OsPOP5, A Prolyl Oligopeptidase Family Gene from Rice Confers Abiotic Stress Tolerance in Escherichia coli

The prolyl oligopeptidase family, which is a group of serine peptidases, can hydrolyze peptides smaller than 30 residues. The prolyl oligopeptidase family in plants includes four members, which are prolyl oligopeptidase (POP, EC3.4.21.26), dipeptidyl peptidase IV (DPPIV, EC3.4.14.5), oligopeptidase B (OPB, EC3.4.21.83), and acylaminoacyl peptidase (ACPH, EC3.4.19.1). POP is found in human and rat, and plays important roles in multiple biological processes, such as protein secretion, maturation and degradation of peptide hormones, and neuropathies, signal transduction and memory and learning. However, the function of POP is unclear in plants. In order to study POP function in plants, we cloned the cDNA of the OsPOP5 gene from rice by nested-PCR. Sequence analysis showed that the cDNA encodes a protein of 596 amino acid residues with Mw ≈ 67.29 kD. In order to analyze the protein function under different abiotic stresses, OsPOP5 was expressed in Escherichia coli. OsPOP5 protein enhanced the tolerance of E. coli to high salinity, high temperature and simulated drought. The results indicate that OsPOP5 is a stress-related gene in rice and it may play an important role in plant tolerance to abiotic stress.

a variety of disorders of the central nervous system. Furthermore, Oligopeptidase B was first isolated from E. coli cells with thadpsin-like specificity and the ability to decompose peptides at lysine and arginine residues [23], but its role in E. coli is unknown. The physiological role of this enzyme is not clear, and its physiological substrate has not identified. Until now, OPB was only discovered in prokaryotes and unicellar eukaryotes. In fact, trypanosome POP and OPB both seem to be important virulence factors [24]. Deletion of the OPB gene results in a marked decrease in trypomastigote virulence and capacity for cell invasion [25].
Different from both prolyl oligopeptidase and oligopeptidase B, Dipeptidyl-peptidase IV is a dimer, an exopeptidase, a glycoprotein and an ectoenzyme bound to the cell membrane. It cleaves dipeptidase at the penultimate proline or to a lesser extent at alanine from the amino terminus of the oligopeptide [26,27]; its cellular localization and enzymatic properties also vary from other dipeptidyl-peptidases. In contrast to prolyl oligopeptidase and oligopeptidase B, acylaminoacyl peptidase is a monomer [9].
In this research, we cloned a rice POP gene and investigated its expression pattern under abiotic stress in rice. To further analyze the characteristics of OsPOP5, we introduced OsPOP5 into the pET32a vector expressed in E. coli. Expression of OsPOP5 enhanced the tolerance of E. coli to dehydration, heat, and high salinity, which suggested that OsPOP5 may play a protective role under stressed conditions.

Cloning and Sequence Analysis of OsPOP5 Gene
The OsPOP5 gene was cloned from cDNA library of rice (Oryza sativa) by nested polymerase chain reaction (PCR) and sequenced ( Figure 1). Sequence analysis indicated that the complete open reading frame of OsPOP5 cDNA is 1791 bp. The predicted protein of OsPOP5 comprises 596 amino acids with the calculated molecular mass of 67,294.7 Da and the isoelectric point (PI) of 6.00. The OsPOP5 cDNA sequence has been submitted to GenBank with accession NM_001053127. InterProscan and MOTIF search results suggested that it has a DPPIV_N (Dipeptidyl Peptidase IV N-terminal region) and Peptidase S9 (prolyl oligopeptidase family) domain, and it might belongs to the prolyl oligopeptidase family.
To investigate the evolutionary relationship of OsPOP5 proteins in plants, a phylogenetic tree was constructed using Neighbor-Joining method with the full-length amino acid sequences. We used the full-length amino acid sequences with BLAST (basic local alignment search tool) [28] and ClustalW [29], conducting multiple sequence analysis. BLASTP (protein blast) [    We then analyzed the upstream cis-acting regulatory element of OsPOP5 and found that stress and defense related elements (Figure 3), such as W-box, Box-W1 (fungal elicitor responsive element), Sp1 (light responsive element), TCA-element (cis-acting element involved in salicylic acid responsiveness), and ABRE (cis-acting element involved in the abscisic acid responsiveness) were enriched in the promoter region of OsPOP5, indicating that these stress related cis-acting elements may be responsive for stress regulated expression of OsPOP5.

Expression of OsPOP5 under Different Stress Treatments
To determine whether OsPOP5 is involved in abiotic stress response, RT-PCR assay was performed to analyze OsPOP5 expression in rice under drought, cold, heat, salt stress and chemical treatment including abscisic acid (ABA), indole-3-acetic acid (IAA) and gibberellin (GA 3 ) in different tissues. The result showed that OsPOP5 was ubiquitously expressed in rice root, stem, and leaf ( Figure 4). In salt treatment, the expression level of OsPOP5 was upregulated after 0.5 h and remained high throughout the treatment in leaf and stem. However, in root, the expression level was up-regulated after 0.5 h and down-regulated at 2 h and maintained this level. In response to drought, the OsPOP5 transcript showed no significant change in leaf and stem, and the transcript was upregulated in root after 0.25 h treatment, meanwhile, the OsPOP5 expression level was downregulated in the control. In cold and heat stress treatment, the OsPOP5 showed similar transcript levels in leaf and stem. However, the OsPOP5 expression level was upregulated in 1 h and downregulated in 24 h in cold stress in root. In heat stress, the OsPOP5 transcript was significantly increased in 1 h and reached its peak at 16 h in root. We also analyzed OsPOP5 response to plant hormones including IAA, ABA and GA 3 . Under IAA treatment, the OsPOP5 transcript level increased after 0.5 h in leaf, which decreased after 0.5 h in stem and increased after 1 h. In response to ABA treatment, the OsPOP5 transcript decreased at 8 h, and there was no obvious change in stem and root. The GA 3 treatment result was similar with IAA. All of these results indicate that the OsPOP5 gene is responsive to all stresses tissue specifically.

Expression Analysis of OsPOP5 in E. coli by SDS-PAGE
The recombinant plasmid pET32a-OsPOP5 and empty vector pET32a as a control were transformed into E. coli BL21. The recombinant protein was induced after 2 h of isopropylthio-β-D-galactoside (IPTG) treatment and reached maximum levels at 6 h as confirmed by SDS-PAGE. The supernatants of treated cell lysates from the IPTG-induced transformed cells were studied. According to the construction of recombinant plasmids, the size of the proteins deduced from BL/pET32a and BL/OsPOP5 cells should be about 21 and 88 kDa, respectively. Figure 5 shows a specific band about 21 kDa in BL/pET32a cells and another thicker band of the 88 kDa fusion protein was obviously observed on gels containing the BL/OsPOP5 cell supenatant ( Figure 5). These results indicated that the OsPOP5 fusion protein was successfully expressed.

Overexpression of OsPOP5 in E. coli Enhances Growth during Abiotic Stresses
To examine the effect of the overexpression of the OsPOP5 protein on the growth of E. coli recombinants under different environment stresses, cultures of BL/pET32a and BL/OsPOP5 were diluted and spread on different plates. Figure 6 showed that recombinant and control cells have similar growth on Luria-Bertani (LB) medium in overnight grown culture, whereas, in high salinity, drought and high temperature supplemented medium, the recombinant cells increased the number of colonies as compared to control cells. Figure 6 shows that the number of colonies varied on plates supplemented with 400 to 600 mM NaCl, and that the viability of BL/OsPOP5 cells were significantly greater than BL/pET32a cells under various NaCl concentrations. Moreover, the cell size of BL/OsPOP5 in the medium supplemented with 400 mM NaCl plates was bigger than that of BL/pET32a on 500 and 600 mM NaCl. When NaCl concentration was increased 600 mM in the mediums, there were small and few BL/OsPOP5 cells found versus nothing in the control. In addition, similar results were found with mannitol treatment; the colony number of BL/OsPOP5 was much higher than that of BL/pET32a at high salt and high mannitol concentrations. These results indicate that the expression of the OsPOP5 gene increased the salt and drought tolerance of E. coli cells. In order to identify the effect of overexpression of OsPOP5 protein on the growth of E. coli recombinants under high-temperature stresses, cultures with IPTG were transferred to 50 °C. After the temperature treatment for different time periods, the number of cells was calculated in diluted culture aliquots. Although the number of cells reduced rapidly in both cultures upon heat shock, BL/OsPOP5 cells were numerous compared with control cells after high temperature treatment and about 2-fold after 1 h at 50 °C. Most of the BL/pET32a cells died after 1.5 h at 50 °C, whereas fewer BL/OsPOP5 cells died under the same condition. These results revealed that the OsPOP5 gene significantly induced tolerance to high-temperature stress. Moreover, we investigated the tolerance of BL/OsPOP5 and BL/pET32a on plates with different concentrations of mannitol. The results showed BL/OsPOP5 and BL/pET32a exhibited almost no difference at 500 mM, but a significant difference was found at 1M. This result indicated that the OsPOP5 gene enhanced tolerance to dehydration stress.

Discussion
A great number of genes in plants are induced after exposure to various abiotic stresses: they encode receptors, kinases, transcription factors, and other signalling molecules. These genes help enhance stress tolerance in plants in various ways [30,31]. The sequence of various cis-acting elements, including ABRE, G-boxes, GCC-boxes, MBSs, TGA-motif, TGAGC-motif and motif IIb, are present in the 1.5 kb promoter region of OsPOP5 ( Figure 1B). The ABRE and MBS are known as recognition sites of the ABRE/ABF transcription factor and the MYB biding site involved in drought response, respectively [32]. These cis-elements and their respective transcription factors have important roles in ABA signaling and abiotic stress responses [33]. The W-box and GCC boxes are known as recognition sites for WRKY and ERF transcription factors, respectively [34,35]. These appear to be involved in the regulation of various plant-specific physiological processes such as pathogen defense and senescence. The TCA-element, TGA-element, TGACG-motif and motif IIb are known as cis-acting elements involved in salicylic acid responsiveness, auxin responsive elements (AuxREs), cis-acting element involved in MeJA responsiveness and abscisic acid responsive elements, respectively. These sequences may function as abiotic and/or biotic stress-responsive cis-acting elements in the OsPOP5 promoter.
ABA plays important roles in adapting vegetative tissues to abiotic stresses such as drought and high salinity, as well as in seed maturation and dormancy. ABA regulates the expression of many genes that might function in dehydration tolerance in both vegetative tissues and seeds [36,37]. The OsPOP5 gene showed no significant induction by various stresses and chemical treatments in RT-PCR (Figure 3). From the promoter analysis we found that the OsPOP5 was a stress related gene, but it was not significantly changed by RT-PCR analysis. There is an ABRE cis-element in the OsPOP5 promoter, and ABRE is a major cis-element in ABA-responsive gene expression. Moreover, a single copy of ABRE is not sufficient for ABA-responsive transcription [32]. ABRE and coupling elements such as CE1 and CE3 constitute an ABA-responsive complex in the regulation of wheat HVA1 and HVA22 genes. Two ABRE sequences are necessary for expressing Arabidopsis RD29B in seeds and for the ABA-responsive expression of RD29B in vegetative tissue. One of these ABRE sequences might function as a coupling element. Most of the known coupling elements are similar to ABREs and contain an A/GCGT motif. Either an additional copy of ABRE or coupling elements are necessary for ABA-responsive gene expression.
POP may play a role in many biological processes, such as maturation and degradation of peptide hormones and neuropathies [38,39], memory and learning [26], signal transduction [40] and protein secretion [41]. Recently, POP was reported to have roles associated with cell cycle regulation [42], angiogenesis [43] and cellular signaling [44]. In addition, there appears to be some consensus that POP possesses a non-hydrolase function and it was demonstrated that POP is located in close association with the cytoskeletal component tubulin [41]. POP has been considered as a housekeeping enzyme, limited to the cytoplasm [6], and experimental data has shown that that expression is neither exclusively cytoplasmic [45,46] nor homogeneous across the tissues [47].
In human and rat, POP enzyme activity has been obtained in different tissues [48], and high activities have been measured in the brain. In the brain, the distribution of immune-reactive POP protein [47] was partly separated from the locations of enzyme activities or POP mRNA levels [49], pointing to endogenous and post-translational regulation of POP. Brain POP has been found also in the membrane and POP protein has also been detected in nuclei. Furthermore, prolyl oligopeptidase enzyme activities have been previously measured in some peripheral tissues such as skeletal muscle, testis, liver, kidney, lung, renal cortex and gut [45]. However, the physiological function of POP and its enzyme activity in plants has remained unclear.
Taken together, owing to our results, it is reasonable to speculate that the protective mechanism of OsPOP5 might be similar in prokaryotes and eukaryotes under environmental stress conditions, and it is expected that heat, salt, drought, and other stress tolerant crops will be developed through overexpression of OsPOP5 from rice. Furthermore, to clarify the mechanism of such stress tolerance in plants, it will be important in the future to determine exactly which proteins interact with OsPOP5.

Plant Material and Abiotic Treatments
The seeds of rice (Oryza sativa L. Sub. japonica cv. Nipponbare) were sterilized in 70% ethanol and 1% NaClO, and germinated at 30 °C in the growth chamber. All stress and chemical treatments were applied to 2-week-old seedlings. Dehydration was induced by placing plants on a dry filter paper in air. Cold and heat treatments were applied by transferring plants to a growth chamber set to 4 and 42 °C, respectively. Salinity treatments were applied by submerging the roots of plants in 250 mM NaCl solution. For chemical treatments, the roots of plants were submerged in an aqueous of abscisic acid (ABA, 10 mg/L), gibberellin (GA 3 , 5 µM), indole-3-acetic acid (IAA, 20 µM), respectively. The leaves, stems and roots were collected at 0, 0.5 (or 1), 2, 4, 8, 16, 24 and 48 h for each treatment. All tissue samples were immediately frozen in liquid nitrogen and stored at −70 °C until use.

RNA Isolation and Reverse Transcription (RT)
Total RNA was extracted from various stress-treated seedlings of rice using Trizol reagent (Invitrogen, ,Shanghai, China) according to the manufacturer's protocol. The RNA was subsequently treated with RNase-free DNase I (Takara, Dalian, China) to remove the remaining genomic DNA.
The first strand cDNA was synthesized using TransScript™ First-strand cDNA Synthesis SuperMix (TransGen, Beijing, China). The harvested complementary cDNA was stored at −20 °C until use.
Homology searches were performed using blast with default parameters at NCBI [28]. Multiple sequence alignment of prolyl oligopeptidase protein was performed using ClustalW [29]. The phylogentic tree was performed using MEGA 5.05 program by neighbor-joining (NJ) method.

Semi-Quantitative PCR Analyse
The primers for OsPOP5 were 5'-GAACTGGTTATGGTCAAGGCTG-3' and 5'-ACACGGAGC GTTGTAGAAG-3' and those for β-actin were 5'-TTCAGCAGCTGTCTCCAGAGAT-3' and 5'-CTCGATTGTCCATCTTCCAAACT-3'. The PCR program includes an initial denaturation at 94 °C for 5 min, 25 cycles at 94 °C for 30 s, 62 °C for 30 s, 72 °C for 30 s, and a final 72 °C for 10 min. In addition, a 250-bp PCR fragment of the rice constitutively expressed actin gene was amplified as an internal control to ensure equal amount of cDNA used in each RT-PCR reaction.

Construction of OsPOP5 Expression Vector
In order to construct the expression vector pET32a-OsPOP5, Primers with single restriction enzyme site for EcoRI were designed: 5'-GATCCGAATTC-ATGATTGCGTATGTGCGGGAT-3', 5'-AGCTCGAATTCCAGGTTCCTCTCTACAAAATC-3'. The pET32a vector using EcoRI digestion and apply by Alkaline CIAP (Takara, Dalian, China) dephosphorylation. The amplified products were cloned into pET32a vector at EcoRI site to express OsPOP5 protein fused with Trx Tag™ thioredoxin at the N-terminus. The pET32a-OsPOP5 plasmid was transformed into E. coli BL21 (DE3). The BL21 cell with pET32a-OsPOP5 plasmid was named as BL/OsPOP5. The transformant of pET32a was used as a control and was named BL/pET32a.

Assays for Abiotic Stress Tolerance of E. coli Transformants
Stress tolerance assays of BL/OsPOP5 and BL/pET32a were analyzed. IPTG induction and cell cultures were prepared as described above. The concentration of all induced cultures in liquid LB was adjusted to OD 600 value of 1.0. To measure salt tolerance of transformed E. coli cells, the samples were diluted by 50-fold, 100-fold, 200-fold and then 10 µL of the diluted samples was spotted on IPTG LB agar plates containing 50 µg/mL ampicillin and 1.0 mM IPTG and supplemented with additional 400, 500, and 600 mM concentration gradient of NaCl, 500 mM, 800 mM and 1 M mannitol for drought treatment, respectively. For the assay of the thermophylactic experiments, 1 mL samples were transferred to 50 °C. Aliquots (100 µL) were taken at different periods 0.5, 1, 1.5, 2, 2.5, and 3 h successively, and then 10 µL of samples diluted by 50-fold, 100-fold and 200-fold was spread onto IPTG LB agar plates. For the assay of cryophylactic experiments, the samples were kept frozen for 24 h at −80 °C and allowed to thaw at ambient temperature (32 °C) for 1 h, which constituted one freeze-thaw cycle. Aliquots (100 µL) were taken after 2, 4, 6, 8, 10, and 12 cycles successively, and then 10 µL of dilutions (1:10) was spread onto IPTG LB agar plates.

Conclusions
In this study, the gene encoding a prolyl oligopeptidase family gene, OsPOP5 was cloned and characterized in rice for the first time. Analysis of the gene structure shows that OsPOP5 encodes a 596 amino acid protein with a PI of 6.00. There are ABRE, G-boxes, GCC-boxes, MBSs, TGA-motif, TGAGC-motif and motif IIb cis-elements in the OsPOP5 promoter. Semi-quantitative RT-PCR analysis results indicate OsPOP5 is a stress related gene. Heterologous expression of OsPOP5 protein in E. coli enhanced E. coli abiotic stress tolerance, and thus implies that OsPOP5 may plays an important role in plant tolerance to abiotic stress.