The DHN proteins, members of the LEA protein family, are characterized by their extreme hydrophilicity (Wise and Tunnacliffe, 2004; Figure S1). Their biased amino acid composition also results in stability in response to heat in solution, which is similar to the recently developed concept of “hydrophilins” (Garayarroyo et al., 2000). The K segment (Figure 1A) as the key position of LEA proteins to bind the membrane (Koag et al., 2003, 2009), which plays a significant role in the cold tolerance effect of DHNs (Eriksson et al., 2011). The results of amino acid sequence analysis indicated the presence of five tandemly repeated motifs in PpDHNA and of three in PpDHNC (Figure 1B) Of course, these repeated amino acid segments are also found in other DHNs (Houde et al., 1992; Neven et al., 1993; Welin et al., 1994). Furthermore, we compared PpDHN with 10 different DHN proteins from Arabidopsis (Hundertmark and Hincha, 2008) and divided them into different groups according to their sequence (Figure 1C). The results indicated that the PpDHN proteins are closely related to the DHNs of Arabidopsis (Figure S2 and Table S1).

For the majority of LEA proteins, both the LEA-GFP and the corresponding GFP-LEA protein fusions were routinely found to give a specific fluorescent pattern typical of a cytosolic location. As indicators of the subcellular localizations of PpDHNA and PpDHNC, the fusion proteins 35S::PpDHNA::GFP mostly accumulated in the cytosol and nuclei, and the 35S::PpDHNC::GFP fusion protein showed more prominent localization in the chloroplast (Figure 2). Thus, it was suggested that PpDHNC conferred tolerance to the chloroplast (Artus et al., 1996). These observations suggest how the DHN proteins confer stress resistance.

There is accumulating evidence that the heterologous expression of DHNs confers significant stress tolerance (Saibi et al., 2015b). To examine the stress tolerance effects of PpDHNA and PpDHNC, we generated transgenic plants by T-DNA insertion of 35S::PpDHNA::GFP, pLEA::PpDHNA::GFP, pLEA::PpDHNA, 35S::PpDHNC::GFP, pLEA::PpDHNC::GFP, and pLEA::PpDHNC into Arabidopsis (Figure S4A).

The ability to adapt to osmotic or salt stress treatments was monitored in PpDHN transgenic and WT plants (seedlings growth on 1/2 MS media for 1 week) exposed for 7 days to concentrations ranging from 100 to 400 mM mannitol or 50 to 150 mM NaCl, respectively (Figure 3 and Figure S4). The PpDHNA and PpDHNC seedlings did show tolerance to the above stress treatments. Instead, the inhibition of primary root growth and substantial bleaching of cotyledons were seen in both WT and vector (Figure 3A and Figure S4). The critical concentrations for inhibition of plant growth were 100 mM for mannitol and 50 mM for NaCl, and the effects were concentration-dependent. After treatment, we confirmed PpDHNA and PpDHNC expression in transgenic plants and WT controls. The overexpressing lines showed significant improvement in the expression of RNA level. Moreover, the transgenic plants using the LEA promoter showed mild improvement in stress tolerance after treatment (Figures S3, S4B,C).

Until a concentration of 400 mM mannitol or 150 mM NaCl was reached, there were significant differences between the transgenic plants and WT controls; most transgenic plants survived, whereas the WT did not (Figure 3E). We further confirmed the phenotypes under conditions of salinity and osmotic stresses by determining chlorophyll contents. We expected that all the transgenic plants would maintain a high chlorophyll content and chlorophyll a/b ratio under the stress conditions (Figures 3F,G). Both strains showed quite different levels of stress response, manifested in the persistent growth phenotypes of the transgenic plants under conditions of stress.

The overexpression of genes encoding DHN proteins confers stress tolerance to transgenic plants, including tolerance to drought (Xu et al., 1996; Sivamani et al., 2000), dehydration (Cheng et al., 2002), and cold (Artus et al., 1996; Hara et al., 2003; Houde et al., 2004). Similarly, we characterized the phenotypes of PpDHNA and PpDHNC lines via whole-plant stress assay in pots (Figure 4A). The seedlings of these lines were transferred to well-watered soil in pots, and watering was withheld for approximately 2 weeks to gradually reduce water content. We chose 200-mM NaCl treatment as salinity stress. Under normal conditions, there were no differences in the growth of the transgenic lines and the WT controls. After induction of salinity stress, almost all the WT plants showed severe reduction of growth (Figure 4E). We observed severe dehydration of the leaves, and the whole plant was wilted (Figure 4E). In contrast, although some of the leaves of most DHN transgenic plants showed chlorosis, as a whole, they showed resistance to salinity stress and were still able to grow. Salinity tolerance in plants is often related to the accumulation of various molecules, such as sugars, including sucrose and trehalose; amino acids, especially proline in plant leaves; and also changes in chlorophyll level (Ben Rejeb et al., 2014). The chlorophyll content and the chlorophyll a/b ratio indicated that the DHN transgenic plants can carry out photosynthesis under conditions of stress (Figures 4B,C).

Meanwhile, DHN transgenic plants also showed characteristics of drought tolerance. After drought treatment, the majority of WT plants exhibited obvious wilting due to severe water deficit. In contrast, most of the PpDHN-overexpressing plants did not show any obvious wilting, and the leaves remained green. The water potential (ψ) of the PpDHN-overexpressing plants tended to be more consistent than that of the WT controls (Figure 4D) water potential. After re-watering for 3 days, the PpDHN lines were largely restored, and the degree of restoration was considerably higher than that of the WT controls (Figure 4E). These results indicated that PpDHN enhances plant tolerance to salinity and drought stress.

To further examine the molecular mechanism underlying the salt and osmotic tolerance conferred by PpDHN, the expression levels of five stress-responsive marker genes in PpDHN transgenic lines and WT plants under treatment with 400 mM mannitol and 150 mM NaCl were examined by qRT-PCR (Figure 5). The five stress-responsive marker genes examined showed significantly upregulated transcription in WT and transgenic plants under conditions of stress. Moreover, the fold changes in upregulation of the five marker genes under each treatment were much higher in PpDHN transgenic lines, especially in PpDHN-overexpressing lines, compared to WT controls.

Abiotic stresses, such as drought and high salinity, can induce the generation of reactive oxygen species (ROS), leading to oxidative damage to plants (Miller et al., 2010; Krasensky and Jonak, 2012). To examine the molecular mechanism of ROS scavenger production underlying the osmotic and salt tolerance conferred by PpDHN, we examined the expression of four ROS scavenger-related genes in PpDHN transgenic lines and WT controls under conditions of treatment with 400 mM mannitol and 150 mM NaCl (Figure 6). The four ROS scavenger-related genes examined were significantly upregulated in WT and transgenic plants under conditions of stress. However, the fold changes in ROS scavenger-related gene upregulation under each treatment condition were much higher in the PpDHN transgenic lines, especially the PpDHN-overexpressing lines, compared to in the WT controls. We speculate that the ROS scavenger-related enzyme activity should be higher in PpDHN transgenic plants.

ROS scavenger analysis of DHN transgenic plants showed that the PpDHN protein conferred stronger stress tolerance to plants due to upregulation of SOD and CAT enzyme activities (Figures 7A,B). These changes result in clearance of excessive ROS accumulated under conditions of stress. MDA is considered an important indicator of plant oxidative stress, a sound membrane structure, and lipid peroxidation in response to salinity and drought (Xu et al., 2010; Wen et al., 2012). In the same way, the accumulation of proline reflects, to a large extent, the plant antioxidant ability (Matysik et al., 2002). MDA and proline contents reflect the ability of plants to resist oxidative damage (Figures 7C,D). The results indicated that PpDHN transgenic plants showed a strong antioxidant capacity. Thus, PpDHN was shown to enhance the ability of plants to resist stress by participating in the antioxidant pathway.

Vector PZP111 (Hajdukiewicz et al., 1994) was used to construct PZP111-eGFP. NOS-terminator (between EcoR1 and SacI restriction sites) and the eGFP fragment (start code ATG artificially mutated to ATC) between KpnI and SacI restriction sites were obtained from vector PBI121 and then recombined into vector PZP111, respectively. The resultant vector was named PZP111-eGFP. 35S CaMV promoter from PBI121 (with HindIII and XbaI) was recombined into vector PZP111-eGFP to obtain vector PZP111-35SeGFP (Figure S7).

Specific primer pair (pLEA-PstI-F and pLEA-SalI-R, Table S2), was designed upstream of the open reading frame to amplify the PpDHNA promoter. The length of the cloned upstream fragment flanked by PstI and SalI restriction sites, as shown by the underlining. A pair of gene-specific primers (PpDHNA-XbaI-F and PpDHNA- BamHI-R, Table S2) was designed to amplify the full length open reading frame of PpDHNA. The CDS without the stop codon and flanked by XbaI and BamHI enzyme sites, was amplified from cDNA. The CDS fragment was cloned into pPZP111-35S- eGFP to obtain 35S::PpDHNA::eGFP.

The native promoter and the CDS of the gene were cloned into pPZP111-eGFP to obtain pLEA::PpDHNA::GFP. To obtain pLEA::PpDHNA, the full gene CDS was amplified using PpDHNA-SalI-F and PpDHNA-BamHI-R (Table S2). The same strategy was adopted to construct pLEA::PpDHNC, pLEA::PpDHNC::GFP, and 35S::PpDHNC::GFP (Information of primers used are listed in Table S2).

Transgenic lines were generated using the Agrobacterium tumefaciens LBA4404 vacuum infiltration method. This method is usually done by stigma infiltration. Transgenic plants were obtained by screening progeny. Seeds of the first-generation transgenic line T1 from infiltrated plants were germinated on 1/2 MS medium containing 50 mg L⁻¹ kanamycin to select for the positive seedlings. Several lines were obtained for each transformation and at least two generations of resistance screening were performed (copy number in Figure S5 and Table S4, Figure S8 shows PCR analysis of the transgenic plants).

All of the Arabidopsis seeds and transgenic plants used in this study were in a Col-0 (Columbia) background. All plants were grown in a controlled growth chamber at 21–22°C under cool-white fluorescent light (80–100 μmol m⁻² s⁻¹) in a long day photoperiod (16 h light/8 h dark).

Arabidopsis seeds were germinated on half-strength Murashige and Skoog (1/2 MS) medium supplemented with 0.6% (w/v) sucrose and 0.7% (w/v) agar. After 7 days, seedlings were transferred to 1/2 MS medium with 100–400 mM mannitol or 50–150 mM NaCl and treated for another 7 days. Mannitol and NaCl were added to different 1/2 MS medium. Seven-day seedlings(normal 1/2 MS medium) were transferred to above-mentioned 1/2 MS medium.

Arabidopsis were also grown in garden soil (Basic substrate No. 1, Pindstrup Mosebrug A/S, Denmark) without additional fertilizer, and were maintained in a greenhouse under standard growing conditions (Weigel and Glazebrook, 2002). Before salinity and drought treatment, young plants were watered once per week. Three-week plants were used for salinity or drought treatment, by watering with 200 mM NaCl solution or depriving of water respectively for 4 weeks. After drought treatment, they are rewaterd for a week.

Chlorophyll content was measured as previously reported (Wolken and Schwertz, 1953). Rosette leaves were taken and weighed at the indicated time, then placed into 5 mL of 90% (v/v) acetone for extraction of chlorophyll a/b. The chlorophyll content of each sample was assayed by measuring the absorbance at 645, 663, and 652 nm using a Beckman DU-640 spectrophotometer.

(V:The final volume of extraction solution) (W: Leaf fresh weight)

Measurement of MDA: 0.05 g of rosette leaves or cauline leaves was ground in liquid nitrogen. Each sample was incubated with 5 ml of 10% (w/v) trichloroacetic acid (TCA) solution, then with the same volume 0.6% (w/v) of 2-thiobarbituric acid (TBA) buffer. The MDA content of each sample was assayed by measuring the absorbance at 450, 532, and 600 nm using a Beckman DU-640 spectrophotometer. MDA content were calculated with the following formula: MDA levels (nmol g−1 FW)=[6.452×(D532−D600)−0.559                                                               × D450]×50

Proline content was measured using the colorimetric determination based on proline's reaction with ninhydrin (Bates et al., 1973). 0.05 g of rosette leaves or cauline leaves were incubated with 2 mL of 3% sulfosalicylic acid solution and boiled for 30 min. The proline solution was mixed with the same volume of ninhydrin acid and glacial acid at 100°C for 30 min. After mixture had cooled down, the proline content of each sample was assayed by measuring the absorbance at 520 nm using a Beckman DU-640 spectrophotometer.

Leaf water potential (ψ) was estimated on rosette leaves after drought or salt treatment. 0.5 g leaves in each plot were used to determine water potential using a DECAGON WP4C pressure chamber.

The antioxidant enzyme activities of superoxide dismutase (SOD), and catalase (CAT) were determined by ELISA using the detection kits following the manufacturer's instructions (Jiancheng Bioengineering Institute, Nanjing, China).

BLASTp was used to search for homologs of PpDHNA and PpDHNC proteins in the complete sequenced genomes of plants from various biological databases (GenBank, protein database, and genomes database). Sequences from several species were aligned with ClustalW, and phylogenetic tree was constructed using the BioNJ software (Gascuel, 1997) with Maximum likehood method (ML) analysis.

Samples were collected from the 2nd true leaves of 7-day-old seedlings. Leaf tips were fixed in 3.5% glutaraldehyde for 1 h (dark; room temperature, RT), then incubated in 0.1 M Na2EDTA (pH 9.0) solution for additional 2 h in dark at RT (Pyke and Leech, 1991). After that, leaf samples were transferred into 1 μg/mL DAPI (Sigma) solution, stained in dark for 20 min and washed with PBS buffer for 5 times before imaging.

GFP signal was examined using a Zeiss LSM 780 laser confocal microscope (Carl Zeiss, Germany). The excitation wavelength used was 488 nm. Emission wavelengths between 503 and 518 nm were used to detect GFP and wavelengths between 590 and 608 nm were used to detect chlorophyll auto-fluorescence. The excitation wavelength and emission wavelengths used for DAPI imaging are 405 nm and between 446 and 475 nm respectively. The obtained images were subsequently analyzed using Adobe Photoshop CS5.1 software.

Total RNA was isolated using Trizol reagent (Invitrogen, Gaithersburg, MD, USA) according to manufacturer's instructions. PrimeScript™ RT Master Mix (Perfect Real Time) was used for RNA purification and reverse transcription following the manufacturer's instructions. Real-time quantitative reverse transcription-PCRs (RT-PCRs) were performed using a QuantStudio™ 6 Flex Real- Time PCR System (Applied Biosystems, Warrington, UK) with SYBR Pre-mix Ex TaqTM (TaKaRa Bio Inc., China) according to the manufacturer's instructions. Using specific primers (Table S3), the expression levels of the genes are presented as values relative to the corresponding control samples at the indicated times or under the indicated conditions after normalization to actin2 (Du et al., 2014) transcript levels.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RJ and handling Editor declared their shared affiliation, and the handling Editor states that the process met the standards of a fair and objective review.