat stress, Azucena (Susceptible to heat) and Co39 (Tolerant to heat), were analyzed by 2-DE (Supplemental Fig. s2). Enlarged images including these five sHsps are given in Fig. 2a. The intensities of these five differentially expressed proteins on the 2-D gels, obtained from three independent experiments, were quantitatively measured. As shown in Fig. 2b and 2c, in response to 12-h and 24-h

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Table 1 Differentially expressed proteins in rice under heat stress identified by MALDI-TOF–TOF-MS. Spot no.a

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Protein identification

Putative mRNA binding protein Ribulosebisphosphate carboxylase large chain Putative uncharacterized protein Putative uncharacterized protein Os01g0791033 protein Putative uncharacterized protein P0036D10.13 Putative mRNA binding protein Allene oxide synthase 3 Putative tyrosine phosphatase 26.7 kDa heat shock protein, chloroplastic 23.2 kDa heat shock protein 17.9 kDa class I heat shock protein Putative uncharacterized protein 16.9 kDa class I heat shock protein 1

Quantitative changesb 12 h heat stressc

24 h heat stressd

– – −0.64 (0.01) −0.87 (0.05) −0.80 (0.05) −0.49 (0.01) – 3.68 (0.01) 2.21 (0.01) ▲ (0.01) ▲ (0.01) ▲ (0.01) ▲ (0.01) ▲ (0.01)

−0.51 (0.01) −0.22 (0.01) −0.44 (0.01) −0.62 (0.01) −0.65 (0.01) −0.32 (0.01) −0.45 (0.01) 2.89 (0.01) −0.64 (0.01) ▲ (0.01) ▲ (0.01) ▲ (0.01) ▲ (0.01) ▲ (0.01)

Acc. noe

Mr (kDa)/pIf

Mg

Cov (%)h

Scorei

Q8GTK8 Q339G9 B8BHK6 A3BG53 C7IXC7 Q9FW29 Q8GTK8 Q6Z6L1 Q9LKK3 Q10P60 Q7XUW5 Q84Q77 A2XEW6 P27777

41.3/7.68 40.8/8.51 30.5/4.74 56.8/6.11 29.1/6.35 30.1/9.93 41.3/7.68 54.52/6.52 27.4/6.73 26.7/6.78 23.2/5.34 17.9/5.80 17.4/6.18 16.9/6.18

7 14 5 10 11 5 11 3 11 15 7 7 8 9

23 20 24 16 25 30 22 10 34 59 42 38 56 47

66 108 67 67 79 66 70 61 83 151 107 86 92 77

a

Numbers correspond to the 2-DE gel in Fig. 1a. The values show the fold-increase of proteins toward control. The minus values (−) show their fold-decrease. Novel protein spots that only appeared at heat treatment were included in the comparison as increasing spots, and their increase is shown by (▲). (–): no change. c 12 h heat stress vs. control. For protein spots that showed statistical significance, the P-values are shown in parentheses (t test). d 24 h heat stress vs. control. For protein spots that showed statistical significance, the P-values are shown in parentheses (t test). e Protein accession in uniprot. f Theoretical pI and molecular weight. g Number of peptides matched by MS. h Sequence coverage. i MASCOT score (PMF). b

heat stress, the levels of expression of the five sHsps in Co39 were all significantly higher than that in Azucena. In addition, the levels of expression of these five proteins in Azucena were significantly lower following 24-h heat stress as compared to 12-h heat stress. However, this did not occur in Co39 (Fig. 2c). We also carried out RT-PCR to detect the level of expression of mRNAs encoding four sHsps (Hsp26.7, Hsp23.2, Hsp17.9A and Hsp16.9A) in these two rice cultivars in response to 12-h and 24-h heat stress. As shown in Fig. 2d, heat stress increased the mRNA abundance of these four sHsps in the two rice cultivars. Not only under 12-h heat stress but also under 24-h heat stress, mRNA abundances of these four sHsps in Co39 were all higher than that in Azucena.

complex gels were denatured and run on second-dimension SDS-PAGE. As shown in Fig. 3b, more than 250 protein spots were identified on each 2-D Native/SDS-PAGE map and most of these were located in the range of MW 10–100 kDa. The 2-D Native-PAGE gels were cut vertically along every distinct 1D protein band to determine the proteins involved in each protein complex. After analyzing, the differential protein spots in 2-D Native-PAGE gels of three rice cultivars with three replications, were marked and identified by MALDI-TOF–TOF-MS. The relative intensities of these differential proteinswere also quantitatively measured (Fig. 3c). One protein complex, named C1, contained four sHsps (Fig. 3b and 3c) including Hsp18.1, Hsp17.9A, Hsp17.7 and Hsp16.9A (Table 3). Interestingly, in response to 12-h and 24-h heat stress, the expressing levels of the four sHsps in Co39 were all significantly higher than that in Azucena and Nipponbare. This pattern was the same as the outcomes in 2-DE gels. Yeast two-hybrid assays were used to validate the interactions among the four sHsps identified in the protein complex C1. Firstly, we fused full-length Hsp18.1 and Hsp17.7 cDNA into the GAL4 DNA binding domain (BD), while the full-length cDNA fragments of Hsp17.7 and Hsp16.9A were fused to the GAL4 activation domain (AD) (Supplemental Fig. s3). As shown in Fig. 4, the BD-Hsp18.1 and AD-Hsp17.7, BD-Hsp17.7 and AD-Hsp16.9A, BD-Hsp18.1 and AD-Hsp16.9A and positive control

3.4. Interactions of sHsps in rice under heat stress The above studies indicated that heat stress induced the expression of sHsps in different rice cultivars and during different developmental stages. Subsequently, we used Native-PAGE and SDS-PAGE techniques to analyze sHsp complexes in rice under heat stress. The 1-D NativePAGE gel maps of the three rice cultivars, Nipponbare, Azucena and Co39, in response to heat stress were shown in Fig. 3a. More than 30 distinct protein complex bands could be detected and these were located between MW 20.0–720.0 kDa. The first-dimension Native-PAGE protein

Table 2 Homologs of unknown differentially expressed proteins in the leaves of rice under heat stress. Spot no

13 N8 a b c

Acc. no

A2XEW6 Q7XR98

Protein name

Putative uncharacterized protein OSJNBa0027H06.1 protein

The accession number of the homologs. Identities. Positives.

Homolog Acc. noa

Protein name

Species

Ident. (%)b

Pos. (%)c

P31673 P51819 Q08277

17.4 kDa class I heat shock protein Heat shock protein 83 Heat shock protein 82

Oryza sativa Ipomoea nil Zea mays

99 88 89

100 94 94

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Fig. 2. Expressing profile of the 5 sHsps (a, b and c) and its mRNA (d) in different rice varieties under heat stress. (a) Expressing profile of the five sHsps (Hsp26.7, Hsp23.2, Hsp17.9A, Hsp17.4 and Hsp 16.9A) in different rice cultivars with different abilities to tolerate heat stress. (b) Enlarged images of the expressing profile of the five sHsps in Azucena and Co39 under control and heat stress. (c) Expressing abundance of these five sHsps showed in (b). The ordinate value represents the relative abundance of protein with three biological replicates (average ± SE). The symbols (**) show that they are significantly different at 0.01 level of Student's t-test. A, Control; B, seedling under 12 h heat stress; C, seedling under 24 h heat stress. (d) mRNA abundance of four of the 5 sHsps in different rice cultivars under heat stress. 1, Control; 2, seedling under 12 h heat stress; 3, seedling under 24 h heat stress.

were all detected by X-gal staining, which suggested that Hsp18.1, Hsp17.7 and Hsp16.9A interacted with each other. Furthermore, the interaction among sHsps was confirmed using Histag pull down. After encoding and inducing (Fig. 5a), His-Hsp16.9A was expressed in E. coli BL21 and purified with Ni-NTA, and then validated by western blotting (Fig. 5b). The His-Hsp16.9A binding proteins were shown with arrow in Fig. 5c, which were identified by LC MS/MS. Undoubtedly, band A was identified as Hsp17.7 (Fig. 5c). In addition, other Hsp16.9A interacting proteins were detected and showed in Table 4. Another three protein complexes containing more than one Hsp, which were induced by heat stress, were named C2, C3 and C4 (Fig. 3c). Hsp 101, malate dehydrogenase and thiamine biosynthesis protein were identified in C2. Protein complex C3 contained OSJNBa0027H06.1 protein (which was highly homologous with Hsp82 in Maize (Zea mays) and Hsp83 in morning glory (Ipomoea nil)) (Table 2), methylmalonate semi-aldehyde hydrogenase and glutamine synthetase; and protein complex C4 contained Hsp 81-1, glucose-1-phosphate adenylyl transferase and fructose-bisphosphate aldolase. Moreover, Hsp 101, Hsp 82 homolog and Hsp 81-1 were up-regulated by 12-h and 24-h heat stress in all three rice cultivars. 3.5. Genetic analysis on the sHsps To explore the evolutionary history of sHsps in the rice genome, MEGA5.2 with neighbor-joining method was used to construct the

phylogenetic tree of 23 sHsps in the rice genome with bootstrap analysis of 1000 replicates. The result showed that, except for Hsp23.2 and Hsp26.7, four sHsps, Hsp17.9A, Hsp18.1, Hsp17.7 and Hsp16.9A, were closely clustered (Fig. 6a). Subsequently, to examine sequence features of these four rice sHsps, we carried out the multiple alignment analysis. Briefly, these four sHsps exhibited highly similar amino acid sequences and contained a conserved α-crystallin domain (PF00011) (Fig. 6b).

4. Discussion 4.1. Expression of sHsps in rice during seedling and anthesis stage under heat stress During seedling or anthesis stages, five sHsps (Hsp26.7, Hsp23.2, Hsp17.9A, Hsp16.9A and Hsp17.4) in Nipponbare were all up-regulated by heat stress. These results were confirmed by the increase in mRNA abundance. The up-regulation of sHsps in rice had also been found in several proteomic studies of heatstress [10–12]. These previous studies noted that sHsps were associated with better tolerance to heat stress [37] and played a crucial and precise role in mitigating heat stress by restoring normal protein synthesis [10]. Therefore, the up-regulation of sHsps was likely to be a key step in protecting rice plants against high temperature stress.

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Fig. 3. Protein complexes of sHsps in rice under heat stress. (a) First-dimension gel maps of Native-PAGE of the proteins extracted from the leaves of 3 rice cultivars under heat stress. (b) Second-dimension gel maps of the above Native-PAGE. (c) Four differential protein complexes C1, C2, C3 and C4, which were cut from second-dimension gels (b). The expressing abundance of differential proteins in the three rice varieties under heat stress was presented under the C1, C2, C3 and C4 protein complex gel maps. The ordinate value represents the relative abundance of protein with three biological replicates (average ± SE). A, Control; B, seedling under 12 h heat stress; C, seedling under 24 h heat stress.

4.2. Expression of sHsps in different rice varieties under heat stress In response to heat stress, the expressing levels of sHsps in heattolerant rice, Co39, were all significantly higher than that in heatsusceptible rice, Azucena. Moreover, the level of expression of the five sHsps in Azucena further decreased with more severe heat stress, while that in Co39 did not decline. Additionally, the expressing levels of sHsps in C1 complex in Co39 were significantly higher than that in Azucena and Nipponbare. Previous study already found that a sHsp was significantly up-regulated in rice cultivar N22 and proposed that this might contribute to the greater heat tolerance of N22 [12]. Taken together, these results seemingly indicated that the relation between the

expression levels of sHsps and the ability of rice plants to tolerate heat stress was positive. Furthermore, the evolutionary relationship and extremely high sequence similarity (Identify N 60%) revealed that Hsp18.1, Hsp17.9A, Hsp17.7 and Hsp16.9A might possess the analogous function. Over-expression of Hsp17.7 in rice plants enhanced not only heat tolerance but also drought tolerance [38,13]; it was therefore reasonable to infer that the other three sHsps might also have an ability to enhance heat tolerance in rice. Thus, the expressing level of these sHsps was positively related to the ability of rice plants to tolerate heat stress and could be regarded as bio-markers for screening rice cultivars with different abilities to avoid heat stress. It was often timeconsuming and costly to screen rice genotypes for the ability to tolerate

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Table 3 Identification of 14 proteins in rice under heat stress, which were separated in 2-D Native/SDS-PAGE gel. Complex

Spot no.a

Protein identification

Acc. nob

Mr (kDa)/pIc

Md

Cov (%)e

Scoref

MS–MS score/peptidesg

C1

N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 N11 N12 N13 N14

18.1 kDa class I heat shock protein 17.9 kDa class I heat shock protein 17.7 kDa class I heat shock protein 16.9 kDa class I heat shock protein 1 Heat shock protein 101 Malate dehydrogenase Putative thiamine biosynthesis protein OSJNBa0027H06.1 protein Methylmalonate semi-aldehyde dehydrogenase Glutamine synthetase, chloroplastic Heat shock protein 81-1 Glucose-1-phosphate adenylyltransferase Glucose-1-phosphate adenylyltransferase Fructose-bisphosphate aldolase

Q84Q72 Q84Q77 Q84J50 P27777 Q6F2Y7 Q7XZW5 Q8GVQ3 Q7XR98 Q6Z4E4 P14655 A2YWQ1 B8AR31 D4AIA3 Q53P96

18.1/6.77 17.9/5.80 17.7/6.18 16.9/6.18 101.0/5.9 37.02/8.13 36.9/5.44 80.25/5.05 57.24/5.98 46.96/5.96 80.19/5.05 55.15/7.01 52.61/6.13 39.56/6.85

4 5 5 5 5 5 9 14 14 4 14 14 5 6

28 36 35 38 10 26 31 20 28 14 23 29 14 30

50 86 81 68 50 66 95 132 118 65 121 141 58 60

– – – 60/1 – 45/1 – – – 109/1 – – 55/1 72/1

C2

C3

C4

a b c d e f g

Spot numbers are marked in Fig. 3b. Protein accession in uniprot. Theoretical pI and molecular weight. Number of peptides matched by MS. Sequence coverage. MASCOT score (PMF). Number of peptides identified by MS/MS.

heat stress by their field phenotype. Traditionally, rice spikelet fertility under high temperatures could be used as a screening marker for heat tolerance during the reproductive phase [39,40], which was the terminal stage of rice development. The level of expression of sHsp could be tested easily and effectively because sHsp expression levels could be investigated at seedling stage, while spikelet fertility could be examined only at a late development stage.

4.3. Interactions of sHsps in rice under heat stress Proteins were often associated with each other, forming temporary or stable large protein complexes because most cellular processes occurring in organism required the action of several enzymes. In response to heat stress, four sHsps (Hsp18.1, Hsp17.9A, Hsp17.7 and Hsp16.9A) were up-regulated and were present in protein complexes. One-to-one yeast two-hybridization was carried out to further determine whether these four sHsps interacted with each other directly. The direct interactions between Hsp18.1 and Hsp17.7, Hsp18.1 and Hsp16.9A, and Hsp17.7 and Hsp16.9A were observed (Fig. 4). Pull down assay also confirmed the interaction between Hsp17.7 and Hsp16.9A in rice under heat stress (Fig. 5). Hsp17.9A did not show direct interaction with any of the other three sHsps, but Hsp18.1, Hsp 17.9A, Hsp17.7 and Hsp16.9A have extremely high similarity in amino acid sequences and all have assembled α-crystallin domain (PF00011)

Fig. 4. Yeast two-hybrid assay map of the interactions among the four sHsps, Hsp18.1, Hsp17.7 and Hsp16.9A, which were identified in the protein complex C1. The interaction of pGBKT7-53 and pGADT7-T was used as the positive control, while the interaction of pGBKT7-Lam and pGADT7-T was used as the negative control.

(Fig. 6b). Several lines of evidence strongly indicate that related proteins with high comparability could form hetero-oligomers, although this was not precise [41,42]. Kim et al. [43] provided the proof that sHsps possess an ability to interact with each other through the α-crystallin domain. Additionally, although Hsp17.9A was found to be located in the nucleus which was different from the cytoplasmic location of Hsp18.1, 17.7 and 16.9A, sHsps were able to shuttle between the nucleus and cytoplasm [44]. Similar sHsp complexes have also been observed in rice, soybean and pea [45]. Jinn et al. [45] reported a high molecular mass complex containing at least 15 polypeptides of the 15–18 kDa class I sHsps in soybean (Glycine max) in response to heat stress. This result demonstrated that the four sHsps were highly likely to aggregate to form larger hetero-oligomeric complexes in rice. The heteromeric formation of sHsps had been found in other species [46]. The hetero-oligomeric complex of sHsps had been confirmed to be more stable and compact than homo-oligomeric complexes of sHsps [47], and the oligomerization of similar but not identical subunits of sHsps potentially offered the opportunity to take advantage of slightly different properties of the individual proteins [48]. Therefore, the large hetero-oligomeric complex assembled from four similar sHsps (Hsp18.1, Hsp17.9A, Hsp17.7 and Hsp16.9A) was a structural prerequisite for chaperone activity of these sHsps and very likely enhanced the ability of rice plants to adapt to high temperature environments. It's worth mentioning that several novel proteins were also identified in the pull down experiment. Out of these eight proteins, six were bound up with energy metabolism, in which four participated in glucose metabolic process and the others belonged to the subunit of ATP synthase. Previous studies had reported that the functions of sHsps were able to protect the respiration of mitochondria and photosynthesis of chloroplast under diverse environmental stresses in vitro, and the interaction between sHsps and other proteins had an ability to reduce the damage in plant cells in response to the stresses and maintain to go on wheels for cell activities [15–18]. It was therefore rational to speculate that sHsps interacted with the enzyme in glycolysis and (or) ATP synthase to take part in the regulation of energy metabolism in rice. In addition, Hsp101, Hsp81-1 and Hsp82 homologs in these three rice cultivars under heat stress were found to be up-regulated and involved in protein complexes. Hsp101 constituted the protein complex C2 together with malate dehydrogenase and putative thiamine biosynthesis protein, indicating that heat stress may affect thiamine biosynthesis in rice plants because the synthesis of thiamine requires NAD+ [49], which is opportunely generated from the pathway catalyzed by malate dehydrogenase.

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Fig. 5. His-pull down assay of Hsp16.9A. (a) Cloning of gene and identification of double enzyme digestion of recombinant plasmid of pET28a. M, DNA marker; 1, amplified gene of PCR; 2, recombinant plasmid of pET28a-Hsp16.9A; 3, double enzyme digestion of recombinant plasmid of pET28a-Hsp16.9A. (b) SDS-PAGE analysis of expressed and purified recombinant His-Hsp16.9A protein. M, Protein marker; 1, expression strain induced by IPTG; 2, purified His-Hsp16.9A protein; 3, validation of His-Hsp16.9A by using anti-His-tagged antibody. (c) SDS-PAGE of pull-down proteins by His-Hsp16.9A, and the arrowhead shows the specific lane of pull-down. M, Protein marker; 1, mixture of recombinant His-Hsp16.9A protein and total protein of rice leaves was purified by Ni-NTA; 2, total protein of rice leaves was purified by Ni-NTA as a negative control.

OSJNBa0027H06.1 protein, which was found to be homologous to Hsp 82 or Hsp83, bound glutamine synthetase (GS) in protein complex C3, indicating that nitrogen metabolism in rice plants under heat stress was regulated by Hsp. In addition, Hsp 81-1, glucose-1-phosphate adenylyl transferases and fructose-bisphosphate aldolase were involved in protein complex C4. Glucose-1-phosphate adenylyl transferase is one of the important enzymes in sugar and starch synthesis, while fructosebisphosphate aldolase is one of the key enzymes in glycolysis or gluconeogenesis in plant. By protein–protein interaction, Hsp 81-1 might prevent the denaturation of key enzymes in sugar or starch metabolism, and thus maintained the synthesis of sugar or starch in rice plants under heat stress.

5. Conclusions In this paper, five sHsps (Hsp26.7, Hsp23.2, Hsp17.9A, Hsp17.4 and Hsp16.9A) were found to be up-regulated by heat stress in rice during seedling and anthesis stages, indicating that up-regulation of sHsp might be a key step in the ability of rice to avoid heat stress. The expression level of these five sHsps was positively related to the ability of rice plants to avoid heat stress, indicating that the abundance of the five sHsps could be regarded as a bio-marker for screening rice genotypes with different abilities to tolerate heat stress. After inducing, Hsp18.1, Hsp17.7, Hsp17.9A and Hsp16.9A were assembled into a heterooligomeric complex and functioned as molecular chaperones. In addition,

Table 4 LC MS/MS identification of proteins interacting with His-HSP16.9A. Protein accessiona Q7X8A1 Q84NW1 Q7XV11 Q9SNK3 Q655W2 P0C2Z6 Q7FAH2 C7IXG8 a b c d e f g

Protein identification Os04g0459500 protein Putative ATP synthase gamma chain 1 Os04g0457000 protein Putative glyceraldehyde-3-phosphate dehydrogenase Putative glyceraldehyde-3-phosphate dehydrogenase ATP synthase F1 sector subunit alpha Glyceraldehyde-3-phosphate dehydrogenase 2, cytosolic Phosphoglycerate kinase

Accession number in Uniprot database. Molecular weight in kDa of the respective protein. Isoelectric point of the protein that calculated from its amino acid sequence. The number peaks that match the trypsin peptides. The MASCOT score of the protein identification. Total of matched peptides. Matched peptides which individual ion scores N 27 (P b 0.05) and only appear in that protein.

MW(KDa)b/pIc 42.72/7.61 39.71/8.60 27.06/6.75 47.11/6.22 43.94/8.78 55.66/5.95 36.77/6.34 39.88/9.00

Matchd f

g

5 (5) 3(3) 3(3) 3(3) 2(2) 2(2) 2(2) 2(2)

Scoree 237 192 171 120 98 92 77 68

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Fig. 6. Genetic analysis of rice sHsps. (a) Phylogenetic analysis of 23 rice sHsp gene family members. Red arrows indicated the six sHsps identified in this study. The tree was generated by using MEGA Version 5.2 with Neighbor-Joining method . Numbers at nodes represent bootstrap percentages (1000 replicates). (b) Sequence alignment of Hsp18.1, Hsp17.9A, Hsp17.7 and Hsp16.9A. The α-crystallin domain (PF00011) was marked by underline.

Hsp101, Hsp81-1 and Hsp82 homologs were found to be up-regulated and involved in the protein complex. By protein–protein interaction, Hsp101 could regulate thiamine biosynthesis, and OSJNBa0027H06.1 protein could regulate nitrogen metabolism, while Hsp81-1 could be involved in the maintenance of sugar or starch synthesis in rice plants under heat stress. Acknowledgement We thank the anonymous referees whose constructive comments were very helpful in improving the quality of this work. This work was supported by the Natural Science Foundation of China and Fujian (grant nos. 31270454, 61163047, 2013J01077 and 2011J01075), a grant from the Education Department of Fujian (grant no. JA12290) and the Key Program of Ecology in Fujian (grant nos. 0608507 and 6112C0600). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2014.02.010.

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