Introduction

Proline is primarily a structural component of proteins; however, it also plays a role as a compatible solute under different environmental stress conditions [1]. Proline accumulation has been associated with the exposure to drought [2], high salinity [3], bright light and UV irradiation [4], heavy metals [5], oxidative stress [6], and in response to biotic stressors [7, 8]. Extensive research has been conducted on the importance of proline metabolism in plants under environmental stress conditions. Several comprehensive studies using transgenic and mutant plants have demonstrated that proline metabolism has a complex effect on development and stress responses [9–12]. Proline catabolism occurs in mitochondria through the sequential action of proline dehydrogenase (ProDH) or proline oxidase (POX) producing P5C (Δ1-pyrroline-5-carboxylate) from proline, and P5C dehydrogenase (P5CDH), which converts P5C to glutamate. Two genes encode ProDH, whereas a single gene has been identified to encode P5CDH in Arabidopsis and Nicotiana tabacum [13–16]. ProDH, a key enzyme that catalyzes the first steps of proline catabolism from l-proline to P5C [17], is a widely distributed enzyme found in bacteria, fungi, plants, and animals. The genes encoding ProDH have been cloned from many organisms, including microorganisms [18, 19], plants [20–23], and mammals [24].

Jatropha curcas L. belongs to the tribe Jatropheae of the subfamily Crotonoideae, under the Euphorbiaceae family. It is a drought-resistant oil plant or small tree. It is widely distributed in tropical and subtropical areas, especially in Central and South America, Africa, India, and Southeast Asia [25]. Because of the high oil content of its seeds, J. curcas is considered to be an important source of energy. As a fuel, the properties of J. curcas biodiesel are close to those of fossil diesel fuel, and it matches both the American and European standards [26].

The understanding of the molecular aspects of this plant’s response to adverse abiotic environmental factors (e.g., cold, drought, and salt stress) is relatively rudimentary. In this study, we report the cloning and characterization of a complementary DNA (cDNA) from J. curcas, which codes for ProDH, in order to characterize the enzyme critical for proline metabolism. Sequence analysis showed that JcProDH is closely related to ProDH from other plants. Semiquantitative analysis revealed that JcProDH was expressed in all the tissues examined, with the highest expression in seeds and flowers. The overexpression of JcProDH in yeast confirmed its resistance to low temperatures, drought, and salt stress.

Materials and Methods

Plant Materials and Treatments

The seeds of J. curcas were collected from Yuanmou, Yunnan Province, China. The seeds were surface sterilized in 1 % CuSO4 for 15 min and rinsed thoroughly with sterilized distilled water according to our previous methods [27] and presoaked for imbibition in distilled water for 24 h. The soaked seeds were sowed on six layers of wet filter paper in trays with covers and germinated at 26 °C in the dark for 5 days. The germinated seeds were selected and transferred to pots containing sterilized soil with perlite, peat, sand (1:2:1), and wetted 1/2 MS [28] basal salts. They were kept in a climate chamber at 26/20 °C (day/night) temperature under a 16-h photoperiod (30 μmol m−2 s−1) and sequentially grown for 7 days. To explore the effect of different stressors on JcProDH expression, the 2-week-old seedlings were subjected to different stress treatments. Some plants were exposed to 5 °C for 2, 4, 6, 8, 12, and 16 h; some plants were exposed to 42 °C for 2, 4, 6, 8, 12, and 16 h; some plants were exposed to salt stress by adding NaCl to 300 mM and exposed for 2, 4, 6, 8, 12, and 16 h; and some plants were exposed to drought stress by adding PEG6000 to 30 % and exposed for 2, 4, 6, 8, 12, 16 h. After these treatments, the young leaves of J. curcas were harvested, promptly frozen in liquid nitrogen, and stored at −70 °C until RNA extraction.

Young seeds, roots, stems, and flowers of J. curcas were collected from Yuanmou, Yunnan Province, China, and instantly frozen in liquid nitrogen and stored at −70 °C until RNA extraction.

Full-Length cDNA Cloning and Sequencing of JcProDH

Total RNA was extracted from young leaves of J. curcas using a plant RNA isolation reagent (Tiangen Biotech, Beijing) following the manufacturer’s instructions. After being treated with DNaseI (Takara, Dalian, China) to remove traces of contaminant genomic DNA, the first-strand cDNA was synthesized using 5 μg of total RNA with oligo(dT)18 (TransScript First-Strand cDNA Synthesis SuperMix). Based on our early transcriptome data and the sequence in GenBank (Accession number GAHK01001669) [29], two primers were designed with Primer Premier (Version 5.0) (Premier Biosoft International, USA) and synthesized for amplifying the full-length cDNA. Their sequences are as follows: JcProDH_F 5′-GTCTTAGGTACCCAAATCCCTTTCTCCAAAAC-3′ and JcProDH_R 5′-GCTACTATCAGTACTCATAGCCACAAACTTCA-3′. A Kpn I site (underlined) was added to the 5′ end of the forward primer (JcProDH_F), and a Spe I site (underlined) was added to the reverse primer (JcProDH_R). The PCR was performed as follows: 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 75 s; and a final extension step of 72 °C for 10 min. After purification, the PCR products were cloned into the pYES2 vector (TaKaRa) and sequenced on both strands (BGI, Shenzhen, China).

Bioinformatics Analysis of JcProDH

The sequence homology and the deduced amino acid sequence comparisons were carried out online using BLAST at NCBI (http://www.ncbi.nlm.nih.gov/blast/) and Expasy (http://cn.expasy.org). The theoretical isoelectric point (pI) and molecular weight (Mw) of the proteins were determined using the ProtParam tool (http://web.expasy.org/protparam/). The signal peptide was predicted with SignalP 3.0 (http://www.dtu.dk/services/SignalP/). The homology-based 3D structural modeling of JcProDH was accomplished by Swiss-modeling and Phyre program (Version 2.0). The VMD program (Version 1.8.7) was used for displaying the 3D structure. Multiple alignment analysis of the full-length plant ProDH amino acid sequence from other species was performed using GenDOC (Version 2.6.002) and ClustalW program. The phylogenetic tree was constructed using the MEGA program (Version 4.0) with neighbor-joining methods.

RT-PCR Analysis of JcProDH Gene Expression

For expression analysis of the JcProDH gene, a reverse transcription PCR (RT-PCR) was conducted. Total RNA was extracted separately from different tissues and young leaves from the different treatments (see “Plant Materials and Treatments”). J. curcas 18S ribosomal RNA (18S rRNA) (GeneBank accession No. AY823528) was used as an internal standard. Gene-specific primers are presented in Table 1. PCR reactions were conducted under the following conditions: one cycle of 94 °C for 5 min, 33 cycles at 94 °C for 30 s, at 55 °C for 30 s, and at 72 °C for 35 s, and 1 cycle of 72 °C for an additional 5 min. All RT-PCR amplifications with the same RNA samples were repeated three times. PCR products (3 μl) were separated on 1.0 % agarose gels stained with 10 μg ml−1 Goldwave and visualized using a UV transilluminator (Bio-Rad Universal Hood II 720BR/02613). The band intensities of the PCR products were analyzed using Quantity One (Version 4.6.2). The ratios between the quantity of messenger RNA (mRNA) in the JcProDH gene and those in 18S rRNA were calculated. The results reflected the relative quantities of JcProDH at the mRNA level. The data were the means of the three different experiments ± SD.

Table 1 Primer sequence used in the RT-PCR experiments
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Expression Analysis of JcProDH in Saccharomyces cerevisiae (Yeast)

The full-length coding region of the JcProDH gene was amplified by PCR using the forward primer JcProDH_F containing the Kpn I site and the reverse primer JcProDH_R containing the Spe I site. The pYES2 vector and the PCR products were digested with Kpn I and Spe I and ligated to obtain the expression plasmids pYES2-JcProDH. The recombinant plasmid pYES2-JcProDH and the empty vector pYES2 were transformed into the yeast strain INVSc1.

The colony PCR was used for the identification of positive clones. Subsequently, a single colony of recombinant and contrast yeast were grown in SC-U liquid medium containing 2 % glucose and cultured overnight at 30 °C and 170 rpm. The cells were then harvested by centrifuging at 4000 rpm for 1 min, dissolved in 20 ml of SC-U liquid medium with 2 % galactose until the optical density (OD600) of 0.4 was reached, and induced expression 36 h at 30 °C and 170 rpm. After centrifuging at 4000 rpm for 1 min, the different stress treatments were evaluated as follows: contrast (no stress), resuspension of yeast with 1 ml sterile water until the OD600 reached 2.0; cold temperature stress, resuspension of yeast in 1 ml sterile water and maintained at 15 °C for 1 h; high salt stress, resuspension of yeast in 1 ml 300 mM NaCl and maintained at 30 °C for 24 h; and drought stress, resuspension of yeast in 1 ml 30 % PEG6000 and maintained at 30 °C for 24 h. The treated and control yeasts were then inoculated into the SC-U solid medium with 2 % glucose and cultured at 30 °C for 2–3 days. In addition, after expression, the yeast cells were diluted by adding SC-U liquid medium with 2 % galactose and then cultured at 15 °C at 170 rpm. The other yeast cells were diluted by adding the SC-U liquid medium with 2 % galactose and containing 300 mM NaCl and 30 % PEG6000 and then cultured at 30 °C and 170 rpm. The initial yeast concentration was modified until the OD600 of 0.2 was reached. The yeast suspension was harvested at different times after inoculation, and the OD600 values were detected using a spectrophotometer. Three independent measurements were carried out simultaneously. The growth state of yeast was examined by comparing their growth curves.

Results

Molecular Cloning of the Full-Length cDNA of JcProDH

The full-length JcProDH cDNA isolated from J. curcas seedlings was 1674 bp and included 5′ and 3′ flanking regions of 40 and 149 bp, respectively. JcProDH contained an open reading frame of 1485 bp encoding a putative protein of 494 amino acid residues with a predicted molecular mass of 54 kDa and a predicted isoelectric point of 8.27 (Figs. 1 and 2). A comparison between the genomic DNA sequence and the isolated cDNA indicated that JcProDH contains four exons and three introns. The full-length cDNA sequence of JcProDH has been deposited in the GenBank database under the accession number of KF879446.1.

Fig. 1
figure 1

Agarose gel electrophoresis for JcProDH cloning by PCR. a Full-length cDNA amplification and b colony PCR of recombinant plasmid (pYES2-JcProDH), Trans 2K Plus II DNA Marker (TransGene, Beijing, China) (lane M)

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Fig. 2
figure 2

Nucleotide and deduced amino acid sequences of JcProDH cDNA from J. curcas. The start codon (ATG) and stop codon (TAG) are underlined

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Bioinformatic Analysis of JcProDH

SignalP 3.0 analysis (http://www.dtu.dk/services/SignalP/) showed that JcProDH had no signal peptide that was synthesized in the cytoplasm. A total of 15 O-glycosylation sites of JcProDH were identified by the YinOYang (http://www.cbs.dtu.dk/services/YinOYang/) analysis, and the greatest possible sites were located in Ser23, Ser24, Ser27, Ser184, Try25, and Try54 along the polypeptide chain. Based on the hierarchical neural analysis by ScanProsite (http://prosite.expasy.org/scanprosite/), JcProDH protein was composed of 52.63 % α-helix (260AA), 6.07 % β-sheet (30AA), 13.56 % extended strand (67AA), and 27.73 % random coil (137AA).

Multiple sequence alignments using GenDOC and BLAST revealed that JcProDH was very similar to many other ProDH from higher plants at the amino acid level, such as Populus trichocarpa (Pt), Vitis vinfera (Vv), Nicotiana tabacum (Nt), and Actinidia deliciosa (Ad), indicating that JcProDH belonged to the ProDH superfamily. However, the N-terminus regions of JcProDH from other species have low sequence identity (Fig. 3). A phylogenetic analysis of various ProDH sequences listed in GenBank was conducted using the MEGA program. A phylogram depicting the relationships between ProDH from several species is shown in Fig. 4.

Fig. 3
figure 3

Sequence alignment of JcProDH from J. curcas with ProDH from other organisms. Alignment was performed by the GenDOC program with the organisms such as Actinidia deliciosa (ProDH_Ad), Arabidopsis thaliana (ProDH_At), Arabis stelleri (ProDH_As), Brassica napus (ProDH_Bn), Brassica oleracea (ProDH_Bo), Capsicum annuum (ProDH_Ca), Glycine max (ProDH_Gm), Jatropha curcas (ProDH_Jc), Medicago sativa (ProDH_Ms), Medicago truncatula (ProDH_Mt), Nicotiana tabacum (ProDH_Nt), Oryza sativa (ProDH_Os), Populus trichocarpa (ProDH_Pt), and Vitis vinifera (ProDH_Vv). Conserved amino acid residues in all the sequences used in this alignment are in black boxes, while similar amino acids are in gray boxes

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Fig. 4
figure 4

Phylogenetic analysis of JcProDH protein with other plants by MEGA from ClustalW alignments. The phylogenetic tree was constructed using MEGA based on the neighbor-joining method. The ProDH used in phylogenetic tree analysis were those from Actinidia deliciosa (GenBank accession No. ABK91948.1), Arabidopsis thaliana (GenBank accession No. NM_113981.5), Arabis stelleri (GenBank accession No. HM013940.1), Brassica napus (GenBank accession No. EU375567.1), Brassica oleracea (GenBank accession No. GU568241.1), Capsicum annuum (GenBank accession No. FJ911549.1), Glycine max (GenBank accession No. NM_001250359.1), Medicago sativa (GenBank accession No. AY556386.1), Medicago truncatula (GenBank accession No. XM_003621569.1), Nicotiana tabacum (GenBank accession No. AY639145.1), Oryza sativa (GenBank accession No. NM_001071853.1), Populus trichocarpa (GenBank accession No. XM_002329277.1), and Vitis vinifera (GenBank accession No. XM_002282733.1)

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The crystal structure of the Geobacter sulfurreducens ProDH has been published (RCSB accession number 4F9I). The homology-based 3D structural modeling of JcProDH was analyzed by Swiss-modeling on the basis of G. sulfurreducens ProDH and displayed by VMD (Version 8.0) (Fig. 5a). The Swiss PDB viewer (Version 3.7) was used to examine the modeling results and to calculate the Ramachandran map. The Ramachandran plot reflected the stereochemical quality. Analysis of the distribution patterns of φ angle and ψ angle indicated whether the simulation structure and natural structure showed the same trend or not. The validation of the JcProDH modeling is in agreement with the stereochemistry and stabilization (Fig. 5b). The 3D structure of eukaryotic ProDH has not yet been determined because of the difficulty in preparing a highly purified enzyme, but it is predicted that the eukaryotic ProDH will also exhibit a distorted (βα)8 barrel fold in the catalytic core [30, 31], like JcProDH. All the results from the bioinformatics analyses strongly suggest that JcProDH is a functional plant ProDH protein involved in the catabolism of proline.

Fig. 5
figure 5

The 3D structure of JcProDH constructed by homology-based modeling. a The 3D structure of JcProDH using the cartoon model. b Ramachandran map of the JcProDH modeling structure generated by PROCHECK

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Expression Analysis of JcProDH

In order to estimate JcProDH mRNA expression levels and distributive patterns among tissues, semiquantitative RT-PCRs were performed on five organs of wild plants, as previously described. The results showed that the JcProDH gene was transcriptionally active in all tested tissues, suggesting that it is a constitutively expressed gene but at varying levels of expression in different tissues. JcProDH is expressed strongly in seeds and flowers (Fig. 6a, b) and is relatively weakly expressed in stems, roots, and leaves.

Fig. 6
figure 6

Expression of the JcProDH gene in different tissues of J. curcas. 18S rRNA from J. curcas was used as an internal control. A representative result of three independent measurements is shown. a The PCR product for the JcProDH gene and 18S rRNA from roots, stems, leaves, flowers, and seeds of J. curcas. b Relative quantities of JcProDH gene mRNAs detected per lane were normalized using 18S rRNA intensities

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Total RNA (3 μg) was subjected to RT-PCR analysis to examine the effect of environmental stress on JcProDH expression in J. curcas. The results showed that the JcProDH gene transcripts of the plants exposed to 5 °C cold stress, 42 °C heat stress, 300 mM NaCl, and 30 % PEG6000 were significantly higher than those of the control plants (Fig. 7A1, B1, C1, D1). The expression level in different stress-treated leaves increased by 310, 130, 340, and 390 %, respectively, as compared to the control (Fig. 7A2, B2, C2, and D2), but downregulated after reaching the highest expression level.

Fig. 7
figure 7

Expression of the JcProDH gene in the leaves of J. curcas seedlings under different stress conditions. 18S rRNA from J. curcas was used as an internal control. One representative result of three independent measurements is shown. A1 cold stress at 5 °C for 2, 4, 6, 8, 12, and 16 h; B1 high temperature stress at 42 °C for 2, 4, 6, 8, 12, and 16 h; C1 salt stress at 300 mM NaCl for 2, 4, 6, 8, 12, and 16 h; and D1 drought stress at 30 % PEG for 2, 4, 6, 8, 12, and 16 h. Relative quantities of the JcProDH gene mRNAs detected per lane were normalized using 18S rRNA intensities and were listed as A2, B2, C2, and D2

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Effect of Different Stressors on the Growth and the Expression of Transformed Yeast Cells

To test whether stressors such as salt, drought, and low temperature affected the expression of JcProDH protein, yeast cells that transformed with the empty vector pYES2 and the recombinant plasmid pYES2-JcProDH were inoculated into fresh SC-U medium (2 % galatose) containing 300 mM NaCl, 30 % PEG6000, and under 15 °C cold condition. As shown in Fig. 8a–d, the growth curves of the two kinds of cells are parallel under no stress condition. The transformed cells with the recombinant plasmid pYES2-JcProDH grew comparatively slower than those with empty vectors under different stress conditions. In this study, growth measurements under different kinds of stress revealed that JcProDH has protective functions in yeast and may be responsible for the improvement in growth. These results indicated that JcProDH, a negative regulator, played an important role in the stress resistance of yeast.

Fig. 8
figure 8

Effect of different stress conditions on the growth of transformed yeast with the recombinant plasmid pYES2-JcProDH and pYES2 empty vector. a Growth curves of yeast cells in liquid medium under normal conditions. b Growth curves of yeast cells in liquid medium at 300 mM NaCl. c Growth curves of yeast cells in liquid medium at 30 % PEG6000. d Growth curves of yeast cells in liquid medium at a low temperature of 15 °C

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Discussion

Proline has long been considered a compatible osmolyte. Recent studies highlight its multiple functions in stress adaptation, recovery, signaling, apoptosis, and programmed cell death. Compartmentalization of proline metabolism implies that intracellular proline transport occurs between the cytosol, chloroplasts, and mitochondria and also indicates the complexity of functional diversification of proline metabolism. Proline metabolism plays an important role in the stabilization of proteins and protein complexes [32], protection of the photosynthetic apparatus and enzymes [33, 34], detoxification of ROS [35] in the chloroplast and cytosol, stabilization of the redox balance, maintenance of cellular homeostasis, programmed cell death [36] in mitochondria, regulation of metabolite pools and the redox balance [37], and expression of numerous genes based on the metabolic signal [38, 39] in the cytosol. This shows that the balance of the proline content in cells is crucial for the growth and development of the plant.

The complexity of the regulation of proline metabolism and the multiple functions of proline pose hurdles in the plant improvement practices for agronomic interest by modifying the expression of genes involved in the metabolism of proline. One of the methods to counter this is to enhance proline biosynthesis by increasing the expression of rate-limiting genes in transgenic plants. Moreover, the inhibition of proline degradation can also lead to proline accumulation. A study associated with antisense ProDH reported to improve salt and cold tolerance [40]. Another study reported that the hypersensitivity of transgenic lines and mutants to proline was due to the altered levels of proline dehydrogenase [41]. In addition, ProDH was detected in the cytosol of soybean nodules, indicating that proline oxidation provides energy for bacteroids during nitrogen fixation [42].

Because of the difficulty in obtaining a highly purified enzyme, the 3D structure of eukaryotic ProDH is yet to be determined. Therefore, the distribution, function, structure, and catalytic mechanism of ProDH need to be investigated further. Detailed research was conducted on hyperthermophilic archaea [43], Escherichia coli [44], and Salmonella typhimurium [45]. PutA from E. coli and S. typhimurium, a membrane-bound dimeric enzyme with a molecular mass of 144 kDa, can catalyze not only proline oxidation to P5C by ProDH but also P5C dehydrogenase to l-glutamate by P5C dehydrogenase (P5CDH), as the two catalytic domains are fused into a single polypeptide chain in this enzyme [46]. By contrast, ProDH from eukaryotes and from some bacteria are monofunctional enzymes, which are separated from P5CDH. To date, three groups of ProDH, Tp-ProDH, Ph-ProDH1, and Pc-ProDH, were classified in hyperthermophilic archaea based on their enzymatic properties such as subunit, structure, substrate, and thermostability. SDS-PAGE analysis of the purified enzyme revealed that Tp-ProDH formed a complex consisting of four different subunits α, β, γ, and δ representing approximately 120 kDa. The genes pdhA, pdhB, pdhF, and pdhX, encoding the α, β, γ, and δ subunits, respectively, were arranged as a gene cluster in tandem [47]. Ph-ProDH is a second novel ProDH complex with a heterooctameric α4β4 structure representing approximately 440 kDa [48]. The β subunit with an adenine-binding motif in the N-terminal region, which functionally combined FAD, was recognized as the site for catalytic action both in Tp-ProDH and Ph-ProDH1. In addition, an adenine-binding motif and [2Fe-2S] iron-sulfur cluster (ISC) motif containing four cysteine residues are conserved in the N-terminal of the α subunit (combined with FAD in the adenine-binding motif of Tp-ProDH, and with ATP of Ph-ProDH). A third type of the ProDH isozyme, Pc-ProDH, forms a homodimer. The gene encoding Pc-ProDH was determined to be Pcal_1655, and it did not form a gene cluster like the αβγδ- and α4β4-type ProDHs. However, the Pc-ProDH exhibits a relatively broad substrate specificity, such as l-proline and l-hydroxyproline. Thus, it differs from the αβγδ- to α4β4-type ProDHs, which contain only l-proline as a substrate.

Two homologous genes have been identified to encode proline dehydrogenases in eukaryotes such as Arabidopsis [41] and tobacco [49]. These results have opposite effects on ProDH1 transcription, which is repressed during daylight and induced in darkness [50, 51]. Proline catabolism is activated in the dark and is controlled by ProDH, whereas ProDH transcription is activated by rehydration but suppressed by dehydration [52]. Genetic analysis has revealed the joint control of ProDH expression and activation by a necessary network of groups—the S basic leucine zipper protein (bZIP) transcription factor [53]. The bZIP transcription factors (AtbZIP-1, AtbZIP−11, AtbZIP−44, AtbZIP−53) have been identified as candidates for binding to the ProDH-activated motif (proline and hypo-osmolarity-responsive element (PRE) motif with ACTCAT) [54, 55], which is necessary for the activation of the ProDH gene. These results suggest that it might not be the actual proline content but the enhanced rate of proline biosynthesis that is important for stress adaptation. Hence, the fact is that proline can act as a signaling, protective, and regulatory molecule, and further studies on the role of key enzymes such as P5CS, P5CR, OAT, ProDH, and P5CDH in proline metabolism are required. A complete understanding of the molecular mechanism of proline metabolism regulation could lead to an improvement in the tolerance of plants to environmental stress conditions. Based on our previous data of the transcriptome and digital gene expression (DGE) of cold-hardened J. curcas [56, 29], as well as the RT-PCR results presented herein (Figs. 6 and 7), JcProDH was found with tissue-differential expression and abiotic stress-induced expression and then gradually depressed expression. Furthermore, JcProDH overexpression in yeast could significantly reduce the abiotic stress resistance of host cells (Fig. 8), probably by increasing the proline degradation. Collectively, these data provided an explicit insight that JcProDH is plausibly involved in the stress response and tolerance in J. curcas and naturally eligible for applications in its correlated genetic engineering [41].