Plant Physiology and Biochemistry 76 (2014) 58e66

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Research article

Identification and functional analysis of a novel parvulin-type peptidyl-prolyl isomerase from Gossypium hirsutum Ping Wang a,1, Xin-Zheng Li a, b,1, Hao-Ran Cui a, Yue-guang Feng c, Xiao-Yun Wang a, b, * a

College of Life Science, Shandong Agricultural University, Shandong, Taian 271018, People’s Republic of China State Key Laboratory of Crop Biology, Shandong Agricultural University, Shandong, Taian 271018, People’s Republic of China c Jinan Academy of Agricultural Sciences, Shandong, Jinan 250300, People’s Republic of China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 July 2013 Accepted 30 December 2013 Available online 8 January 2014

Plants have developed a variety of adaptive mechanisms to cope with stresses. A novel salt-induced gene was isolated during the screening of a NaCl-induced cDNA library of cotton seedlings. The gene was registered as accession number AY972810 in GenBank. Phylogenetic analysis suggested that the protein encoded by the gene belongs to the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases, EC 5.2.1.8). Northern blot analysis indicated that the mRNA accumulation of GhPPI was induced by salt stress. Subcellular localization revealed that GhPPI (Gossypium hirsutum peptidyl-prolyl isomerase) was localized in the nucleus. The purified recombinant GhPPI could accelerate the initial velocity of the cis-trans conversion of peptidyl-prolyl bonds of a tetrapeptide in a GhPPI concentration-dependent manner. Recombinant GhPPI also suppressed protein aggregation under denaturing conditions using Gdn-HCl (guanidine hydrochloride), suggesting an additional chaperone activity. Several amino acid residues in GhPPI were speculated to be involved in substrate binding or catalysis based on molecular modeling and docking results. The activity of the peptidyl-prolyl isomerase was affected when the relevant amino acids were mutated. Among the 11 mutants, five amino acids mutations led to the enzyme activities decreased to 30% as that of wild type, and two reduced to approximately 60%. To the best of our knowledge, this is the first report of a plant parvulin PPIase involved in the salt stress response. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Gossypium hirsutum Key amino acids PPIase Salt stress

1. Introduction High salinity and drought are common abiotic stresses that adversely affect plant growth and crop production (Glenn et al., 1999; Zhu, 2001). To cope with salt stress, plants have developed a variety of adaptive mechanisms in the long course of evolution (Hans J. Bohnert, 1995; Incharoensakdi and Karnchanatat, 2003). Recent studies have suggested that chaperones and foldases may be involved in the response to stress because they can stabilize the native conformation of proteins to resist denaturation. Understanding these mechanisms is important to improve crop stress tolerance using rational breeding and transgenic strategies.

Abbreviations: PPIase, peptidyl prolyl cis/trans isomerase; GFP, green fluorescence protein; IPTG, isopropyl thio-b-D-galactoside; Gdn-HCl, guanidine hydrochloride; GhPPI, Gossypium hirsutum peptidyl prolyl isomerase; CK, creatine kinase. * Corresponding author. College of Life Science, Shandong Agricultural University, Shandong, Taian 271018, People’s Republic of China. Tel.: þ86 538 8242656x8430; fax: þ86 538 8242217. E-mail address: [email protected] (X.-Y. Wang). 1 These authors contributed equally to this work. 0981-9428/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.12.020

Peptidyl-prolyl cis/trans isomerases represent an important type of protein foldase that can accelerate the energetically unfavorable cis-trans isomerization of the peptide bond of proline (Hunter, 1998). Four structurally distinct subfamilies of PPIases have been characterized: cyclophilins, FK506-binding proteins, parvulins, and PP2A phosphatase activators (Lu et al., 2007). Parvulins constitute an important PPIase subfamily. The first member of the parvulin subfamily of PPIases was identified in Escherichia coli in 1994 and consists of only 92 amino acids, hence named parvulin (Rahfeld et al., 1994). Subsequently, parvulin homologs were found in both prokaryotic and eukaryotic organisms, such as SurA and Ess1/Ptf1 and Pin1 and Par14/Par17, respectively (Eisenstark et al., 1992; Hanes et al., 1989; Ping Lu et al., 1996). Eukaryotic parvulins can be subdivided into two groups according to their substrate specificity: phosphorylation-specific and nonspecific. Human Pin1 (hPin1) belongs to the phosphorylationspecific group and is essential for the cell cycle and growth regulation (Ranganathan et al., 1997; Yaffe et al., 1997). Pin1-like parvulins have been identified as ESS1 in yeast, which has been shown to be essential for yeast survival (Hanes et al., 1989). Human parvulin hPar14 and hPar17 are encoded by one gene and exhibit a

P. Wang et al. / Plant Physiology and Biochemistry 76 (2014) 58e66

substrate specificity that is independent of phosphorylation (Mueller et al., 2006). hPar14 possesses a Lys-rich N-terminus and was found to act as a novel rRNA processing factor and be involved in DNA binding (Fujiyama-Nakamura et al., 2009; Reimer and Fischer, 2002; Rulten et al., 1999; Surmacz et al., 2002). hPar17 was shown to promote microtubule assembly and also be involved in DNA binding (Kessler et al., 2007; Mueller et al., 2006; Thiele et al., 2011). In addition to PPIase activity, many PPIases also possess chaperone-like activity. The mammalian FKBP52 and cyclophilin 40 (Cyp40) were shown to function as molecular chaperones by their ability to assist in the refolding of b-galactosidase and suppress the aggregation of citrate synthase. Several plant parvulins have been identified thus far. The first plant parvulin member is a Pin1 homolog in Arabidopsis called AtPin1, which was shown to regulate flowering time, suggesting that parvulins may play an important role in plant growth and development (Landrieu et al., 2000; Wang et al., 2010). Two other Pin1 homologs identified in plants are MdPin1 from Malus domestica and LjPar1 from Lotus japonicus (Kouri et al., 2009; Yao et al., 2001). These three plant parvulins exhibit phosphorylationspecific substrate specificity. For phosphorylation-nonspecific plant parvulins, the catalytic mechanism and biological function are not fully understood, although some members have been identified such as AtPin2 and LjPar2 (Kouri et al., 2009). Cotton (G. hirsutum) is one of the oldest and most important fiber and oil crops. The growth and yield of cotton are severely inhibited by high soil salinity, especially at the germination and emergence stages in some cultivation areas (Ashraf, 2002). The identification and characterization of genes correlated with salt induction in seedlings of a salt-tolerant cotton cultivar are important to reveal the salt-tolerance mechanism in cotton and improve the salt tolerance of cotton using genetic engineering techniques. Here, we isolated a novel plant phosphorylation-nonspecific parvulin designated GhPPI from a NaCl-induced cDNA library of cotton seedlings. We demonstrated that the expression of GhPPI in cotton seedlings was induced by salt stress. The purified recombinant GhPPI could accelerate the initial velocity of the cis-trans conversion of peptidyl-prolyl bonds in tetrapeptides and suppress the aggregation of CK during CK refolding, suggesting that it exhibits PPIase and molecular chaperone activity. In addition, we presented here the active site of GhPPI using site-directed mutagenesis combined with the results of molecular modeling and docking. To our knowledge, this is the first report of parvulins involved in the salt stress response in plants. Studies on GhPPI will provide insight into the diverse physiological function of parvulins in plants and the molecular mechanisms underlying plant salt tolerance. 2. Materials and methods 2.1. Plant materials and salt treatments Seeds of upland cotton (G. hirsutum) ZM3 were bought from the Chinese Academy of Agricultural Sciences. Seedlings were grown in a growth chamber for 20 d with 300 mmol m2 s1 light intensity and a day/night temperature of 28  C/20  C. For salt treatment, the uniformly developed seedlings were transferred into media containing the indicated concentrations of NaCl for 24 h. The leaves and cotyledons were harvested directly into liquid nitrogen and stored at 80  C for subsequent use. 2.2. RNA isolation The total RNA used in this report was isolated by the RNeasy Plant Mini Kit (QIAGEN, U.S.A.).

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2.3. cDNA library construction and screening Poly(A)þ RNA (0.5 mg) isolated from the cotyledons of ZM3 seedlings treated with 300 mM NaCl for 24 h was used to synthesis first-strand cDNA then amplified by LD PCR according to the manufacturer’s protocol (Clontech, SMARTÔ cDNA Library Construction Kit). The double-stranded cDNA was digested by the SfiI restriction enzyme and then fractionated by Chroma Spin-400 columns. Fragments longer than 500 bp were cloned into SfiIdigested dephosphorylated lTripIEx2 arms with T4 DNA ligase. The recombinants were packaged in vitro with Packagene (Promega). The original cDNA library was estimated to contain 5  105 independent recombinants and then amplified with E. coli XL1-Blue cells before screening. The cDNA library was screened by differential hybridization (one with an NaCl-untreated cotyledon cDNA probe, one with an NaCl-treated cotyledon cDNA probe). The second-strand cDNA was used to synthesize two kinds of probes and labeled using random primers with the incorporation of [a-32P] dCTP (Promega). Plaques at a density of 104 plaques/plate (15-cm diameter) were transferred onto a colony/plaque screen. The DNA on the membrane was denatured with 0.2 M NaOH containing 1.5 M NaCl and neutralized with 2  SSPE. Prehybridization, hybridization, and washing were performed as previously described (Zheng et al., 1998). Positive clones were plaque purified by two additional rounds of plaque hybridization with identical probes. Clones exclusively or preferentially hybridized by the NaCl-treated cotyledon cDNA probe were selected. Among these, one cDNA clone, GhPPI, is described in this report. 2.4. Northern blot analysis Northern blot analyses were performed using approximately 20 mg of total RNA. The total RNA was extracted from different treated leaves and cotyledons of the salt tolerant cotton cultivar ZM3. The hybridization procedure was performed as previously described (Wu et al., 2004). The GhPPI cDNA fragment containing a partial coding region and the 30 untranslated region was labeled with [a-32P] dCTP by Priming, which is a gene labeling system from Promega, and used for the hybridization probe. The blots were autoradiographed at 80  C for up to 6 d. The ethidium bromidestained rRNA band in the agarose gel was shown as a loading control. 2.5. Subcellular localization The Agrobacterium tumefaciens strain LBA4404 and the pBI121 Ti-based binary vector pBI121-GFP were used. A full-length GhPPI cDNA was cloned into the pBI121-GFP vector and fused to the N-terminus of GFP. The resulting 35S:GhPPI-GFP fusion plasmid construct, which was verified by sequence analyses, was introduced into the Agrobacterium tumefaciens strain LBA4404. Onion transformation was performed essentially as previously described (Varagona et al., 1992). Inner epidermal peels of white onions (Allium cepa L.) were placed inside-up on modified MS medium (1  MS salts, 1  Gamborg’s B5 vitamins (Sigma), 30 g/L sucrose, and 2% agar, pH 5.7) containing 50 mg/mL kanamycin for 24 h at 28  C in the dark. The Agrobacterium strain LBA4404 containing the GhPPI-GFP plasmid was cultured for 12 h at 28  C on MS medium with 20 mg/L acetosyringone (AS) and cocultivated at 28  C with onion epidermal peels for 30 min. Afterwards, the onion epidermal peels were grown on MS agar (25  C, 16 h light/8 h dark). The expression of the introduced genes was

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examined after 48 h using fluorescence microscopy and light microscopy. 2.6. Expression and purification of GhPPI in E. coli To study the catalytic properties of GhPPI, the coding regions of GhPPI were isolated from the cDNA using the following primers: GhPPI ExF (50 -TGAATTCATGGGGAAGGACTCAAAATC-30 ) and GhPPI ExR (50 -AGCGGCCGCTTCACTGCCTGACTGTATC-30 ). The PCR products were subcloned into the pET-30a expression vector (Qiagen). The combined vector was transformed into the E. coli strain BL21 (DE3). After growth at 37  C to an OD600 of 0.6 in LuriaeBertani medium containing kanamycin (100 mg/mL), the expression of the recombinant polypeptides was induced by the addition of 0.5 mM IPTG, and the cultures were grown for an additional 5 h at 28  C. Recombinant proteins were purified under native conditions using nickel-affinity chromatography (GE) according to the manufacturer’s instructions for His-tagged proteins.

3. Results 3.1. Identification and characterization of cDNA clones encoding parvulin-type PPIases in G. hirsutum To identify genes of G. hirsutum that play important roles in the salt stress response, we constructed a NaCl-induced cDNA library. Many genes were cloned from the cDNA library using differential screening based on reverse Northern blot analysis. Among them, a 729-bp gene encodes a protein of 143 amino acids containing the characteristic peptidyl-prolyl isomerase domain (from residues 41e140) was speculated to be a parvulin-type peptidyl-prolyl isomerase according to BLAST results. The gene was registered as AY972810 in GenBank and encodes a protein of an estimated molecular mass of 15.1 kDa and a pI of 9.48. The phylogenetic relationship of GhPPI to the other parvulin-type PPIases was investigated by constructing a dendrogram generated from a multiple amino acid sequence alignment (Fig. 1 and Fig. S1). Sequence alignment with the other members of the parvulin-type

2.7. PPIase and chaperone activity assay Determination of the PPIase activity of recombinant GhPPI was performed as previously described (Fischer et al., 1984). The assay was conducted as follows: A 1 mL reaction mixture containing achymotrypsin and recombinant parvulin in 50 mM TriseHCl, pH 8.0, was prechilled to 10  C and then rapidly added into a cuvette containing the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The absorbance at 390 nm was observed over 4 min. Gdn-HCl-denatured creatine kinase (60 mM) was diluted 30-fold with refolding buffer (30 mM TriseHCl/1 mM EDTA, pH 8.0) containing 0 mM, 1.5 mM and 3.0 mM GhPPI. The reactions all occurred at 37  C. Aggregation was monitored by measuring the turbidity at 400 nm. 2.8. Homology modeling of GhPPI The three-dimensional structure of GhPPI was obtained by homology modeling using the website SWISS-MODEL (http://www. expasy.org/tools). 2.9. Molecular docking AutoDock 4.0 was used in combination with the Lamarckian genetic algorithm (LGA) for the docking study to search for a globally optimized conformation. The LGA was applied to model the interaction/binding between the ligand, the cis Ala-Ala-Pro tripeptide, and GhPPI to describe the relationship between the ligand and the macromolecule by the translation, orientation, and conformation of the ligand. First, all hydrogens and merged nonpolar hydrogens were added to the GhPPI PDB file, and Gasteiger charges were computed. Simultaneously, all the torsion angles of the ligand were defined with the AutoDockTools program to allow them to be explored during molecular docking. Then, the 3D grid was created using the genetic algorithm to generate the grid parameter file. The Lamarckian genetic algorithm in AutoDock 4.0 was applied to search the conformational and orientational space of the ligand. During each docking experiment, 20 runs were performed. At the end of each multiple-run docking experiment, cluster analysis was performed. Docking solutions with ligand allatom root mean square deviations within 2.0 nm of each other were clustered together and ranked by the lowest docking energy. The best binding modes were selected according to the binding energy and ligand binding position.

Fig. 1. Phylogenetic relationships of the GhPPI amino acid sequence to other parvulin family members. Full-length amino acid sequences were aligned using the ClustalW program then manually adjusted to optimize the alignment. The neighbor-joining tree was built using MEGA version 4.0 (Tamura et al., 2007) adopting the Poisson correction distance. The tree was drawn to relative scale with branch lengths in identical units as those of the evolutionary distances used to infer the phylogenetic tree. The bootstrap values from 1000 replicates are given at each node. Sequences included are as follows: AtPin1, Arabidopsis PPIase Pin1 (AAD20122); ScESS1, S. cerevisiae ESS1 (P22696); NcSSP1, Neurospora crassa SSP1 (CAA06818); Dmdodo, Drosophila melanogaster DODO (AAC28408); hPin1, human Pin1 (AAC50492); Par10, E. coli Par10 (P0A9L7); AtPin2, Arabidopsis putative PPI (AAG48777); hPar14, human Par14 (BAA82320); MmPin4 M. musculus Pin4 (NP_081457); LmPin1, Leishmania major Pin1 (CAJ07069); SurA, E. coli SurA (P0ABZ8); BsPrsA, Bacillus subtilis PrsA (P24327); LjPar2, Lotus japonicus Par2 (CAM59672); GhPPI, Gossypium hirsutum PPI (AAY41234); AfPin4, Anoplopoma fimbria Pin4 (ACQ58402); SjPPI, Schistosoma japonicum PPI (AAW26716); TcPar14, Trypanosoma cruzi Par14 (ABD78868); ZmPin4, Zea mays Pin4 (ACG31834); and EhPPI, Entamoeba histolytica PPI (XP657226).

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Fig. 2. Northern blot analysis of GhPPI expression induced by 300 mM NaCl for different times. The total RNA was extracted from the salt-tolerant cultivar ZM3 treated with 300 mM NaCl for different times. Approximately 20 mg of total RNA was analyzed by RNA gel blotting. The blot was hybridized with the 30 -cDNA fragment of GhPPI. The ethidium bromide-stained rRNA band in the agarose gel is shown as a loading control.

PPIase family indicated that GhPPI is highly homologous to parvulin from plants, demonstrating 80% and 91% identity with AtPin2 and LjPar2, respectively, in contrast to 55% identity with human parvulin, hPar14. GhPPI also contains a Lys-rich extension in its Nterminus, which has been shown to be involved in DNA binding (Surmacz et al., 2002). The PPIase activity of GhPPI was speculated to be independent of substrate phosphorylation based on the absence of a recognition cluster of three basic amino acids, Lys63, Arg68, and Arg69, in the S1eH1 loop that specifically recognizes phosphorylated substrates (Ranganathan et al., 1997).

3.2. The GhPPI mRNA level is upregulated under NaCl stress To determine the mRNA level of GhPPI under stress, we extracted the total RNA from the leaves of 20-day-old seedlings of the salt-tolerant cotton variety ZM3 (a salt-tolerant cultivar) for Northern blot analysis. The GhPPI mRNA levels were assayed in seedlings treated with 300 mM NaCl at different time points. The results indicated that GhPPI gene expression was strongly induced by salt stress. The amount of mRNA significantly increased after 6 h of treatment and plateaued at 12 h at a level approximately 10 times higher than that before salt stress (Fig. 2). The relative GhPPI gene expression decreased slightly from 12 h to 24 h.

Fig. 3. Subcellular localization of the transiently expressed GhPPI-GFP fusion protein in onion epidermal cells. Expression of the introduced genes was examined after 48 h by fluorescence microscopy and light microscopy. GFP localization is observed in onion epidermal cells transfected with a 35S:GhPPI-GFP construct (A) and a control 35S:GFP construct (B).

Fig. 4. PPIase activity of GhPPI. Spontaneous, 0 mM PPI; and GhPPI, 14 mM GhPPI.

3.3. Subcellular localization of GhPPI To investigate the subcellular localization of GhPPI, we assayed the transient expression of a fusion construct of GhPPI-GFP. We fused GhPPI to the N-terminus of GFP and transiently expressed the fusion protein in onion epidermal cells under the control of the constitutive 35S promoter. The subcellular localization of the fusion protein was observed using fluorescence microscopy (Fig. 3). The GhPPI-GFP fluorescence signal was observed only in the nucleus (Fig. 3A), whereas the control of GFP alone demonstrated a ubiquitous distribution in the cell (Fig. 3B). These results suggested that GhPPI was localized in the nucleus and might exert its function in the nucleus of the cell.

3.4. GhPPI exhibits PPIase activity To determine the catalytic activity of GhPPI, the encoded cDNA was cloned with an N-terminal His tag in the pET-30a vector and

Fig. 5. The relationship of the initial velocity with the concentration of GhPPI.

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Fig. 6. Effect of GhPPI on the aggregation of CK during refolding.

transformed into E. coli BL21 (DE3) cells. The cells were grown in LB medium to an OD600 of 0.6 at 37  C and were induced for 6 h with 0.5 mM IPTG at 28  C. The recombinant protein was purified by nickel-affinity chromatography to homogeneity. PPIase activity was determined in a coupled assay with chymotrypsin as previously described using the synthetic peptide N-Suc-Ala-Ala-Pro-Phe-pnitroanilide (Bachem, Bubendorf, Switzerland) as the substrate (Blecher et al., 1996; Fischer et al., 1984). Compared with the control (without GhPPI in the assay system, spontaneous), the slope with GhPPI present was more steep and suggested that the initial velocity of the hydrolysis of the substrate increased significantly when GhPPI was present (Fig. 4). The initial velocity of the catalytic reaction demonstrated a linear relationship with the concentration of GhPPI (Fig. 5).

Fig. 8. Putative substrate binding pocket of GhPPI. The PPIase domain (white) is represented as a space-filling model, and the cis Ala-Ala-Pro tripeptide (red) is represented as a ball-and-stick model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.5. GhPPI suppresses the aggregation of CK during refolding Some PPIases possess chaperone activity, but some do not. To determine whether GhPPI possesses chaperone activity, we examined the chemical refolding and aggregation of creatine kinase (CK), which allowed a quantitative analysis of the number of intermediates formed during refolding and the distribution between native and aggregated forms of CK. CK was incubated in the absence or presence of GhPPI. When GhPPI was absent, the turbidity increased significantly in a time-dependent manner. When GhPPI was present, the aggregation decreased. As the concentration of GhPPI increased, the turbidity decreased, which clearly indicated that GhPPI suppresses the aggregation of CK (Fig. 6).

Fig. 7. Comparison between the PPIase domains of GhPPI and hPin1. The PPI domains of GhPPI and hPin1 are shown in a similar orientation, and their residues in the catalytic site are represented as sticks and labeled. (A) The PPI domain of GhPPI (residues 41e140); and (B) the PPIase domain of hPin1 (PDB ID 1PIN, residues 54e163).

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3.6. Prediction of key amino acids using homology modeling and molecular docking To gain insight into the interactions between GhPPI and its substrate, a molecular model of the PPIase domain of GhPPI

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(residues 41e140) was constructed based on the known crystal structures of hPin1 (Bayer et al., 2003). GhPPI folds into a typical ba3bab2 parvulin-type PPIase structure (Fig. 7) (Bayer et al., 2003; Bitto and McKay, 2002; Kühlewein and Voll, 2004; Landrieu et al., 2002; Li et al., 2005; Ranganathan et al., 1997; Sekerina et al.,

Fig. 9. Substrate binding site of GhPPI. The cis Ala-Ala-Pro tripeptide substrate is shown as a ball-and-stick model, and the relevant residues of GhPPI are labeled. (A) The substrate binding residues of GhPPI in one direction. (B) The substrate binding residues of GhPPI in another direction.

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2000; Terada et al., 2001). According to the homology model of GhPPI, seven residues (His48, Cys90, Lys99, Met107, Phe111, Thr130, and His134) may participate in prolyl isomerization, as they are located in relatively similar positions as in the other parvulins. Computer-assisted molecular docking was also performed to examine the interactions between the substrate and GhPPI. The results provided the most rational binding conformation, including the most probable substrate binding site and the relative parameters. As shown in Fig. 8, the substrate is located within a hydrophobic pocket of the enzyme. Eleven amino acid residues were found to be involved in substrate binding, as shown in Fig. 9A and B, which include the aforementioned seven residues predicted by homology modeling. 3.7. PPIase activity of GhPPI mutants According to the homology modeling and molecular docking results, site-directed mutagenesis of possible active site residues was performed to confirm the critical amino acids. Eleven mutants were obtained and their PPIase and chaperone activity were evaluated. As shown in Fig. 10A, compared with that of the wild type, mutations at five amino acid positions led to a significant decrease in the PPIase activities. The remaining activities of six mutants (five amino acid positions) His48Ala, His131Ala, Phe111Tyr, Phe111Ala, His134Ala, and Cys90Ala were 7.8%, 19%, 20%, 30%, 30% and 35%, respectively. Two other mutants, Thr130Ala and Met107Ala, showed decreased activities to 68% and 73% of wild type respectively (Fig. 10B). Three additional mutants, Cys51Ala, Cys138Ala, and Cys41Ala, demonstrated nearly unaltered PPIase activity, which were 93%, 93%, and 104%, respectively, of the wild type activity (Fig. 10B). The results indicated that five amino acids, His48, His131, Phe111, His134 and Cys90 play crucial roles in the PPIase activity.

Fig. 10. PPIase activity of wild type GhPPI and mutants. (A) The activity of wild type GhPPI and six mutants. (B) The activity of wild type GhPPI and five additional mutants.

4. Discussion In our study, a G. hirsutum cotyledon cDNA library was established using differential screening based on reverse Northern blot analysis to identify mRNAs whose abundance changed under salt stress in cotton seedlings. We isolated a cDNA encoding a PPIase belonging to the parvulin family, which was named GhPPI. Phylogenetic analysis indicated that GhPPI is grouped in the same clade as hPar14, AtPin2, and LjPar2, which PPIase activity is independent of substrate phosphorylation (Ranganathan et al., 1997). GhPPI is a single-domain protein containing the characteristic PPIase domain (from residues 41e140), the conserved N-terminal region of hPar14, and the N-terminal Lys-rich extension that is present in hPar14. These results indicate that GhPPI is a member of the phosphorylation-nonspecific parvulin subfamily. Northern blot analysis indicated that GhPPI was overexpressed when induced by 300 mM NaCl, and its expression level increased as the time of NaCl induction increased until 12 h and remained elevated from 12 h to 24 h under a salt stress of 300 mM NaCl, which suggests that this gene is involved in the response to salt stress. Other PPIase subfamily members such as cyclophilins were shown to play important roles in plant biology, stress response and the anti-pathogen immune response (Ahn et al., 2010; Kim et al., 2012; Romano et al., 2004). Subcellular localization revealed that GhPPI was localized in the nucleus using a GhPPI-GFP fusion protein expressed in onion epidermal cells. In addition, our comprehensive comparison indicated that GhPPI contains a similar C-terminal PPIase domain and the conserved N-terminal region of hPar14. hPar14 was shown to be localized primarily in the nucleus, and its basic N-terminal domain was shown to be involved in its nuclear localization and the binding of DNA and may be involved in transcriptional regulation, chromatin remodeling and cell cycle regulation (Surmacz et al., 2002). hPar14 could combine with precursor ribonucleoprotein particles and is a rRNA processing factor (Fujiyama-Nakamura et al., 2009; Fujiyama et al., 2002). GhPPI may possess a similar function as human hPar14 in plants. To characterize the GhPPI active site, a homology model of GhPPI was generated, and the substrate was docked to the model by molecular simulation using the software AutoDock 4.0. From the 3D model, we can see that GhPPI folds into a ba3bab2 structure. GhPPI possesses a proline binding pocket consisting of Leu99, Met107, and Phe111 that is similar to hPar14 and hPin1 (Ranganathan et al., 1997; Sekerina et al., 2000). GhPPI also contains a similar catalytic pocket consisting of His48, His134, and Cys90, which correspond to His59, His157 and Cys113, respectively, in hPin1. The catalytic pocket participates in covalent catalysis during the cis-trans isomerization catalyzed by hPin1. The major difference between GhPPI and hPin1 lies in a cluster of three basic amino acids present in the S1eH1 loop of hPin1 that determine the specificity for phosphorylated substrates (Ranganathan et al., 1997; Sekerina et al., 2000; Terada et al., 2001). The S1eH1 loop in GhPPI is very short and does not contain equivalent residues for the basic amino acids Lys63, Arg68, and Arg69 in hPin1 (Ranganathan et al., 1997). Although GhPPI contains two basic amino acids, Lys53 and Lys56, they cannot bind the substrate because of the orientations observed in the model. The absence of the cluster of three basic amino acids suggests that GhPPI does not specifically recognize phosphorylated substrates, which is similar to hPar14 (Sekerina et al., 2000; Terada et al., 2001). Phylogenetic analysis also indicated that GhPPI was more related to hPar14 than to hPin1. The structural studies indicated that GhPPI may preferentially catalyze the cis-trans isomerization of peptidyl-prolyl bonds in substrates with positive or neutral amino acids preceding the proline.

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To determine the roles of specific amino acid residues in the GhPPI active site, site-directed mutagenesis was used based on the results of molecular modeling and docking. Among the eleven mutants, when the amino acids were mutated at five positions, His48, His131, Phe111, His134, and Cys90, the PPIase activities of GhPPI reduced to 30% as that of the wild type. For additional two mutants, Met107Ala and Thr130Ala, the PPIase activities reduced to approximately 60% as in the wild type. Several plant parvulins have been identified so far, such as AtPin1 from Arabidopsis, MdPin1 from Malus domestica, and LjPar1 from Lotus japonicus (Kouri et al., 2009; Landrieu et al., 2000; Wang et al., 2010; Yao et al., 2001). These three characterized plant parvulins belong to the phosphorylation-specific PIN1-type parvulins. For the phosphorylation-nonspecific plant parvulins such as AtPin2 and LjPar2, the catalytic mechanisms and biological functions in plants are thus far unknown (Kouri et al., 2009). All of the above results suggested that GhPPI was a novel member of the phosphorylation-nonspecific plant parvulins that could act as a foldase and/or chaperone in the cell nucleus and might play an important role in the salt stress response in plants. Contributions Ping Wang performed the experiments and analyzed data, XinZheng Li performed the experiments and wrote the manuscript, Hao-Ran Cui performed the experiments and participated in the writing and revising, Yue-guang Feng designed the study, Xiao-Yun Wang designed the study and wrote the manuscript. Acknowledgments This work was supported by Special Research Fund of Public Welfare of China agricultural Ministry (201303093), Shandong Province System of Modern Agriculture Industrial Technology (SDAIT-07-011-02), the Science and Technology Development Project of Shandong Province (2012GGB010026). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2013.12.020. References Ahn, J., Kim, D.-W., You, Y., Seok, M., Park, J., Hwang, H., Kim, B.-G., Luan, S., Park, H.S., Cho, H., 2010. Classification of rice (Oryza sativa l. Japonica nipponbare) immunophilins (FKBPs, CYPs) and expression patterns under water stress. BMC Plant Biol. 10, 253. Ashraf, M., 2002. Salt tolerance of cotton: some new advances. Crit. Rev. Plant Sci. 21, 1e30. Bayer, E., Goettsch, S., Mueller, J.W., Griewel, B., Guiberman, E., Mayr, L.M., Bayer, P., 2003. Structural analysis of the mitotic regulator hPin1 in solution: insights into domain architecture and substrate binding. J. Biol. Chem. 278, 26183e26193. Bitto, E., McKay, D.B., 2002. Crystallographic structure of SurA, a molecular chaperone that facilitates folding of outer membrane porins. Structure 10, 1489e 1498. Blecher, O., Erel, N., Callebaut, I., Aviezer, K., Breiman, A., 1996. A novel plant peptidyl-prolyl-cis-trans-isomerase (PPIase): cDNA cloning, structural analysis, enzymatic activity and expression. Plant Mol. Biol. 32, 493e504. Eisenstark, A., Miller, C., Jones, J., Leven, S., 1992. Escherichia coli genes involved in cell survival during dormancy: role of oxidative stress. Biochem. Biophys. Res. Commun. 188, 1054e1059. Fischer, G., Bang, H., Mech, C., 1984. Determination of enzymatic catalysis for the cis-trans-isomerization of peptide binding in proline-containing peptides. Biomed. Biochim. Acta 43, 1101e1111. Fujiyama-Nakamura, S., Yoshikawa, H., Homma, K., Hayano, T., TsujimuraTakahashi, T., Izumikawa, K., Ishikawa, H., Miyazawa, N., Yanagida, M., Miura, Y., Shinkawa, T., Yamauchi, Y., Isobe, T., Takahashi, N., 2009. Parvulin (Par14), a peptidyl-prolyl cis-trans isomerase, is a novel rRNA processing factor that evolved in the metazoan lineage. Mol. Cell. Proteom: MCP 8, 1552e1565.

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Identification and functional analysis of a novel parvulin-type peptidyl-prolyl isomerase from Gossypium hirsutum.

Plants have developed a variety of adaptive mechanisms to cope with stresses. A novel salt-induced gene was isolated during the screening of a NaCl-in...
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