Arabidopsis thaliana ecotype Colombia-0 was used as the wild type (WT) for all experiments. Arabidopsis transgenic lines overexpressing empty vector (EV/At) or IbOr (IbOr-OX/At) were generated as described previously (Park et al., 2016), and grown in a growth chamber at 22 ± 1°C under a photoperiod of 16 h light/8 h dark.

Transgenic sweetpotato plants [Ipomoea batatas (L.) Lam. cv. Sinzami] overexpressing empty vector (EV/Ib) or IbOr (IbOr-OX/Ib) were generated as described previously (Park et al., 2015). Sweetpotato plants were propagated from stem cutting, and grown in a growth chamber at 25 ± 1°C under a photoperiod of 16 h light/8 h dark. Sweetpotato calli were subcultured on MS1D medium [0.43% basic salt Murashige & Skoog medium supplemented with 1 mg L^-1 2,4-dichlorophenoxyacetic acid (2,4-D), 3% sucrose, and 0.4% Gelrite] at intervals of 2 weeks, and samples were taken after two rounds of propagation.

For two-dimensional electrophoresis (2-DE) analysis, transgenic Arabidopsis lines (EV/At, IbOr-OX/At) were cultured for 7 days on 1/2 MS medium (0.23% Murashige & Skoog medium including vitamins, 3% sucrose, and 0.6% phyto agar) in incubation chambers at 22 ± 1°C, followed by heat treatment at 42°C for 40 min. To investigate the expression of IbPsbP, 1-month-old sweetpotato plants with six fully expanded leaves were subjected to various stresses [38 and 47°C (heat), 4°C (cold), 100 mM methyl viologen (MV), 10 mM H₂O₂, and 200 mM NaCl] (Kim et al., 2014; Park et al., 2016), and then samples were taken from the fully expanded leaves third from the top of treated plants at the indicated time. Two-month-old sweetpotato plants were used for acquisition of samples of leaves from shoot apical meristem, according to position-dependent development stage (Ji et al., 2016). To test the abiotic stress tolerance of transgenic sweetpotato, 1-month-old IbOr-OX/Ib and EV/Ib plants with six fully expanded leaves were subjected to heat treatment at 47°C for 24 h, and then plants were recovered in a growth chamber at 25 ± 1°C. All samples were frozen in liquid nitrogen and stored at -80°C after processing. All experiments were repeated as three biological and three technical replicates.

Total protein extractions were performed with trichloroacetic acid (TCA)/acetone, as previously described (Wang et al., 2008) with some modifications. Arabidopsis seedlings were ground in liquid nitrogen into a fine powder. Total protein was isolated from 200 mg of fine powder treated with 1.3 mL of cold TCA/acetone buffer (10% TCA and 0.07% 2-ME), mixed with SDS extraction buffer [30% sucrose, 2% SDS, and 0.1 M Tris-HCl (pH 8.8)] and saturated phenol, and finally precipitated by addition of 0.1 M ammonium acetate in methanol. The resultant protein was dissolved in lysis buffer [9 M urea, 4% CHAPS, 50 mM DTT, 1 mM PMSF, and 0.5% immobilized pH gradient (IPG) buffer (Amersham Biosciences, San Francisco, CA, United States)], vortexed for 1 min, and centrifuged for 1 h at 15°C, 40000 × g. The supernatant was collected and centrifuged again (15 min at 15°C, 40000 × g). Total protein was quantitated by the Bradford method (Kruger, 1994) using the Bio-Rad Quick Start^TM Bradford 1X Dye Reagent. Protein solutions were prepared and stored at -80°C. Aliquots (150 μg) of total protein were loaded on isoelectric focusing electrophoresis (IEF) gels. For first-dimension separation, IEF was carried out on a 17 cm long, pH 5–8, IPG strip on the Bio-Rad PROTEAN IEF system. Application settings were as follows: 250 V for 15 min, 10000 V for 3 h, and 80000 V for 8 h. Prior to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the gel strips were reduced in equilibrating solution [30% glycerol, 50 mM Tris-HCl (pH 8.8), 6 M urea, 2% SDS] containing 1% DTT, and then alkylated with 2.5% iodoacetamide. Second-dimension SDS-PAGE separation was carried out on the Bio-Rad Protean II, with typical run conditions as follows: initial power, 2.5 W for 30 min, followed by 6 W/gel for 5–6 h in 13% resolving gel. After electrophoresis, proteins were visualized by silver staining. All experiments were repeated twice, and similar results were obtained.

Silver-stained gels were scanned on a GS-800 Imaging Densitometer (Bio-Rad, Hercules, CA, United States) at an optical resolution of 300 dpi, and the intensities of protein spots were calculated from scanned digitized images. 2-D gel electrophoresis data analysis software (Bio-Rad PDQuest 7.2.0; Bio-Rad, Hercules, CA, United States) was used for analysis of differentially expressed protein spots. After spots were detected and quantified, matching and editing were carried out. Silver-stained protein spots were de-stained, and in-gel trypsin digestion was carried out as described in Supplementary Material.

Total RNA was extracted from the indicated plant tissues (Arabidopsis seedlings; sweetpotato callus, root, stem, and leaf) using the GeneAll Ribospin Plant^TM kit (GeneAll, Seoul, South Korea). For cDNA production, 2 μg of total RNA was reverse-transcribed to 20 μL of cDNA with 0.1 pmol of gene-specific primers using a RT-PCR kit (Enzynomics, Daejeon, South Korea). The reaction mixture was diluted 1:5 with sterilized water, and 2 μL of each reaction was subjected to real-time qRT-PCR. The gene-specific primers used in this study are listed in Supplementary Table S1. All quantitative RT-PCR (qRT-PCR) analyses were performed on a CFX real-time PCR system with the CFX system software (Bio-Rad) using Ever-Green 20 fluorescent dye (BioFACT, Daejeon, South Korea). The program used for PCR was as follows: initial denaturation for 15 min at 95°C, followed by 40 cycles of 95°C for 20 s, 60°C for 40 s, and 72°C for 30 s. Three biological repeats and three technical repeats were performed for each data point.

ORF sequences of IbPsbP and IbOr, containing the attB site and lacking a stop codon, were amplified with the indicated primers (Supplementary Table S1) to generate entry vectors based on pDONR207 using BP Clonase (Invitrogen, Carlsbad, CA, United States). For subcellular localization analysis, IbPsbP-PDONR207 was cloned into destination vector PGWB5 using LR Clonase (Invitrogen) to create the corresponding GFP fusion proteins. For bimolecular fluorescence complementation (BiFC), IbPsbP was transferred from the entry vectors to destination vector pDEST-VYNE (R) GW (containing the N-terminal fragment of Venus fluorescent protein), and IbOr was transferred from the entry vectors to destination vector pDEST-VYCE (R) GW (containing the C-terminal fragment of Venus fluorescent protein), using LR Clonase (Gehl et al., 2009). For protein expression, IbOr was transferred from the entry vectors to destination vector pDEST17 using LR Clonase to generate His-tag fusion proteins, and IbPsbP was transferred from the entry vectors to destination vector pDEST15 using LR Clonase to generate GST-tag fusion proteins.

Sequences of PsbP family members were identified by BLAST searches in GenBank (UniProt and NCBI website). The published sequences were collated and converted to predicted amino acid sequences using BioEdit. Alignments were carried out using BioEdit and BoxShade server. Phylogenetic trees were constructed using Molecular Evolutionary Genetics Analysis Version 6. Neighbor-joining phylogenetic trees were created with 1,000 bootstrap replicates.

Agrobacterium tumefaciens GV3101 strains carrying the indicated constructs and P19-silencing suppressor gene cloned into pCAMBIA 1304 vector (Voinnet et al., 2003) were grown in YEP medium supplemented with the appropriate antibiotics overnight. Cultures were spun down and resuspended in infiltration solution (10 mM MES, 10 mM MgCl₂, and 100 μM acetosyringone). The cultures were grown in infiltration solution to a final OD₆₀₀ = 0.5. Agrobacterium carrying the indicated construct or combinations of constructs and P19 were co-infiltrated into 6-week-old Nicotiana benthamiana leaves for subcellular localization analysis. After 3 days of growth in a greenhouse at 25°C under LDs, the infiltrated parts of leaves were cut and subjected to fluorescence signal detection under a Leica TCs SP2 confocal microscope (Leica Microsystems, Heidelberg, Germany) with the appropriate filter sets, as described by Gehl et al. (2009).

For protein expression, Escherichia coli BL21 (DE3) cells were induced to express GST fusion proteins (GST-IbPsbP and GST-IbOr) or 6× His fusion proteins (His-IbPsbP and His-IbOr) by treatment with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) overnight at 15°C (Miroux and Walker, 1996; Park et al., 2016). The cells were disrupted by sonication and shaken rapidly on ice for 1 h after the addition of 1% Triton X-100. The samples were then centrifuged, and the supernatant was transferred to a new tube. The supernatants were incubated overnight with prewashed GST beads (GE Healthcare, Uppsala, Sweden) or Ni-NTA beads (Qiagen, Hilden, Germany) with gentle rotation at 4°C. The beads were extensively washed, and the fusion protein was eluted with 10 mM glutathione (Sigma–Aldrich, Ann Arbor, MI, United States) for GST-tag fusion proteins, or 250 mM imidazole (Fluka, Buchs, Switzerland) for His-tag fusion proteins (Ke et al., 2017). The concentration of each fusion protein was determined by the Bradford method, as described previously (Kruger, 1994).

IbOr and IbPsbP were fused with Venus-C and Venus-N, respectively, using Gateway cloning technology. pDONR207-IbOr and pDONR207-IbPsbP were subjected to site-specific recombination into pVyCE and pVyNE, which contained the cauliflower mosaic virus 35S promoter and the C-terminal or N-terminal of Venus protein improved YFP derivative (Chen et al., 2008). Constructs were transformed into A. tumefaciens EHA105, and Agrobacterium-mediated transient expression was performed. Three days after infiltration, N. benthamiana leaves were cut off into small squares. The samples were examined for Venus fluorescence by confocal microscopy.

In vitro holdase chaperone activity was evaluated using GST:IbPsbP as substrates. The substrates were incubated in 50 mM HEPES-KOH (pH 8.0) buffer (Park et al., 2016) at 45 or 50°C with the indicated concentrations of His:IbOr. Substrate stability was determined by SDS-PAGE and western blotting using the appropriate antibodies.

In vivo holdase chaperone activity was evaluated using GFP:IbPsbP as substrates. Through Gateway cloning system, IbPsbP-pDONR207 and IbOr-pDONR207 were subjected to site-specific recombination into pGWB5 (35S promoter, C-sGFP) and pGWB11 (35S promoter, C-FLAG), respectively. IbPsbP and IbOr were fused with GFP and FLAG. GUS was amplified from the pENTE^TM GUS vector (Invitrogen, Carlsbad, CA, United States), after the PCR product was cloned into T-blunt and sequenced, clones were ligated into the pCAMBIA1300-multi vector by digestion with restrictive enzyme. All three plasmid constructs were transformed into A. tumefaciens strain GV3101 and transient expressed in N. benthamiana with or without FLAG:IbOr (as shown in Figure 6B). Infected part of tobacco leaves were used for total proteins isolation and western blotting with corresponding antibodies.

Photosynthetic activity in leaves was evaluated based on chlorophyll fluorescence determination of photochemical yield (Fv/Fm), which represents the maximal yield of the photochemical reaction in PSII (Butler and Kitajima, 1975), using a portable chlorophyll fluorescence meter (Handy PEA, Hansatech, England) after 30 min of dark adaptation. Chlorophyll contents were measured with a portable chlorophyll meter (SPAD-502, Konica Minolta, Japan). Total chlorophyll contents after stress treatment were compared with those under normal conditions. Both of these values were detected using the third through fifth intact, fully expanded leaves (counting from shoot apical meristem) of individual plants.

Our previous results suggested that IbOr plays important roles in protecting photosynthesis from abiotic stress via regulation of carotenoid biosynthesis and stabilization of PSII (Kim et al., 2013; Park et al., 2015, 2016). However, no photosynthetic proteins have been reported to interact with IbOr. To identify proteins that bind IbOr, we incubated 7-day-old transgenic Arabidopsis seedlings (EV/At and IbOr-OX/At) at 42°C for 40 min, and then extracted total proteins from the heat-stressed seedlings and subjected the samples to 2-DE analysis. We detected around 850 and 875 proteins in EV/At and IbOr-OX/At seedlings, respectively (Supplementary Figure S1).

In total, we identified 18 protein spots that were reproducibly differentially expressed in IbOr-OX/At transgenic Arabidopsis seedlings (Supplementary Figure S1B) in comparison with EV (Supplementary Figure S1A). Among them, five proteins were up-regulated and 13 were down-regulated in IbOr-OX/At transgenic plants (Figure 1). All protein spots exhibiting statistically significant differences between EV and IbOr-OX/At transgenic plants were selected for further characterization. To investigate the functions of these proteins, we excised all 18 protein spots from silver-stained gels and digested them with trypsin, and subjected the digestion products to MALDI-TOF/TOF-MS analysis. The list of differentially expressed proteins is summarized in Table 1. SPX domain-containing protein 3 (Uni), oxygen-evolving enhancer protein 2-1 (PsbP), thioredoxin reductase 1, fructose-bisphosphate aldolase, and ribulose bisphosphate carboxylase large chain were up-regulated by 1.75-, 2.82-, 2.97-, 2.7-, and 2.3-fold, respectively. To investigate whether IbOr, which is involved in resistance to heat stress, can directly interact with these up-regulated proteins, we performed yeast two-hybrid assays. Among five up-regulated proteins, PsbP protein only interacted with IbOr (Supplementary Figure S2). Therefore, we focused on PsbP in our subsequent investigation of how IbOr regulates heat stress tolerance and photosynthesis. PsbP is an extrinsic subunit of PSII and participates in the normal function of photosynthetic water oxidation (Ifuku, 2014). However, its physiological roles have not been well-defined in higher plants.

We isolated the cDNA fragment of IbPsbP from orange-fleshed sweetpotato (cv. Sinhwangmi) by homology-based BLAST searches against the sweetpotato transcriptome database (unpublished data). Phylogenetic analysis of the deduced plant PsbP amino acid sequences from 11 species indicated that IbPsbP clusters closely with other PsbP homologs (Figure 2A). The predicted protein IbPsbP contains 268 amino acids, and comparison of the deduced amino acid sequence revealed that IbPsbP shares 73–80% amino acid identity with other PsbP homologs (Figure 2B). These results suggest that IbPsbP shares a conserved role with other PsbP homologs.

To investigate the subcellular localization of IbPsbP, we transiently expressed GFP-fused IbPsbP in N. benthamiana leaves by Agro-infiltration. Three days after infiltration, the epidermal cells of infiltrated leaves were observed by confocal laser scanning microscopy. As shown in Figure 2C, GFP fluorescence produced by IbPsbP-GFP fusion proteins overlapped with chlorophyll fluorescence, indicating that IbPsbP is a chloroplast protein.

We next examined the expression of the IbPsbP gene in various tissues of sweetpotato. IbPsbP transcripts were most abundant in leaves among the tissues examined (leaf, stem, and fibrous root) of 1-month-old sweetpotato plants and in calli (Figure 3A). We then investigated IbPsbP gene expression at every position along the leaf (from shoot apical meristem) of 2-month-old sweetpotato plants. As shown in Figure 3B, IbPsbP was expressed preferentially in older leaves (most abundant in leaf position 12) in comparison with tender leaves.

In addition, we investigated whether IbPsbP transcripts levels were affected by multiple abiotic stresses, including heat (38 and 47°C), cold (4°C), MV (100 μM), H₂O₂ (10 mM), and salt (200 mM NaCl). For this purpose, we continually monitored IbPsbP expression in the third expanded leaves from treated plants over 48 h. As shown in Figure 4, IbPsbP was induced by multiple abiotic stresses, but the expression pattern differed depending on the specific nature of the stress. IbPsbP transcript levels were elevated 1.4- and 1.7-fold in the early phase of heat stress. IbPsbP was also up-regulated fourfold at 12 h by MV treatment and reached maximum expression (2.6-fold) at 24 h by NaCl treatment. Interestingly, IbPsbP was dramatically induced after 3 h of cold treatment and reached the highest expression level (10.7-fold) at 12 h, followed by a decrease. In addition, IbPsbP was up-regulated sixfold by H₂O₂ treatment (Figure 4). These results suggest that IbPsbP transcripts are tissue-specifically expressed, and may play an important role in response to abiotic stress in sweetpotato plants.

Our previous results indicated that IbOr functions as a holdase chaperone to stabilize phytoene synthase (IbPSY) in sweetpotato (Park et al., 2016). To determine whether IbOr plays a similar role in protecting IbPsbP from heat stress induced denaturation, we first examined the interaction between IbOr and IbPsbP in plant cells using BiFC assays. Cell suspensions of A. tumefaciens carrying constructs encoding N-terminal Venus fluorescent protein NV-IbPsbP and C-terminal Venus fluorescent protein CV-IbOr were infiltrated into N. benthamiana leaves. We detected strong Venus fluorescence in chloroplasts when NV-IbPsbP and CV-IbOr were used in combination indicating that IbOr interacts with IbPsbP, predominantly in chloroplasts (Figure 5A). This interaction was confirmed by in vitro pull-down assays (Figure 5B).

We next tested whether the IbOr–IbPsbP complex affects the stability of IbPsbP upon heat stress. To this end, we purified bacterially expressed recombinant GST:IbPsbP protein and evaluated its stability under heat stress. Specifically, GST:IbPsbP was incubated in the presence or absence of purified recombinant His:IbOr protein at the indicated temperatures, and then IbPsbP levels were analyzed on SDS-PAGE by immunoblotting. Previous work showed that IbOr is stable even at 70°C (Park et al., 2016), whereas GST:IbPsbP protein aggregated at 50°C after 30 min. However, when purified recombinant His:IbOr protein was included in Tris-HCl (pH 8.0) buffer, IbPsbP was protected from aggregation (Figure 6A). These results indicated that IbOr holdase chaperone activity can stabilize IbPsbP during heat stress in vitro. To confirm IbOr holdase chaperone activity for IbPsbP in planta, further investigation was carried out. A. tumefaciens carrying GFP:IbPsbP fusion protein and the expression control GUS protein with or without FLAG:IbOr fusion protein were transiently co-expressed in well-conditioned N. benthamiana in a consistent state. Three days after infiltration, infiltrated plants were subjected to heat stress at 38°C for 1 h. Total proteins were isolated from infected leaves before and after heat treatment, and were analyzed by western blotting with anti-GFP, anti-FLAG, anti-GUS. GFP:IbPsbP protein shows parallel stability in the presence or absence of FLAG:IbOr fusion protein co-expression under normal condition (Figure 6B). However, after heat treatment (38°C, 1 h), GFP:IbPsbP protein was severely degraded in infiltrated plants without FLAG:IbOr protein, but was fairly consistent with FLAG:IbOr protein co-expression (Figure 6B). Taken together, these results indicated that IbOr holdase chaperone activity can stabilize IbPsbP under heat stress condition in planta.

In higher plants, PsbP protein is essential for the regulation and stabilization of PSII, and is required for normal thylakoid architecture (Yi et al., 2007, 2009). Heat or oxidative stress induced damage frequently occurs in the OEC of PSII (Allakhverdiev et al., 2008). To determine whether IbOr overexpression confers increased PSII efficiency upon exposure to heat stress, we developed transgenic sweetpotato plants harboring empty vector (EV/Ib) or IbOr overexpression construct (IbOr-OX/Ib), and then subjected 1-month-old pot-cultured EV/Ib and IbOr-OX/Ib plants of the same health status to high-temperature (47°C) treatment. Because oxygen-evolving activity of PSII started to decline at 40°C with half inactivation at 46–47°C (Allakhverdiev et al., 2007), we treated the EV/Ib and IbOr-OX/Ib plants with heat stress at 47°C. After 24 h of heat stress, EV/Ib plants exhibited wilting and chlorosis, whereas IbOr-OX/Ib plants grew well and exhibited only slight yellowish leaf coloration (Figure 7A). IbOr-OX/Ib plants maintained approximately 4.6- and 5.1-fold higher PSII efficiency and chlorophyll content, respectively, than EV/Ib plants (Figures 7B,C), consistent with their high temperature resistant phenotype. These results indicate that the holdase chaperone function of IbOr, which regulates IbPsbP stability, stabilizes PSII, thereby conferring heat stress tolerance on sweetpotato plants.