Functional Characterization of the Stipa purpurea P5CS Gene under Drought Stress Conditions

Free proline has multiple functions in plant cells, such as regulating osmotic potential and protecting both proteins and cell membranes. The expression of Δ1-Pyrroline-5-carboxylate synthase (P5CS), a key enzyme in the proline biosynthetic pathway, increases under drought, salt and cold stress conditions, causing plant cells to accumulate large amounts of proline. In this study, we cloned and identified the P5CS gene from Stipa purpurea, which has a full-length of 2196 bp and encodes 731 amino acids. A subcellular localization analysis indicated that SpP5CS localized to the cytoplasm. The ectopic overexpression of SpP5CS in Arabidopsis thaliana resulted in higher proline contents, longer roots, higher survival rates and less membrane damage under drought stress conditions compared with wild-type controls. SpP5CS-overexpressing A. thaliana was more resistant to drought stress than the wild type, whereas the deletion mutant sp5cs was less resistant to drought stress. Thus, SpP5CS may be a potential candidate target gene for increasing plant resistance to drought stress.


Introduction
Almost half of the agricultural area in the world is at risk of drought, which has become the most important problem in global agriculture, seriously affecting crop yields [1]. Increasing crop yields under drought conditions is a major goal of plant breeding, and physiological and molecular mechanisms of plant drought resistance have been studied to develop targets for molecular breeding. Drought can cause plant cells to produce excess reactive oxygen species (ROS) and free radicals. The accumulation of ROS and free radicals leads to membrane lipid peroxidation and subcellular structure damage, which destroys the integrity of plant cell membranes [2]. Water is an important solvent and reaction substrate in cells, and many biological macromolecules become permanently deformed and functionless after dehydration. To survive, plants under drought stress conditions accumulate some low molecular weight metabolites called osmolytes, including proline, glycine betaine, mannitol and quaternary amines. These osmolytes act as solutes to reduce the water potential in cells to improve the water absorption or water retention capacity of the cell while maintaining the cell volume and protecting the cell membrane [3].
The amino acid proline, as a common osmolyte, has many effects on plant cells under drought conditions. Under osmotic stress conditions, large quantities of proline accumulate in plant cells and the proline accumulation level varies among species, with levels in some 2. Results

Cloning and Structural Analysis of SpP5CS
Based on the EST sequence obtained by transcriptome sequencing, we used RACE (rapid amplification of cDNA ends) technology to obtain the CDS sequence of SpP5CS. The SpP5CS (GenBank: MZ710734) contains an open reading frame of 2196 bp that encodes 731 amino acids. The molecular weight of the SpP5CS protein is 78.95 kDa, and the theoretical isoelectric point is 5.95. The SMART protein structural analysis of SpP5CS revealed two functional domains: γ-glutamyl kinase (GK) and glutamic-γ-semialdehyde dehydrogenase (GSA DH) (Figure 1a). To analyze the relationship between SpP5CS and P5CS genes from other species, we extracted the P5CS gene sequences of Brachypodium distachyon, Lolium perenne, Triticum aestivum, Aegilops tauschii, O. sativa, Oryza brachyantha, Zea mays, Sorghum bicolor, A. thaliana, Helianthus tuberosus, L. regale, M. truncatula, P. vulgaris, B. napus and Setaria italica through the BLAST tool of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 6 August 2021). A phylogenetic tree was constructed using the BLAST tool of MEGA X and the neighbor-joining method. SpP5CS is highly similar to P5CS from B. distachyon (Figure 1b). The results indicate that SpP5CS belongs to the P5CS2 isoform.

The Expression of SpP5CS and the Proline Content Analysis
qRT-PCR was used to detect the expression of the SpP5CS in S. purpurea leaves after exposure to different treatments. After growing for 2 weeks, S. purpurea was exposed to different abiotic stress conditions (cold, salt, PEG and soil drought) for different times. After 6 h of the cold-stress treatment at 4 • C, SpP5CS expression increased to two times that of the control, but after 12 h of continuous treatment, the expression level decreased to 1.7 times that of the control. Additionally, after being treated with 150-mM NaCl for 6 h, SpP5CS expression increased to 1.6 times that of the control, and it continued to slowly increase to 1.9 times that of the control by 12 h. After 12 h of treatment with 30% PEG 6000, the expression of SpP5CS increased slightly compared to the control. After 14 d of soil drought, the expression of SpP5CS increased 3.8 times compared with the control, and after 1 d of rehydration, the expression increased significantly, to 85.7 times that of the control ( Figure 2). The SpP5CS gene was upregulated under the cold, salt and PEG stress conditions. The expression of the SpP5CS gene was strongly and significantly upregulated during soil drought and rehydration treatment. that of the control, but after 12 h of continuous treatment, the expression level decreased to 1.7 times that of the control. Additionally, after being treated with 150-mM NaCl for 6 h, SpP5CS expression increased to 1.6 times that of the control, and it continued to slowly increase to 1.9 times that of the control by 12 h. After 12 h of treatment with 30% PEG 6000, the expression of SpP5CS increased slightly compared to the control. After 14 d of soil drought, the expression of SpP5CS increased 3.8 times compared with the control, and after 1 d of rehydration, the expression increased significantly, to 85.7 times that of the control ( Figure 2). The SpP5CS gene was upregulated under the cold, salt and PEG stress conditions. The expression of the SpP5CS gene was strongly and significantly upregulated during soil drought and rehydration treatment.

Identification of Ectopically Expressed 35S:SpP5CS-GFP Transgenic Plants
To further explore SpP5CS's functions, we cloned the gene and transformed it into A. thaliana, with the expression being driven by the 35S Cauliflower mosaic virus promoter. The primers SpP5CS-GFP-F and SpP5CS-GFP-R were used to successfully identify the transgenic plants (Figure 3a). We received six transgenic lines, and the transformation efficiency was about 0.1%. We observed the SpP5CS-GFP fusion protein in the cytoplasm from transgenic A. thaliana using a laser confocal microscope (Figure 3b). The experiment has three biological replicates, and the error bar denotes the standard deviation. The data are analyzed by one-way ANOVA (Tukey's test). Different letters indicate significant differences among the relative expression levels of the genes at p < 0.05.

Identification of Ectopically Expressed 35S:SpP5CS-GFP Transgenic Plants
To further explore SpP5CS's functions, we cloned the gene and transformed it into A. thaliana, with the expression being driven by the 35S Cauliflower mosaic virus promoter. The primers SpP5CS-GFP-F and SpP5CS-GFP-R were used to successfully identify the transgenic plants ( Figure 3a). We received six transgenic lines, and the transformation efficiency was about 0.1%. We observed the SpP5CS-GFP fusion protein in the cytoplasm from transgenic A. thaliana using a laser confocal microscope ( Figure 3b).

SpP5CS Overexpression Enhances Drought Tolerance
To analyze whether SpP5CS expression enhances the plant's drought resistance func-

SpP5CS Overexpression Enhances Drought Tolerance
To analyze whether SpP5CS expression enhances the plant's drought resistance function, the SpP5CS gene was first transformed into a P5CS deletion mutant to obtain a p5cs/SpP5CS complemented plant. Then, the WT, mutant, p5cs/SpP5CS-complemented and OE-SpP5CS A. thaliana seeds were cultured on 1/2 Murashige and Skoog (MS) medium supplemented with 5% PEG 6000 and ordinary 1/2 MS medium, and the phenotypes were observed. On 1/2 MS medium containing 5% PEG 6000, the average root length of the OE-SpP5CS plants was 2.17 cm, and the average root length of a mutant plants was 0.69 cm. The root lengths of OE-SpP5CS plants under drought conditions were significantly longer than those of the other types of plants, whereas the mutant plants themselves were significantly shorter than the other types of plants (Figure 4a,b). The average root lengths were 1.18 cm and 1.54 cm, respectively, on 1/2 MS medium, with no significant differences among the root lengths of the various plants.

SpP5CS Overexpression Enhances Drought Tolerance
To analyze whether SpP5CS expression enhances the plant's droug tion, the SpP5CS gene was first transformed into a P5CS deletion m p5cs/SpP5CS complemented plant. Then, the WT, mutant, p5cs/SpP5 and OE-SpP5CS A. thaliana seeds were cultured on 1/2 Murashige an dium supplemented with 5% PEG 6000 and ordinary 1/2 MS medium, a were observed. On 1/2 MS medium containing 5% PEG 6000, the ave the OE-   Seeds were grown on the 1/2 MS medium and 1/2 MS medium supplemented with 5% PEG 6000 for 10 d; (b) Root lengths of WT, OE-SpP5CS, p5cs/SpP5CS-complemented and p5cs mutant plants grown under normal and drought stress conditions. Data were obtained from three independent experiments. Each column represents the mean ± SD, and the error bar denotes the standard deviation. Different letters indicate significant differences in the root length (one way ANOVA, Tukey's test, p < 0.05).
To further identify the functions of SpP5CS in improving plant drought resistance, WT, mutant, p5cs/SpP5CS-complemented and OE-SpP5CS A. thaliana seedlings were planted in square plastic pots and subjected to the soil drought treatment. After 15 d of the drought treatment, the growth status of OE-SpP5CS A. thaliana was the best among all the plant types ( Figure 5a). Then, their survival rates and electrical conductivity levels were measured. As shown in Figure 5c, after 5 d of drought treatment, the survival rate of OE-SpP5CS A. thaliana was 87.4%, and the plants showed the lowest degree of damage at 6.4%, while the survival rate of the mutant A. thaliana was 66.1%, and the plants showed the greatest degree of damage at 33%. After 12 d of drought treatment, the survival rate of OE-SpP5CS A. thaliana was 63.6%, and the damage degree was 36.4%, while the survival rate of the mutant A. thaliana was 25.8%, and the damage degree was 74.2%. After 15 d of drought treatment, the survival rate of the OE-SpP5CS A. thaliana was 56.1%, and the damage degree was 43.9%, while the survival rate of the mutant A. thaliana was 13.7%, and the damage degree was 86.3% (Figure 5b,c). The survival rates and degrees of damage for the WT and p5cs/SpP5CS-complemented A. thaliana were at intermediate levels, with little differences between the two lines. Therefore, the overexpression of SpP5CS improved the drought resistance of transgenic A. thaliana.
of OE-SpP5CS A. thaliana was 87.4%, and the plants showed the lowest degree of damage at 6.4%, while the survival rate of the mutant A. thaliana was 66.1%, and the plants showed the greatest degree of damage at 33%. After 12 d of drought treatment, the survival rate of OE-SpP5CS A. thaliana was 63.6%, and the damage degree was 36.4%, while the survival rate of the mutant A. thaliana was 25.8%, and the damage degree was 74.2%. After 15 d of drought treatment, the survival rate of the OE-SpP5CS A. thaliana was 56.1%, and the damage degree was 43.9%, while the survival rate of the mutant A. thaliana was 13.7%, and the damage degree was 86.3% (Figure 5b,c). The survival rates and degrees of damage for the WT and p5cs/SpP5CS-complemented A. thaliana were at intermediate levels, with little differences between the two lines. Therefore, the overexpression of SpP5CS improved the drought resistance of transgenic A. thaliana.  The soil drought treatment adopts continuous withholding water. The experiment has three biological and technical replicates. The data are expressed as the mean ± SD from three independent experiments. Different letters indicate significant differences in the degree of damage or survival rate (one-way ANOVA, Tukey's test, p < 0.05).

Variation in the Proline Content
To investigate the effects of SpP5CS, the proline content was determined for WT, p5cs mutant, p5cs/SpP5CS-complemented and OE-SpP5CS A. thaliana using the acidicninhydrin assay. After 5 d of drought treatment, the proline content of the OE-SpP5CS A. thaliana was significantly higher than the other plant types and more than five times that of the WT. At this time, the proline content of the mutant was the lowest among all the plants at approximately half the WT value. After 12 d of drought treatment, the proline content of the OE-SpP5CS A. thaliana was still the highest, but the differences compared with the WT decreased. The mutant still had the lowest content. After 15 d of drought treatment, the proline contents of the plants were nearly the same ( Figure 6). These results indicate that the SpP5CS gene improves the drought resistance of plants by increasing their proline content. of the OE-SpP5CS A. thaliana was still the highest, but the differences compared w WT decreased. The mutant still had the lowest content. After 15 d of drought trea the proline contents of the plants were nearly the same ( Figure 6). These results in that the SpP5CS gene improves the drought resistance of plants by increasing their p content.

Molecular Characterization of SpP5CS
As an important gene involved in proline synthesis, P5CS has been reported sativa, P. vulgaris, A. thaliana and S. bicolor, and it contains the conserved domains G GSA DH [28,29]. In this study, we cloned the full-length CDS sequence of SpP5C from S. purpurea. Then, we performed multiple sequence alignment and phylog analysis of the P5CS protein of S. purpurea and other species. We found that they similar structural domains (GSA DH and GK), and SpP5CS and P5CS2 of multiple s are in the same branch. Due to their structural similarity, SpP5CS may have fun similar to P5CS2 in other species. According to previous reports, the research of mainly focused on growth and development, drought resistance and salt resistanc 22,30]. We speculate that SpP5CS has a similar effect. Previous subcellular locali studies have shown that P5CS2 are localized in the cytoplasm [30]. The subcellular ization results show that SpP5CS is localized in the cytoplasm, which is consisten the previous report.

Expression Analysis of SpP5CS
The gene expression analysis shows differences in the gene expressions under ent environmental conditions, which helps predict the gene function. The P5CS ge sponds to osmotic stress and increases the proline content of plants to improve o

Molecular Characterization of SpP5CS
As an important gene involved in proline synthesis, P5CS has been reported in O. sativa, P. vulgaris, A. thaliana and S. bicolor, and it contains the conserved domains GK and GSA DH [28,29]. In this study, we cloned the full-length CDS sequence of SpP5CS gene from S. purpurea. Then, we performed multiple sequence alignment and phylogenetic analysis of the P5CS protein of S. purpurea and other species. We found that they have similar structural domains (GSA DH and GK), and SpP5CS and P5CS2 of multiple species are in the same branch. Due to their structural similarity, SpP5CS may have functions similar to P5CS2 in other species. According to previous reports, the research of P5CS2 mainly focused on growth and development, drought resistance and salt resistance [18][19][20][21][22]30]. We speculate that SpP5CS has a similar effect. Previous subcellular localization studies have shown that P5CS2 are localized in the cytoplasm [30]. The subcellular localization results show that SpP5CS is localized in the cytoplasm, which is consistent with the previous report.

Expression Analysis of SpP5CS
The gene expression analysis shows differences in the gene expressions under different environmental conditions, which helps predict the gene function. The P5CS gene responds to osmotic stress and increases the proline content of plants to improve osmotic resistance. In S. italica, SiP5CS2 is remarkably upregulated after 9 d of drought treatment [31]. After 10 d of salt stress, the expression of Opuntia streptacantha P5CS increases [32]. Salt-tolerant varieties of O. sativa have more OsP5CS mRNA transcripts and proline under high-salt than normal conditions [14]. The expression of Setaria viridis P5CS2 is upregulated after 6 and 10 d of an osmotic stress treatment in the form of an external PEG 8000 application [33]. In addition to the response to osmotic stress, the P5CS gene is also upregulated during cold stress. After 24 h of a cold treatment at 4 • C, the transcription level of the P5CS gene in L. perenne is significantly upregulated [34]. The expression of SpP5CS was significantly upregulated under soil drought, cold and salt stress, which was consistent with the results of the other species. According to reports, different isoforms of P5CS genes are induced by different stresses. In Sorghum bicolor, the transcription levels of SbP5CS1 and SbP5CS2 are both upregulated under drought and salt stress. Meanwhile, the transcription level of SbP5CS2 increased faster and more obviously than that of SbP5CS2 under the induction of methyl jasmonic acid [29]. In our study, SpP5CS strongly responds to soil drought, which may be the result of S. purpurea adapted to arid environment for a long time. In addition, the proline content of S. purpurea increased significantly after 14 days of soil drought and 1 day after rehydration. We speculate that S. purpurea accumulates proline by increasing the expression of the SpP5CS gene to improve the drought resistance after drought stress.

Potential Mechanism of SpP5CS for Improving Plant Resistance to Drought Stresses
There are various drought resistance mechanisms in plants, among which, an increasing root length and increasing osmotic pressure regulatory substances are very common. As a key enzyme in the proline biosynthesis pathway, P5CS also enhances root growth, and it plays an important role in plant resistance to drought. The electrical conductivity of cell leakage indirectly evaluates the degree of cell membrane damage [35]. OE-SpP5CS A. thaliana showed the lowest degree of membrane damage compared with WT. P5CS transgenic rice increase their proline contents under water shortage conditions and have increased fresh shoot weights, by 50-95%, after PEG treatments [23]. The overexpression of SpP5CS in A. thaliana increases the proline content to combat drought stress. Combining the results of the heterologous expression in A. thaliana and qPCR in S. purpurea, SpP5CS can enhance the drought resistance of plants by increasing the proline production.
S. purpurea is widely distributed in the precipitation gradient of the Qinghai-Tibet Plateau, and it can still survive in extremely arid areas. Previous studies have revealed that S. purpurea in arid regions have a higher content of proline than S. purpurea in humid regions, and a transcriptome analysis has shown that the P5CS gene is upregulated at a higher level in arid regions [36]. The proteomic analysis also suggests that the P5CS protein in S. purpurea from more arid areas has a high level of upregulation [37]. Furthermore, we verified the single gene function of SpP5CS to show that SpP5CS improves the drought resistance of plants. These studies all indicate that the upregulation of SpP5CS gene leads to an increase in the proline content to enhance osmotic regulation, which is an important part of the drought-resistant mechanism of S. purpurea.

Plant Materials
Mature S. purpurea seeds were collected from Nyima County, Tibet (32 • 00 05 N, 86 • 50 54 E), China. The seeds were planted in square plastic pots (10 cm × 10 cm × 8 cm) containing nutrient soil (Pindstrup Mosebrug A/S, Ryomgaard, DK) and placed in the dark to germinate. After that, the seedlings were moved into a greenhouse and grown for about 2 weeks (trefoil stage) for cold and soil drought treatments. After 0, 6, and 12 h of cold treatment in a plant incubator (SAFU EXPERIMENTAL APPARATUB TECHNOLOGY, China) at 4 • C, the second and third leaves of S. purpurea were collected. For the soil drought treatment, the second and third leaves of S. purpurea were selected after continuously withholding water for 0 d and 14 d and rehydration (fully watered) for 1 day. For salt and PEG treatment, S. purpurea seeds were soaked in water overnight and germinated on moist filter paper in the dark. After germination, the seedlings were transplanted into a plastic pot (10 cm × 10 cm × 8 cm) containing the same amount of vermiculite and 1/2 liquid MS in a greenhouse for about 2 weeks (trefoil stage). After that, the 1/2 MS was replaced with 1/2 MS containing 150-mM NaCl or 30% polyethyleneglycol 6000 (PEG 6000). After 0, 6 and 12 h of salt or PEG treatment, the second and third leaves of S. purpurea were collected [38,39]. The samples were frozen in liquid nitrogen and stored at −80 • C for subsequent RNA isolation. A. thaliana plants (ecotype: Columbia ecotype, Col-0) were cultured for ectopic expression experiments on 1/2 solid MS medium. After 10 days, the seedlings were transferred to nutrient soil. The p5cs mutant (SALK_045245) was purchased from The Arabidopsis Information Resource (https://www.arabidopsis.org/, accessed on 11 June 2016). All the plants were grown in a greenhouse at 21 • C, with 75-80% relative humidity and a light intensity of 200-µmol photons m −2 s −1 , under a 16-h light/8-h dark photoperiod. For untreated A. thaliana and S. purpurea, the normal irrigation situation was 30 mL of water every two days [37].

RNA Isolation and SpP5CS Gene Cloning
Total RNA was isolated from leaves of S. purpurea using an Eastep ® Super Total RNA Extraction Kit (Promega, Madison, WI, USA), and the DNA digested with DNase I. RNA was quantified using a NanoDrop1000 (NanoDrop Technologies, Wilmington, DE, USA), and the integrity was checked on a 0.8% agarose gel. Then, RNA (5 µg) was reverse-transcribed into first-strand cDNA using the GoScript™ Reverse Transcription System (Promega, Madison, WI, USA). We used RACE technology to obtain the full length of the SpP5CS sequence. Based on the transcriptome data (the Genome Sequence Archive in the BIG Data Center, Beijing Institute of Genomics (BIG), accession numbers CRA002018), the required EST fragments were screened out, and specific primers SpP5CS-RACE-1 (5 -TCTCGCGAAGCTGGTTCCCAAAACT-3 ) and SpP5CS-RACE-2 (5 -GGGCCTGTTGCACATGACACACTGAA-3 ) were designed. Then we used SMART RACE cDNA kit (BD Biosciences Clontech, MountainView, CA, USA) to amplify the 5 and 3 sequences of the target gene. Then specific primer SpP5CS-F (5 -ATGGGTAGAGGAGGGATCGGA-3 ) and SpP5CS-R (5 -TCATTGCAACGGAAGCTCCCT GTGG-3 ) were used to obtain the full-length CDS sequence of SpP5CS. We constructed the full length of SpP5CS into the pMD 18-T vector (Takara Bio, Kusatsu, Japan), and applied Sanger sequencing for paired-end sequencing.

Sequence Analysis of the SpP5CS Gene
The full-length SpP5CS cDNA sequence was translated into a protein using DNAMAN (Lynnon Biosoft, San Ramon, CA, USA). The protein was analyzed using the online SMART tool (http://smart.embl-heidelberg.de/smart/show_motifs.pl, accessed on 5 October 2018) and ExPASy ProtParam tool (https://web.expasy.org/protparam/, accessed on 8 October 2018). The protein sequences of other species were obtained using the BLAST tool of NCBI and aligned using the Clustal X program [40]. Then, a maximum parsimony analysis was performed using MEGA X with the neighbor-joining method and the 1000 bootstrap test replicates [41].

Generation and Screening of Transgenic A. thaliana Plants
The full-length SpP5CS CDS was inserted into the vector pRI 101 green fluorescent protein (GFP) and driven by the 35S Cauliflower mosaic virus promoter. Then, the recombinant plasmid was transformed into Agrobacterium tumefaciens stain GV3101 that was used to transform WT and p5cs mutant A. thaliana by the floral dip method [43]. The harvested seeds were grown on MS medium supplemented with 50-mg L −1 kanamycin to select for positive transgenic plants. The transgenic A. thaliana were examined by RT-PCR using SpP5CS-GFP-F (5 -GACCCCGGGGGTACCGGATCCATGGGTAGAGGAGGGATCGGA-3 ) and SpP5CS-GFP-R (5 -TCAGAATTCGGATCCTCATTGCAACGGAAGCTCCCTGTGG-3 ). The A. thaliana leaves of the same period were taken to extract RNA and reverse to obtain cDNA (according to the method in Section 4.2). Atactin2 (NM_001338358) were used to normalize the A. thaliana samples. The PCR reaction was performed under following conditions: 25 cycles at 95 • C for 30 s, 55 • C for 30 s, 72 • C for 2 min.

Subcellular Localization of SpP5CS
The SpP5CS-overexpression (OE-SpP5CS) and WT A. thaliana lines were germinated on 1/2 MS medium until the root lengths were approximately 2 cm, and then, the roots were cut for subcellular localization. Leaf mesophyll protoplasts were isolated from ten-day-old cotyledons of OE-SpP5CS transgenic A. thaliana. The method of protoplast isolation was based on previous research [44]. A laser scanning confocal microscope (Olympus Optical, Tokyo, Japan) was used to observe the GFP expression.

Drought Stress, Survival Rate, and Root-Length Measurements
For drought tests, seeds of WT, p5cs mutant, p5cs/SpP5CS-complemented and OE-SpP5CS A. thaliana plants were sown on 1/2 MS medium and 1/2 MS medium supplemented with 5% PEG 6000. Root lengths were measured after 10 d. To determine the transgenic phenotypes under drought and normal irrigation conditions (30 mL of water every two days), WT, mutant, p5cs/SpP5CS-complemented and OE-SpP5CS A. thaliana seedlings were planted in plastic pots filled with nutrient soil and grown in a greenhouse under normal irrigation conditions. Four-week-old seedlings with about 17 true leaves were used for drought stress treatment. Drought conditions were simulated by withholding water for 5, 12 or 15 d. After each treatment, the plants were sampled, quickly frozen in liquid nitrogen and stored in a refrigerator at −80 • C for subsequent experiments. During the drought treatment process, the survival rate of A. thaliana at each drought stage was calculated.

Relative Electrical Conductivity Measurement
The relative conductivity of A. thaliana at different treatment periods was measured simultaneously. The fresh leaves (4th to 6th leaves) were weighed (about 0.3 g), cut into 0.5-mm lengths and placed in a tube containing 10 mL of distilled water. To extract the air between the cells, the leaves in the distilled water were pumped in a vacuum for 20 min. Then, the samples were placed at room temperature for 1 h, and the conductivity of the solution were measured (S1). The solution was then heated to 100 • C for 10 min, cooled to room temperature and the final electrical conductivity (S2) was measured. The relative electrical conductivity (REC) was calculated as follows: REC = S1/S2. The degree of damage was calculated as follows: (REC TREATMENT − REC CONTROL )/(1 − REC CONTROL ) × 100%. The measurement method was based on the previously described method [35,45].

Proline Content Measurement
The proline content was measured by a previously described method [46]. The sample was weighed (about 0.4 g) and put in a 10-mL centrifuge tube containing 5 mL of 3% (w/v) 5-sulfosalicylic acid dihydrate and boiled for 10 min. Two milliliters of boiling supernatant were reacted with 2 mL of glacial acetic acid and 2 mL of ninhydrin and boiled for 30 min. When the temperature of the reaction mixture reached room temperature, it was extracted with 4 mL of toluene, and the supernatant was measured at 520 nm. At the same time, the same method was used to determine the absorbance values of different standards of proline to construct a standard curve. Finally, the proline content was calculated according to the standard curve.

Conclusions
In this study, the P5CS gene cloned from S. purpurea had the same structure as the P5CS genes of other plant species. The expression level of SpP5CS was significantly upregulated under drought stress conditions, and SpP5CS localized to the cytoplasm. The overexpression of SpP5CS in A. thaliana increased the drought resistance and root lengths, and it reduced damage to the cell membrane. We speculate that, under drought stress conditions, the SpP5CS expression level increases, which further increases the proline contents of plant cells, thereby improving the drought resistance of the plants.