Identification and Characterization of an OSH1 Thiol Reductase from Populus Trichocarpa

Interferon gamma-induced lysosomal thiol reductase (GILT) is abundantly expressed in antigen-presenting cells and participates in the treatment and presentation of antigens by major histocompatibility complex II. Also, GILT catalyzes the reduction of disulfide bonds, which plays an important role in cellular immunity. (1) Background: At present, the studies of GILT have mainly focused on animals. In plants, GILT homologous gene (Arabidopsis thaliana OSH1: AtOSH1) was discovered in the forward screen of mutants with compromised responses to sulphur nutrition. However, the complete properties and functions of poplar OSH1 are unclear. In addition, CdCl2 stress is swiftly engulfing the limited land resources on which humans depend, restricting agricultural production. (2) Methods: A prokaryotic expression system was used to produce recombinant PtOSH1 protein, and Western blotting was performed to identify its activity. In addition, a simplified version of the floral-dip method was used to transform A. thaliana. (3) Results: Here, we describe the identification and characterization of OSH1 from Populus trichocarpa. The deduced PtOSH1 sequence contained CQHGX2ECX2NX4C and CXXC motifs. The transcript level of PtOSH1 was increased by cadmium (Cd) treatment. In addition, recombinant PtOSH1 reduced disulfide bonds. A stress assay showed that PtOSH1-overexpressing (OE) A. thaliana lines had greater resistance to Cd than wild-type (WT) plants. Also, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in PtOSH1-OE plants were significantly higher than those in WT A. thaliana. These results indicate that PtOSH1 likely plays an important role in the response to Cd by regulating the reactive oxygen species (ROS)-scavenging system. (4) Conclusions: PtOSH1 catalyzes the reduction of disulfide bonds and behaves as a sulfhydryl reductase under acidic conditions. The overexpression of PtOSH1 in A. thaliana promoted root development, fresh weight, and dry weight; upregulated the expression levels of ROS scavenging-related genes; and improved the activity of antioxidant enzymes, enhancing plant tolerance to cadmium (Cd) stress. This study aimed to provide guidance that will facilitate future studies of the function of PtOSH1 in the response of plants to Cd stress.


Introduction
Interferon-γ-inducible lysosomal thiol reductase (GILT) has been discovered in many species [1,2]. The precursor of GILT is a soluble glycoprotein, which enters the lysosome by interacting with the mannose-6-phosphate receptor and is transformed into the mature form by cleavage (POPTR_0016s12980) gene from P. trichocarpa. Recombinant PtOSH1 catalyzed the reduction of disulfide bonds. Additionally, the expression of PtOSH1 was high in leaves and roots and responded to Cd. The enhanced resistance to Cd caused by PtOSH1 may be mediated by the reparation of antioxidant enzymes and promotion of ROS homeostasis.

Plant Cultivation, Gene Isolation, and Vector Construction
Poplar (Populus trichocarpa) and Arabidopsis thaliana were cultivated in half-strength Murashige and Skoog (1/2 MS) medium (23 • C, humidity 74%, and 16 h light/8 h darkness). The young leaves, mature leaves, stems, roots, and petioles of P. trichocarpa were collected (Supplementary Figure S1) and total RNA from different tissues was extracted based on the manufacturer's instructions (Biomiga, San Diego, CA, USA). In addition, P. trichocarpa seedlings treated with 200 µM ABA, 2 mM CdCl 2 , and 2 mM H 2 O 2 and P. trichocarpa young leaves treated with the different abiotic stresses were collected at 0, 2, 4, 6, 8, 12, 24, and 48 h. Forward and reverse primers were designed based on the sequence of P. trichocarpa GILT obtained from the National Center for Biotechnology Information, and PtOSH1 was amplified by polymerase chain reaction (PCR). The PCR product was inserted into the PEASY-T3 vector (TransGen Biotech, Beijing, China), which was sequenced by Nanjing Genscript Co. (Nanjing, China). The PCR product with EcoRI and XhoI restriction sites was inserted into the PET-28a vector and the PCR product with XbaI and BamHI restriction sites was inserted into the PBI121 vector by double digestion and T4 ligation. The primers used are shown in the Supplementary Table S1.

Prokaryotic Expression, Purification, and Activity of PtOSH1 In Vitro
The recombinant plasmid PET-28a-PtOSH1 was transformed into Escherichia coli BL21 (DE3) and cultured in Luria-Bertani (LB) medium. The optical density of E. coli BL21 (DE3) at 600 nm was determined and the isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce synthesis of recombinant PtOSH1. We evaluated the effect of temperature (37 and 16 • C) and rotational speed (220 and 110 rpm) on PtOSH1 synthesis. The PtOSH1 level was determined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A nickel-nitrilotriacetic acid (Ni-NTA)-chelating column was used to purify PtOSH1. The supernatant of recombinant E. coli BL21 (DE3) cultures was added to the column, followed by wash buffer (20 mM Tris-HCl, 0.15 M NaCl, and 20 mM imidazole; pH 8.0) until the absorbance reached baseline, and finally elution buffer (20 mM Tris-HCl, 0.15 M NaCl, and 250 mM imidazole; pH 8.0).
To assay PtOSH1 activity, 0.2% SDS was used to denature immunoglobulin G (IgG) at 100 • C for 5 min. Dilution buffer (50 mM NaCl, 0.1% Triton X-10) was used to dilute the denatured IgG. PtOSH1 was dissolved in enzyme activity solution (100 mM NaCl, 0.1% Triton X-10, 50 mM CH 3 COONa, 25 mM DTT; pH 4.5), and the mixture was incubated at 37 • C for 10 min to pre-activate PtOSH1. Also, 10 µL of denatured IgG and 100 µL of PtOSH1 were incubated at 37 • C for 1 h and subjected to non-reducing SDS-PAGE.

Overexpression of PtOSH1 in Arabidopsis Thaliana (A. Thaliana)
The recombinant plasmid PBI121-PtOSH1 was transformed into Agrobacterium tumefaciens GV3101, and subsequently transformed into A. thaliana by the floral-dip method [39]. Putative T1 seedlings were screened in 1/2 MS medium containing 50 µg mL −1 kanamycin, and the putative plants were grown in soil. Subsequently, the T2 seedlings were screened as above. The DNA and RNA of T2 plants were extracted to confirm the insertion of PtOSH1 into the A. thaliana genome. Three independent biological experiments were performed.
For CdCl 2 treatment, wild-type (WT) and PtOSH1-OE A. thaliana plants were cultivated in 1/2 MS medium, and 1-week-old WT and PtOSH1-OE A. thaliana plants were planted in 1/2 MS medium with 0, 20, 40, 60, and 80 µM CdCl 2 . WT and PtOSH1-OE A. thaliana plants were grown in MS medium containing 0-80 µM CdCl 2 for 20 days, and the root length, fresh weight, and dry weight of WT and PtOSH1-OEs were determined. Three independent biological experiments were performed. In addition, the transcript levels of genes related to ROS scavenging were evaluated in WT and PtOSH1-OEs. A microplate reader (Bio-Rad, Hercules, CA, USA) was used to analyze the CAT, SOD, and POD activities before and after treatment with 60 µM CdCl 2 based on the protocol of the Nanjing Jiancheng Bioengineering Institute.

Polymerse Chain Reaction (PCR) and Quantitative Reverse Transcription-PCR
PCR was performed as follows: 95 • C for 5 min; 35 cycles of 95 • C for 30 s, 58 • C for 40 s, and 72 • C for 30 s; and 72 • C for 10 min. SYBR Green Mix (Roche, Basel, Switzerland) was added to the PCR mixture, and quantitative reverse transcription-PCR (qRT-PCR) was conducted as follows: initial incubation at 95 • C for 5 min; 40 cycles of 95 • C for 30 s and 60 • C for 30 s; and 72 • C for 30 s. β-Actin of P. trichocarpa and A. thaliana were used as internal controls. Three independent biological experiments were analyzed with three technical repeats. The primers used are shown in the Supplementary Table S1.

Molecular Characterization of a Poplar OSH1
We cloned an OSH1 from P. trichocarpa. The open reading frame (ORF) of PtOSH1 (POPTR_0016s12980) contained 813 nucleotides and encoded 270 amino acids. The predicted signal peptide of PtOSH1 was 'MGSSPLLSFLVLTSLVVLFVTPSHS' and located at the N-terminus of PtOSH1, this petide was necessary for its transportation to the lysosome. (Supplementary Figure S2). Also, PtOSH1 was predicted to be located in the lysosome. The amino acid sequence of PtOSH1 had two characteristic motifs (CXXC and CQHGX 2 ECX 2 NX 4 C), and two glycosylation sites (NNT and NTS) (Supplementary Figure S2). We speculated that PtOSH1 had sulfhydryl reductase activity. The homology of PtOSH1 with GILTs from other species was analyzed using Clustal Omega and BOXSHADE software. All of the GILTs had the characteristic CXXC and CQHGX 2 ECX 2 NX 4 C motifs and ten highly conserved cysteine residues (Supplementary Figure S3), as well as sulfhydryl reductase activity, suggesting that PtOSH1 also has sulfhydryl reductase activity.

Transcript Levels of PtOSH1 in Tissues and Under Cd 2+ Stress
The highest transcript level of PtOSH1 was in young and mature leaves, followed by roots, and the lowest was in petioles ( Figure 3A). Treatment with 2 mM CdCl 2 significantly increased the transcript level of PtOSH1 from 6 to 48 h, with a peak at 12 h ( Figure 3B). Treatment with 200 µM ABA caused significant accumulation of PtOSH1 mRNA from 6 to 24 h ( Figure 3C). The expression of PtOSH1 was enhanced from 1 to 24 h of 2 mM H 2 O 2 treatment ( Figure 3D). Plant roots and leaves absorb exogenous substances. During plant growth and development, nutrient transport involves various signaling pathways, in which oxidoreductases play an important role. Therefore, the differential expression of GILT in different tissues may be related to normal physiological functions. The mRNA transcript level of PtOSH1 in response to H 2 O 2 stress. Three independent biological experiments were performed with three technical repeats. One-way analysis of variance (ANOVA) and Tukey's test were used to evaluate significant differences. Data are 2 -∆∆Ct levels relative to the petiole and normalized to that of PtActin. Vertical bars represent the means ± standard deviation (SD, n = 3). *, Significant difference at p < 0.05; **, Significant difference at p < 0.01; ***, significant difference at p < 0.001.

Expression, Purification, and Functional Analysis of Recombinant PtOSH1
The ORF of PtOSH1 was cloned into the plasmid PET-28a between the EcoRI and XhoI sites. Analysis by 12% SDS-PAGE showed that the production of recombinant PtOSH1 was induced by 1 mM IPTG at 110 rpm at 16 • C, but not at 37 • C ( Figure 4A). Recombinant PtOSH1 was purified using Ni-IDA resin ( Figure 4B) and Western blotting showed that recombinant PtOSH1 was specifically recognized by rabbit antiserum ( Figure 4B).
Arunachalam et al. [3] showed that GILT proteins can catalyze the reduction of disulfide bonds and exhibit sulfhydryl reductase activity under acidic conditions. We used human IgG as a substrate to assay the activity with PtOSH1 in vitro. The disulfide bond of IgG was intact (lane 2) following treatment of DTT ( Figure 4C

Characterization of Transgenic A. Thaliana Lines
To further study the function of PtOSH1 in transgenic A. thaliana lines, the PBI121-PtOSH1 vector was introduced into WT A. thaliana. Subsequently, PtOSH1-OE lines were obtained (Supplementary Figure S4). The total genome of the WT and PtOSH1-OE lines was extracted and amplified by PCR. The lanes of the PtOSH1-OE lines, but not that of the WT, had the expected band (Supplementary Figure S5A). Also, qRT-PCR analysis showed that PtOSH1 was stably expressed in A. thaliana (Supplementary Figure S5B). In addition, the transcript levels of Arabidopsis homologs At1G07080 and At5G01580 were identified. The expression of At1G07080 in PtOSH1-OE lines was increased significantly (p < 0.05) (Supplementary Figure S6A). However, there are no significant difference in expression of At5G01580 between WT and PtOSH1-OE lines (Supplementary Figure S6B).

Response of Transgenic Plants to Cd 2+ Stress
WT and PtOSH1-OE A. thaliana plants were grown in 1/2 MS medium containing 20, 40, 60, and 80 µM CdCl 2 and their growth was evaluated after 20 days. The growth of WT and transgenic A. thaliana lines was little affected by 20 µM CdCl 2 ( Figure 5A,B and Figure 6). When exposed to 40-60 µM CdCl 2 , the root length and biomass of plants decreased significantly, and the root length and biomass of the PtOSH1-OE A. thaliana lines were significantly higher than those of WT A. thaliana ( Figure 5C,D and Figure 6), suggesting that transgenic plants have a growth advantage. At 80 µM CdCl 2 , the germination and growth of A. thaliana were inhibited; leaf yellowing, root length, and the fresh weight of the PtOSH1-OE A. thaliana lines were significantly higher than those of WT A. thaliana; and the number of lateral roots increased significantly (Figures 5E and 6). Therefore, PtOSH1-like transgenic A. thaliana plants showed greater tolerance to CdCl 2 stress than did WT plants.  Three independent biological experiments were performed. One-way ANOVA and Tukey's test were used to evaluate significant differences. Vertical bars represent the means ± SD (n = 3). **, Significant difference at p < 0.01. Cd stress triggers the production of ROS in plants, the accumulation of which hinders photosynthesis. In addition, the metalloproteins related to electron transfer in plants are compromised by Cd, disrupting respiration [12]. The plant antioxidant defense system eliminates ROS to prevent the damage caused by oxidative stress [40]. qRT-PCR analysis showed that the expression levels of ascorbate peroxidase (APX), CAT, glutathione S-transferase (GST), POD, and SOD in WT and PtOSH1-OE plants were not significantly different under normal conditions but were higher in PtOSH1-OE plants than in WT plants under CdCl 2 stress (Figure 7). To determine whether overexpression of PtOSH1 increased tolerance to CdCl 2 stress, we analyzed the activities of POD, SOD, and CAT under 60 µM CdCl 2 stress. Under normal conditions, PtOSH1-OE A. thaliana lines had slightly higher activities of POD, SOD, and CAT than WT A. thaliana; however, PtOSH1-OE A. thaliana lines had significantly higher activities of POD, SOD, and CAT than WT A. thaliana after 20 days of CdCl 2 stress (Figure 8). Therefore, PtOSH1 promotes ROS scavenging by POD, SOD, and CAT to alleviate the oxidative damage to membranes caused by CdCl 2 stress, increasing the tolerance to CdCl 2 of the PtOSH1-OE plants.  thaliana lines before and after exposure to 60 µM Cd 2+ . Three independent biological experiments were performed with three technical repeats. One-way ANOVA and Tukey's test were used to evaluate significant differences. Vertical bars represent the means ± SD (n = 3). **, Significant difference at p < 0.01; ***, significant difference at p < 0.001.
In addition, GILT has a signature sequence (CQHGX 2 CX 2 NX 4 C), multiple N-glycosylation sites, and 10-11 conserved cysteine residues [48]. However, the function of plant GILTs was unknown. In this study, we characterized PtOSH1 of P. trichocarpa. PtOSH1 has the same motifs and a similar structure to human GILT, indicating that these proteins have similar functions.
During the processing and presentation of antigens, disulfide bonds are denatured, renatured, and reduced; the latter is particularly important. GILT is the only reductase that catalyzes the reduction of disulfide bonds at low pH and exhibits high activity in the acidic lysosome [49][50][51]. Ohkama-Ohtsu et al. [37] demonstrated that recombinant At5g01580 protein expressed by E. coli had thiol reductase activity under neutral conditions. However, the GILT of humans has the highest thiol reductase activity at acidic conditions [3]. In this study, we demonstrated that PtOSH1 cleaves IgG into heavy and light chains by catalyzing the reduction of disulfide bonds under neutral conditions. PtOSH1-catalyzed reduction of disulfide bonds may alter the structure and function of proteins in poplar. The reduction of disulfide bonds in some proteins restores their normal physiological function. Therefore, PtOSH1 may affect homeostasis of poplar by catalyzing the reduction of disulfide bonds.
Plants under stress conditions produce large amounts of ROS, including superoxide anions and free radicals [52,53]. To decrease the resulting oxidative damage, the antioxidant systems of plants not only scavenge ROS and limit their production but also repair oxidative damage [54,55]. Thioredoxin reductase (Trx) has a WCGH/PCK domain, which catalyzes the reduction of disulfide bonds and is involved in a variety of biochemical reactions [56], including regulation of redox potential, antioxidants, signal-transduction pathways, transcription factors, and the response to heavy metal stress [30,57,58]. In this study, the expression of PtOSH1 was upregulated by CdCl 2 treatment. Also, overexpression of PtOSH1 enhanced the resistance of A. thaliana to CdCl 2 stress. These findings implicate PtOSH1 in the response to CdCl 2 stress. Trx scavenges ROS and activates proteins inactivated by oxidative stress, which are important for maintaining the physiological function of cells under oxidative stress [59][60][61]. In WT and PtOSH1-OE plants, APX, CAT, GST, POD, and SOD transcript levels were increased by CdCl 2 stress, and the magnitude of the increase was higher in PtOSH1-OE plants than in WT plants.
In addition, the activities of SOD, POD, and CAT were higher in PtOSH1-OE A. thaliana lines than in WT A. thaliana. Therefore, PtOSH1 might be important for defense against CdCl 2 stress. However, the underlying mechanism is unknown; therefore, a mechanistic investigation of the antioxidant activity of PtOSH1 is required to determine its effect on ROS.
Previous studies showed that Cd poisoning can lead to oxidative stress and protein denaturation in plants. However, plants have defense mechanisms to alleviate the damage from oxidative stress, including increasing the ability to remove oxidized proteins, improving the synthesis of antioxidant molecules and molecular chaperones, and changing the composition of plant cell walls and xylem sediments [17]. In addition, Salt et al. [62] found that Cd was mainly distributed in the leaf epidermis and epidermal hairs of mustard. Cd in plants may be a defense mechanism to prevent damage because the substances needed for photosynthesis, growth, and development are mainly in mesophyll cells, and leaf epidermis and epidermal hairs play a role in isolation and protection. Due to the lack of GILT, the activity and stability of SOD2 were decreased in the animal cells. Recombinant GILT improved the activity and stability of SOD2 and maintained a relatively steady level of ROS; thus, GILT promotes maintenance of the redox state in cells. GILT also promotes intracellular oxidative stress in cells, which can accelerate autophagy and decompose damaged mitochondria [9]. In this study, the transcript level of PtOSH1 was improved under Cd treatment, and PtOSH1 catalyzed the reduction of disulfide bonds. Two cysteines are located in active sites of GILT. One cysteine is nucleophilic; it attacks the disulfide bond on the substrate to form a disulfide compound and intermediate substrate, and then the two cysteines on GILT carry out an internal attack to cause the substrate to escape so that the disulfide bond of the substrate is opened [51]. GILT catalyzes disulfide reduction, which is accompanied by changes in protein structure and function in cells. Some disulfide bonds can be reduced to restore their normal physiological functions. GILT regulates ROS homeostasis by repairing antioxidant enzymes and ultimately maintains the redox state in cells. Together, our results demonstrate that PtOSH1 is essential for Cd stress and that PtOSH1 may restore antioxidant enzymes by catalyzing disulfide reduction as well as regulating the steady state of ROS.

Conclusions
In conclusion, when plants are stressed by heavy metals, the activity of the antioxidant system in plants is improved, and antioxidant enzymes can scavenge ROS and play a protective role. When stressed by heavy metals for a long time, the antioxidant enzyme system of plants is destroyed, and plants are poisoned by ROS. In addition, although GILT from animals has been well characterized and its function evaluated, there have been fewer studies of the function of GILT-like in plants. Therefore, a more thorough understanding of GILT, including how it is regulated, is important for maintaining the mechanism of ROS underlying Cd treatment. In this study, we isolated PtOSH1 from P. trichocarpa, which can respond to Cd treatment; transgenic experiments in A. thaliana provided further evidence related to the response to Cd stress. Collectively, our results demonstrate that PtOSH1 catalyzes the reduction of disulfide bonds and may repair the antioxidant enzymes resulting from Cd stress and regulate the ROS-scavenging system, which is important for the steady state of plant cells.