Identification and Functional Analysis of a Protein Disulfide Isomerase (AtPDI1) in Arabidopsis thaliana

Protein disulfide isomerase (PDI) catalyzes the conversion of thiol-disulfide and plays an important role in various physiological events in animals. A PDI (OaPDI) from a tropical plant was detailed studied and it was found to be involved in response of biotic stress (Gruber et al., 2007). However, the activities of PDI related to physiological functions in plants are poorly understood. In the present study, a homolog of human PDI in Arabidopsis (AtPDI1), encoded by the gene (At3g54960), was characterized. The recombinant AtPDI1 protein had disulfide isomerase activity in vitro and two pairs of conservative cysteines in catalytic domains play a crucial role in the PDI activities. Expression of AtPDI1 in Escherichia coli significantly enhanced stress tolerance of cells and the mutations of critical cysteines almost lose this function. In plants, AtPDI1 was strongly induced by abiotic stresses and exogenous abscisic acid. An Arabidopsis AtPDI1 knockdown mutant (pdi1) and overexpression lines of transgenic plants obtained by this investigation were used to further examine the function of AtPDI1. The mutant line was more sensitive to stresses than the wild-type, while overexpressing AtPDI1 increased tolerance of seedlings to abiotic stresses, with a higher germination ratio and longer length of roots than the wild-type. Our results suggested AtPDI1 played roles in anti-stresses in Arabidopsis, which relate to the activities of PDI.

Identification and Functional Analysis of a Protein Disulfide Isomerase (AtPDI1) in Arabidopsis thaliana INTRODUCTION Protein disulfide isomerase (PDI,EC 5.3.4.1) is a thiol-disulfide oxidoreductase, catalyzing oxidation, reduction, and isomerization of target proteins in protein folding and unfolding (Wilkinson and Gilbert, 2005;Ali and Mutus, 2014;Meng et al., 2015). Some PDIs also display chaperone activities, helping protein correct folding (Narindrasorasak et al., 2003;Wilkinson and Gilbert, 2005). However, some play opposite functions, for example, human PDI (hPDI) catalyzing extensive intermolecular disulfide cross-linking of lysozyme into large, inactive aggregates (Wilkinson and Gilbert, 2005;Khan et al., 2016). As a classical PDI, hPDI (Gene ID: 5034) is composed of four modular domains (a, b, b , a ) and endoplasmic reticulum (ER) retention signal (KDEL sequence) at carboxyl terminus (Alanen et al., 2003). The domain a and a are catalytic domains sharing homology to thioredoxin domain with a di-cysteine motif (CXXC motif), respectively, while domain b and b play roles in keeping the tertiary structures of the whole molecule in U-shape (Kemmink et al., 1997;Byrne et al., 2009). Generally, more than a dozen members of PDIs exist in animal cells and some of them are involved in unfolded protein response (UPR) of persistent stress in ER, which is closely related to the development and progression of diseases like diabetes and neurodegenerative diseases (Schröder and Kaufman, 2005;Honjo et al., 2014). Elevated expressions of some PDIs have been found in patients suffering from lung cancer or melanoma (Xu et al., 2012).
Family members of plant PDIs are also identified in the genome in different plants, for example, 12 members for Oryza sativa, 9 for Triticum aestivum L., 32 in Brassica rapa ssp. pekinensis, and 22 for Zea mays (Houston et al., 2005;D'Aloisio et al., 2010;Onda and Kobori, 2016;Kayum et al., 2017). Similar to how animal PDIs catalyze the exchange of disulfide, plant PDIs are found to be involved in the unfolded and refolded protein response (Takemoto et al., 2002;Lu and Christopher, 2008;Kimura et al., 2015;Onda and Kobori, 2016;Peng et al., 2017), maturation of the embryo sac , the programmed cell death of endothelial cells in developing seeds, and response to biotic stress (Gruber et al., 2007;Ondzighi and Staehelin, 2008). The multiplicity and structural difference of plants PDIs suggest that they may serve both specialized and overlapping functions.
Though plants do not suffer animal-specific diseases, plants are more susceptible to stresses in the process of growth and development, due to their immobility. Individual plant PDIs are found to be up-regulated under abiotic stresses. PDIs from Brachypodium distachyon L. and Brassica rapa ssp. pekinensis were up-regulated under abiotic stresses, suggesting their involvement in multiple stress responses (Zhu et al., 2014;Kayum et al., 2017). A PDI from Jatropha curcas could notably enhance cold resistance of yeast cells . PDI from Oldenlandia affinis (OaPDI), with activity of PDI, is involved in the oxidative folding of a cystine knot defense protein (kalata B1), suggesting OaPDI takes part in antibiotic stress (Gruber et al., 2007).
As a model plant, Arabidopsis thaliana has 12 or more PDIrelated members (Houston et al., 2005;Lu and Christopher, 2008). Arabidopsis PDIs are classified into three groups based on polypeptide length, presence of signal peptide and ER retention signal, and composition of thioredoxin domains (Lu and Christopher, 2008). There are three members group III (AtPDI9-AtPDI11) and two members in group I (AtPDI7, AtPDI12), all of them lacking b and b domains. Group II is the largest group with six members, all of them containing classical four domains. AtPDI8 contains only three domains (a, b, b ), not belonging to any of the above groups.
As a member in group II, AtPDI1, which contains classical four domains (a, b, b , a ) with C-terminal ER-anchoring signal. Different from hPDI, AtPDI1 has an additional sequence at N-terminus which is predicted as possible signal peptide targeting to chloroplast. GFP-fused experimental evidences showed that AtPDI1 is located in ER (Yuen et al., 2013), while AtPDI1 is also identified in chloroplast by 2D-proteome (Armbruster et al., 2009;Kieselbach, 2013). Up-regulation of AtPDI1 induced by stress suggested it may play roles in stress responses (Lu and Christopher, 2008). Whether the potential anti-stress function of AtPDI1 is related to its activity of PDI is unknown. In this study, the purified recombinant AtPDI1 and cysteine-mutations were used to examine enzymatic activity. To further investigate the function in plants, overexpression lines of AtPDI1 were obtained by transgenic technique. Different responses to stresses were observed among wild-type, overexpression lines, and a knockdown mutant (pdi1). This study will provide a clue about the function of the AtPDI1 in anti-stresses.

Bioinformatics Analyses of AtPDI1
The sequences of AtPDI1, plant PDIs, and hPDI were obtained from the NCBI database 1 . Multiple sequence alignment was performed with CLC sequence viewer. The three-dimensional structure of AtPDI1 was obtained by homology modeling using the website SWISS-MODEL 2 . The crystal structure of the hPDI was downloaded from the PDB database 3 . All structure images were output by PyMOL software.

Cloning of AtPDI1 cDNA and Construction of Expression Plasmids
The cDNA sequence of AtPDI1 was amplified by the RT-PCR method, using Arabidopsis leaves treated by NaCl. The sequence was inserted into pET-30a expression plasmid which contained His-tagged. Six mutations of conserved cysteines were amplified using the recombinant vector as the template, which change cysteine to alanine at positions 128, 131, 467, and 470 of AtPDI1 amino acid sequence. All the primers used in this investigation were listed in Supplementary Table S1. All recombinant plasmid were confirmed by Shanghai Sunny Biotechnology Co. (Shanghai, China), and the recombinant vectors were transformed into Escherichia coli BL21 cell.

Assay Protein Disulfide Isomerase of Recombinant Proteins
The disulfide bond isomerase activity of recombinant proteins was measured as described previously (Walker et al., 1996). The denatured RNase (reduced RNase) was prepared as described  previously (Shin and Scheraga, 2000;Fu and Zhu, 2009). Scrambled RNase A (20 µg/mL) was incubated with or without recombinant proteins into 1 mL solution containing 1 mM GSH, 0.2 mM GSSG, 0.5 mM cCMP, 2 mM EDTA, and 100 mM Tris-HCl performed at 25 • C. The formation of active RNase was measured spectrophotometrically by continuous monitoring hydrolysis of the RNase substrate, cCMP, at 296 nm (Walker et al., 1996).

Cell Growth in Stress Medium
Survival test on LB solid medium was carried out to ascertain the function of AtPDI1 in E. coli. Transformed E. coli of BL21 cell were grown till OD 600 of 0.6, induced by 0.5 mM IPTG for 2 h at 37 • C (Du et al., 2014). Cultures were diluted to OD 600 of 0.6, and then diluted to 10 −2 , 10 −3 , and 10 −4 . For stress treatments, different dilution cultures were spotted on LB medium supplemented with 0.5 M NaCl, 0.3 mM H 2 O 2 , and 0.6 M mannitol, and then incubated at 37 • C for 16 h.
Growth analysis in LB solution culture was used to further determine cell growth under abiotic stress. Transformed E. coli of BL21 cell were grown till OD 600 of 0.6, induced by 0.5 mM IPTG for 2 h at 37 • C and then 100 µl cultures were inoculated in 20 mL LB solution medium containing mannitol, H 2 O 2 or NaCl, and incubated at 37 • C on a rotary shaker (200 rpm). The bacterial suspension was harvested at every 2 h till 12 h and OD 600 was measured. Each experiment was carried out in three biological replicates.

Stress Treatments to Plants, RNA Extraction, and Quantitative Real-Time PCR (qRT-PCR) Analysis
Normally grown 3-week-old Arabidopsis seedlings were treated with solution containing 10 mM H 2 O 2 , 20% PEG, 200 mM NaCl, or 100 µM exogenous ABA, respectively, for different times, with water at room temperature as a control. The leaves were collected at different treatment times and the total RNA was extracted from the material. Qualified RNA was used to synthesize cDNA with a cDNA synthesis kit. The GAPDH gene from Arabidopsis was used as an internal control for expression normalization in the quantitative real-time PCR (qRT-PCR) as similar investigations did and the relative expression levels were calculated by the method of 2 − Ct . The specific primers for amplifying GAPDH and AtPDI1 genes were used for qRT-PCR and are listed in Supplementary Table S1. The relative expression levels of AtPDI1 in stressed samples (12, 24, and 36 h) were compared to the controls (0 h) with Student's t-test at significance levels of * P < 0.05 and * * P < 0.01. Each reaction was carried out in three biological replicates.

Plasmids for Plant Transformation
The full-length sequence of AtPDI1 was fused to plant transformation plasmid pROKII-GFP. The promoter sequence of AtPDI1 (2 kb upstream the gene) was cloned and β-glucuronidase (GUS) gene was fused to plant transformation plasmid pBI121. Plant transformation plasmid was transformed into Agrobacterium GV3101, which was used for floral dip transformation. Transgenic plants were selected for kanamycin resistance and verified by genomic PCR with specific primers (Supplementary Table S1). The homozygous lines were obtained. For the GUS assays, 2-weeks-old transgenic Arabidopsis T3 seedlings were exposed to 100 µM ABA, 20% PEG, 200 mM NaCl or 10 mM H 2 O 2 for 9 h, as done in similar studies (Jia H. et al., 2015).  overexpressing lines and a knockdown AtPDI1 mutant (pdi1-1) by T-DNA insertion line purchased from the Arabidopsis biological resource center 4 were used for analysis of plants under stresses. For growth on 1/2 MS medium, the seeds of wildtype, T-DNA insertion mutant and overexpression lines were surface-sterilized with 70% ethanol and 2.6% bleach for 5 and 10 min, respectively. Then, seeds were washed for more than 10 mins with sterilized water containing detergent Tween-20. The washed seeds were allowed to germinate on 1/2 MS agar medium, or added 0.6 µM ABA, 200 mM NaCl, 300 mM mannitol, and 10 mM H 2 O 2 , respectively. The concentrations of ABA, NaCl, mannitol, and H 2 O 2 were determined based on the similar investigations Gallino et al., 2018). The concentrations used in the present study were stronger than general studies (Brini et al., 2007;Wang et al., 2017). In the root length experiment, the washed seeds were laid in 1/2 MS as above described. All the seeds were photographed after 10 days of growth and the seeds germination rate. The seedling root lengths were counted after 10 days of growth. Each experiment was carried out in three biological replicates.

Bioinformatics Analyses on Subcellular Location of AtPDI1
The cDNA sequence of AtPDI1 (also named as AtPDIL1-3) was downloaded from the Arabidopsis information resource  a, b, b , a ) and C-terminal ER-anchoring signal (-KEDL-sequence). Multiple sequence alignment of AtPDI1 with plant PDIs and hPDI showed considerable conserved amino acid sequences, with 50 and 70% similarity of hPDI and OaPDI, respectively ( Figure 1A). However, it was a different case with OaPDI, as AtPDI1 contains additional sequence in N-terminus, which is predicted as signal peptide. Threedimensional modeling structure of the AtPDI1 suggested that four domains (a, b, b , a ) of AtPDI1 were arranged in the U-shape, similar with other animal and plant classical PDIs. The a and a domains of AtPDI1 were at the open end of the U-shape with several α helix and β sheets, while b and b were located at the bottom, maintaining the U-shape. The catlytic sites of C-X-X-C motif of AtPDI1were located in a and a domains, respectively ( Figure 1B). This modeling structure of AtPDI1 was reasonable based on checking dihedral angle, since 94% of amino acid residues were located in the energy stable regions, using Ramachandran plot ( Figure 1C).
As to the subcellular localization of AtPDI1, we expressed the recombinant plasmids 35S:AtPDI1-GFP in epidermal cells of tobacco leaf transiently. The GFP signals were almost co-localized with ER-specific label (mCherry) (Supplementary Figure S2), indicating that AtPDI1 protein was predominantly retained within the ER in epidermal cells of tobacco leaves.

Recombinant AtPDI1 Had Activities of Protein Disulfide Isomerase
The cDNA of AtPDI1 was successfully cloned by the RT-PCR method, using RNA extracted from leaves treated by NaCl, instead of that from normal growth leaves, which suggested the expression of AtPDI1 was up-regulated under stresses. The qRT-PCR experiment showed the expression of AtPDI1 was up-regulated sixfold under NaCl treatment ( Figure 5A). The purified recombinant AtPDI1 expressed in E. coli was used to examine enzymatic activity by using scramble RNase A. The initial rate of the reaction was promoted as the concentration of AtPDI1 increased, which was AtPDI1 concentration dependent (Figure 2A). When Cys in thioredoxin motif (Cys-X-X-Cys) are mutated to alanines, the isomerase activities of AtPDI1 decreased 30-70%, which suggested that four Cys play an important role in the isomerase activities of AtPDI1 (Figure 2B).

Heterogeneous Expression of AtPDI1 in E. coli Enhanced Cell's Resistance to Abiotic Stresses
The stress medium containing different concentrations of osmolytes or H 2 O 2 was used to check the resistance of E. coli cells transformed the plasmid containing AtPDI1 gene, the FIGURE 6 | Germination phenotype of the wild type (WT), pdi, and AtPDI1-overexpressing lines under 1/2 MS medium containing ABA (0.6 µM), H 2 O 2 (10 mM), NaCl (200 mM), or mannitol (300 mM), respectively. Three independent experiments were conducted and each phenotype included 50 seeds. empty vector as the controls. No significant differences were observed regarding the growth of the cells on non-stress medium among AtPDI1 and the empty vector. On the medium with NaCl, mannitol or H 2 O 2 , the colony number showed significant differences, much more colonies with AtPDI1 than that with empty vector, when same concentration of cultures were inoculated on stress medium plates, respectively ( Figure 3A). In liquid fermentation, the growth rate of the E. coli with AtPDI1 also remarkably outperformed the control, the time to reach the logarithmic growth phase was much shorter than that with vector ( Figure 3B). The results suggested that AtPDI1 conferred strong stress-tolerance to cells against abiotic stresses. Defective mutations of cysteines of AtPDI1 almost lose the anti-stress activities (Figure 4), which suggested that AtPDI1 isomerase activity and anti-stress function were closely related.

The Expression of AtPDI1 Was Up-Regulated by Stresses in Plants
To determine the response of AtPDI1 in plants, 3-week-old Arabidopsis seedlings were treated with H 2 O 2 , PEG, NaCl or exogenous ABA, respectively. The expression of AtPDI1, checked by qRT-PCR, was up-regulated under different stresses, approximately four-to ninefold compared with control condition in leaves ( Figure 5A). The expression peak of AtPDI1 was reached at 12-24 h under different treatments.
To further test the response of the AtPDI1 to stresses, the promoter sequence of AtPDI1 (2 kb upstream the gene) was cloned and linked with GUS gene and transformed into Arabidopsis. Four independent transgenic Arabidopsis T3 lines were used for GUS histochemical staining assays. The GUS signal of seedlings were significantly enhanced after NaCl, PEG or H 2 O 2 treatments, respectively, compared with H 2 O treatment ( Figure 5B). Both the above results suggest that AtPDI1 was a stress-inducible gene and up-regulated expression may participate in defensive responses to stresses.

Up-and Down-Expression of AtPDI1
Affected Abilities of Anti-resistance of Seedlings Independent T3 transgenic plants of overexpressed AtPDI1 driven by the CaMV 35S promoter were generated. A knockdown AtPDI1 mutant (pdi1-1) by T-DNA insertion line was purchased from the Arabidopsis biological resource center 5 . Under normal conditions, no significant differences were observed in the growth among the wild-type, transgenic, and pdi1-1 plants (Figure 6). However, under stress treatments (0.6 µM ABA, 200 mM NaCl, 300 mM mannitol, and 10 mM H 2 O 2 ), the seeds of the overexpression lines (OE1 and OE2) germinated much earlier and showed significantly higher germination rates than that of FIGURE 7 | The roots lengths of WT, pdi, and AtPDI1-transgenic lines in stress-free (Control) or stress-containing MS medium. The root lengths of wild-type, pdi, and AtPDI1 transgenic seedlings were determined after 14 days on basal medium supplemented with ABA, H 2 O 2 , NaCl, or mannitol. Each experiment was carried out in three biological replicates.
wild type (WT), while the pdi1-1 mutant was later and lower than that of WT under same stress treatments (Figure 6 and Supplementary Figure S3). Similar tendency of root length of young seedlings were observed among OE lines, WT, and pdi1-1 mutant (Figure 7 and Supplementary Figure S4). However, no obvious differences of phenotype were observed among OE lines, WT, and pdi1-1 mutant at mature growth stages under stresses (data was not shown). The results showed that AtPDI1 play important roles in stress-tolerance during seed germination and the roots growth of young seedlings.

DISCUSSION
When animals and plants are exposed to various stresses, misfolded or unfolded proteins are accumulated, while a series of signal transduction pathways are activated, such as UPR to response the stress (Schröder and Kaufman, 2005). In animals, PDIs existing in ER are key proteins during UPR (Schröder and Kaufman, 2005;Ali and Mutus, 2014). In plants, the relations between ER stress and PDIs is poorly studied. Lu and Christopher (2008) treated seedlings with chemical inducers of ER stress and found that six of twelve Arabidopsis PDIs, including AtPDI1, were up-regulated. Three of them (AtPDI1, AtPDI5, and AtPDI6) have classical constitution of domains (a, b, b , a ) (Supplementary Figure S5). No homologous b and b fold domain could be found in the other three members (AtPDI9, AtPDI10, and AtPDI11). All six members have signal peptide sequence at N-terminus and predominantly retained within the ER (Supplementary Figure  S5), while they belong to different groups (Lu and Christopher, 2008). Our results showed that AtPDI1 predominantly existed in ER and can catalyze disulfide bond formation, which suggested that it played roles in UPR. The T-DNA knockout Arabidopsis mutant (pdi1-1) was sensitive to stresses, but overexpression AtPDI1 in WT could significantly enhance the stress resistance of plants. Discovering which proteins interact with AtPDI1 and then alleviate the protein damages of stress-elicited oxidative stress needs to be elucidated.
Proteomic analyses of chloroplasts showed that AtPDI1 was also localized in the chloroplast (Wittenberg and Danon, 2008). In chloroplast, there are at least one third proteins whose activities are regulated by thiol-disulfide conversions (Kieselbach, 2013). Compared with intensive studies about reduction of disulfide bonds in chloroplasts, only one thylakoid protein vitamin K epoxide reductase (AtVKOR/LTO1) has been demonstrated to catalyze the formation of the disulfide bonds in luminal proteins (Feng et al., 2011;Lu et al., 2011;Kieselbach, 2013). However, in chloroplast stroma, which protein catalyzes the formation of the disulfide bond has been unclear until now. AtPDI1 is regarded as the most likely candidate (Kieselbach, 2013). In this study, AtPDI1 was confirmed to have activities of PDI and two pairs of cysteins in domain a and a were critical for catalyzing function, just like other classical animal and plant PDIs. AtPDI1 may have alternative form without ER retention signal by different edit processing according to the National Center for Biotechnology Information (NCBI). However, our results showed that this form of AtPDI1 was still predominately targeted to ER (Supplementary Figure S2). Whether AtPDI1 play important roles in chloroplast needs to be elucidated.
In summary, AtPDI1 was involved in the abiotic stress processes, which was related to its activity of disulfide isomerase. The mechanisms of AtPDI1 used to regulate antistress ability and related physiological functions have yet to be investigated.

AUTHOR CONTRIBUTIONS
XW, ZZ, and XL designed the experiments. ZZ, XL, RL, and LY performed the experiments. XL, RL, and YD analyzed the data. XW, XL, and ZZ wrote the paper. All authors read and approved the manuscript.