Overexpression of a Barley Aquaporin Gene, HvPIP2;5 Confers Salt and Osmotic Stress Tolerance in Yeast and Plants

We characterized an aquaporin gene HvPIP2;5 from Hordeum vulgare and investigated its physiological roles in heterologous expression systems, yeast and Arabidopsis, under high salt and high osmotic stress conditions. In yeast, the expression of HvPIP2;5 enhanced abiotic stress tolerance under high salt and high osmotic conditions. Arabidopsis plants overexpressing HvPIP2;5 also showed better stress tolerance in germination and root growth under high salt and high osmotic stresses than the wild type (WT). HvPIP2;5 overexpressing plants were able to survive and recover after a 3-week drought period unlike the control plants which wilted and died during stress treatment. Indeed, overexpression of HvPIP2;5 caused higher retention of chlorophylls and water under salt and osmotic stresses than did control. We also observed lower accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), an end-product of lipid peroxidation in HvPIP2;5 overexpressing plants than in WT. These results suggest that HvPIP2;5 overexpression brought about stress tolerance, at least in part, by reducing the secondary oxidative stress caused by salt and osmotic stresses. Consistent with these stress tolerant phenotypes, HvPIP2;5 overexpressing Arabidopsis lines showed higher expression and activities of ROS scavenging enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) under salt and osmotic stresses than did WT. In addition, the proline biosynthesis genes, Δ1-Pyrroline-5-Carboxylate Synthase 1 and 2 (P5CS1 and P5CS2) were up-regulated in HvPIP2;5 overexpressing plants under salt and osmotic stresses, which coincided with increased levels of the osmoprotectant proline. Together, these results suggested that HvPIP2;5 overexpression enhanced stress tolerance to high salt and high osmotic stresses by increasing activities and/or expression of ROS scavenging enzymes and osmoprotectant biosynthetic genes.


INTRODUCTION
Aquaporins belong to major intrinsic proteins (MIPs) that are present from prokaryotes to plants and animals. These proteins facilitate the transport of water and small uncharged molecules across biological membranes (Park and Saier, 1996;Heymann and Engel, 1999;Engel and Stahlberg, 2002;Zardoya et al., 2002;Maurel et al., 2008Maurel et al., , 2015. MIPs are categorized into two groups: aquaporins that show water-specific channel activity and glycerol-uptake facilitators (GLPs or aquaglyceroporins) that have channel activity for additional small molecules such as glycerol or urea (Park and Saier, 1996;Heymann and Engel, 1999;Engel and Stahlberg, 2002;Zardoya et al., 2002;Zardoya, 2005;Maurel et al., 2008). In plants, all MIPs, except for GlpF-like intrinsic proteins (GIPs), exhibit water-specific channel activity; therefore, most plant MIPs are collectively called aquaporins (Maurel et al., 2008(Maurel et al., , 2015. Recently, it has been shown that plant MIPs can transport other small molecules such as CO 2 and ammonia (Uehlein et al., 2003;Jahn et al., 2004).
The high diversity in plant aquaporins suggests variation of their physiological roles. Indeed, aquaporins were shown to be associated with vital physiological processes such as photosynthesis, nitrogen fixation, nutrient uptake and other environmental stress responses (Li et al., 2014;Hove et al., 2015;Sun et al., 2015). Plant aquaporins are classified into five subgroups, i.e.,: the plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and the recently identified uncategorized (X) intrinsic proteins (XIP) (Maurel et al., 2015). Based on sequence divergence, PIPs are further divided into PIP1 and PIP2 subclasses each consisting of several isoforms which play important roles in determining hydraulic conductivity particularly in roots (Martre et al., 2002;Siefritz et al., 2002;Javot et al., 2003;Postaire et al., 2010). Analyses on PIP1 and PIP2 from barley and maize revealed that the PIP2 proteins had higher water transport activity than PIP1 proteins in Xenopus oocytes (Chaumont et al., 2000;Horie et al., 2011). When PIP2 was co-expressed with functional or even nonfunctional PIP1 proteins, water transport activity of PIP2 was enhanced (Chaumont et al., 2000;Fetter et al., 2004;Horie et al., 2011). This enhanced water transport was attributed to their ability to form heterotetramers for proper trafficking to the plasma membrane (Fetter et al., 2004;Zelazny et al., 2007).
Dynamic changes in the expression levels of many PIP genes were observed in response to drought stress, suggesting their involvement in stress responses by regulating water balance (Afzal et al., 2016). Studies with PIP-defective mutants or overexpressing plants also linked the roles of PIP proteins to water-deficit stress tolerance. For example, when expression of a tobacco PIP1 member (NtAQP1) was reduced by antisense technology, the NtAQP1-downregulated tobacco plants showed reduced root hydraulic conductivity and wilting phenotypes under water stress (Siefritz et al., 2002). Physcomitrella patens PIP2;1 or PIP2;2 knockouts showed low water permeability with drought-sensitive phenotypes (Lienard et al., 2008). Reduction in water permeability of protoplasts and root hydraulic conductivity were observed respectively in Arabidopsis PIP1;2 and PIP2;2defective mutants but without clear developmental defects (Kaldenhoff et al., 1998;Javot et al., 2003). Overexpression of several PIP genes from various plants including Oryza sativa, Vicia faba, Nicotiana tabacum, and Triticum aestivum successfully enhanced water stress tolerance in transgenic plants (Lian et al., 2004;Cui et al., 2008;Sade et al., 2010;Zhou et al., 2012). Interestingly, some contrasting results (i.e., stress sensitive phenotypes in PIP overexpressing plants) have also been reported, implying the complexity of PIP function in plants (Aharon et al., 2003;Katsuhara et al., 2003;Jang et al., 2007;Li et al., 2015).
Barley (Hordeum vulgare L.) is one of the most agronomically cultivated crops; it is more adaptable to drought, salinity and cold than other cereal crops (Katsuhara et al., 2014;Hove et al., 2015). These characteristics would possibly make the barley gene pool, including barley aquaporins, as one of stress-adaptive genetic resources. Although, several PIPs have been identified in barley, only few of them have been functionally characterized thus far.
In this study, we overexpressed barley PIP2;5 (HvPIP2;5) in yeast and Arabidopsis and characterized these lines to understand the functions of the barley PIP gene under high salt and high osmotic stress conditions.

Transformation of Yeast and Stress Analysis
The HvPIP2;5 coding sequence under control of GAL1 promoter in pYES2 yeast expression vector was introduced into yeast FY3 cells. As controls, FY3 cells containing pYES2 vector only (vector control) and FY3 strain only were used in stress assays. The yeast strain FY3 was transformed with a pYES2 empty vector or pYES2:HvPIP2;5 recombinant vector by lithium acetate method (Kawai et al., 2010) and selected on SC medium devoid of uracil. Yeast cells expressing HvPIP2;5 along with control cells were grown on YPD solid medium (1% Yeast extract, 2% peptone, and 2% glucose).
For stress analysis, transformed yeast cells were propagated in SC-U medium containing 2% galactose for 12 h and cell density was adjusted to 1.0 of OD600 followed by serial dilutions. Yeast cell were spotted on YPD medium supplemented with PEG (4%) or NaCl (200 mM), respectively. Plates were maintained at 30 • C and growth was monitored after 2 days.
For germination analysis, the seeds of WT and homozygote HvPIP2;5 OE Arabidopsis lines were plated on half strength Murashige Skoog (MS) media and allowed to germinate at 22 • C and 60% relative humidity with a 16/8 h light-dark photo cycle after 3 day stratification at 4 • C.
Final concentration of 100-200 mM NaCl (for high salt stress) or 10-20% PEG (for high osmotic stress) was supplemented to the MS media for stress administration. Hereby salt stress or osmotic stress indicates high salt stress or high osmotic stress, respectively. Emergence of cotyledons was used as a germination criterium. The number of germinated seeds was expressed as a percentage of total number of seeds planted after 7 days. For root elongation analysis, vertically grown seedlings with 1-1.5 cm long root were transferred onto a MS vertical plate supplemented with or without stress agents (200 mM NaCl and 20% PEG). Root length was measured 15 days after transfer. Root length of seedlings under stress conditions was expressed as a percentage of their respective controls grown on normal MS medium. All stress analysis experiments were conducted three times and each contained 3 biological repeats. For drought test with soil-grown plants, 4 week old seedlings were subjected to drought stress by withholding water supply for 21 days and then re-watered. For salt and osmotic stress, 3 week old plants were irrigated with either half strength MS liquid (control) or half strength MS liquid supplemented either with 200 mM NaCl or 20% PEG to impose stress for 15 days at 2 day intervals and then harvested for analysis. The chlorophyll, proline, and malondialdehyde (MDA) content in control or stress treated plants were determined by the methods reported previously by Lichtenthaler (1987), Bates et al. (1973), and Heath and Packer (1968), respectively.

Water Loss and Relative Water Content Analysis
Rosette leaves of 3 week old seedlings were detached, weighed and placed on paper under the fume hood to administrate drought stress. Fresh weights of rosette leaves were measured each hour for 5 h. Water loss was calculated as the loss in fresh weight of the samples. For relative water content analysis, the root from WT and the HvPIP2;5 OE lines were excised and treated either with 200 mM NaCl or 20% PEG or deionized water after measuring the fresh weight. After 24 h of incubation, the tissues were weighed again for their turgid weight and then dried completely to obtain dry weight. The root RWC was calculated by the following formula; RWC (%) = [(fresh weight − dry weight)/(turgid weight − dry weight)] × 100 (Weatherley, 1950).

Reactive Oxygen Species Analysis and Antioxidant Enzyme Activity Assay
In situ detection of superoxide by nitro blue tetrazolium (NBT) staining and hydrogen peroxide by 3, 3'diaminobenzidine (DAB) staining was performed according to methods previously described by Rao and Davis (1999) and Ramel et al. (2009). For this, 3 week old seedlings were grown on MS medium with 0, 100, 200 mM NaCl and the third leaf from the top was used. Superoxide radicals were visualized as blue color produced by NBT precipitation while hydrogen peroxide spots were visualized as brown color due to DAB polymerization.
For ROS quantification and antioxidant enzyme activity assay, 3 week old plants were irrigated with either half strength MS liquid (control) or half strength MS liquid supplemented either with 200 mM NaCl or 20% PEG to impose stress for 15 days at 2 day intervals and then harvested for analysis. Contents of superoxide and hydrogen peroxide were estimated by methods previously described by Elstner and Heupel (1976) and Sagisaka (1976), respectively. For antioxidant enzyme assay, the samples (200 mg tissue) were homogenized in 1.5 mL of 0.1 M phosphate buffer (pH 6.8) containing 1 mM ethylenediamine tetra-acetic acid (EDTA) and 1% polyvinylpyrrolidone (PVP) in a chilled pestle and mortar. The homogenate was then centrifuged at 17,000 g for 15 min at 4 • C. The supernatant was quantified by the Bradford method (Bradford, 1976) and used for the assay of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) (Chance and Maehly, 1955;Smith et al., 1988;Gupta et al., 1993). For the ascorbate peroxidase (APX) activity assay, a final concentration of 1 mM ascorbic acid was added to the assay buffer (Nakano and Asada, 1981).

Gene Expression Analysis
Plants were treated with water for control or 300 mM NaCl for 6 h or 20% PEG for 6 h, and total RNAs were extracted using RNeasy Plant mini Kit (Qiagen, Germany). cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, USA), and resulting cDNAs were used for semi-quantitative RT or qRT-PCR experiments for selected genes. Primer pairs used in experiments are shown in Supplementary Table 1.

Statistical Analysis
All statistical comparisons between variances were determined by ANOVA (Analysis of variance) and least significant differences (LSD) between variants were calculated using Statistix 8.1 computation software. Statistically significant mean values were denoted as * (P ≤ 0.05).
It has been recently reported that the barley genome comprises at least 40 aquaporin genes with 5 PIP1 genes and 9 PIP2 genes (Hove et al., 2015). Multiple sequence alignment using all reported HvPIP2 isoforms was performed using Mega 6 software (http://www.megasoftware.net). As expected, HvPIP2 proteins showed high degree of homology among them, and HvPIP2;5 was located in the same clade as HvPIP2;1 with 85% identity to HvPIP2;1 (Supplementary Figure 1D).

Increased Tolerance of
HvPIP2;5-Expressing Yeast under High Salt and Osmotic Stresses A yeast expression system was employed to examine the functions of the HvPIP2;5 gene under high saline and high osmotic conditions. As shown in Figure 1A, yeast strains transformed with HvPIP2;5 or empty vector and FY3 cells could grow up to 10 −6 dilution on YPD plates. In the presence of 200 mM NaCl, pYES2 vector-containing FY3 cells grew only until 10 −3 dilution, whereas the yeast strain transformed with HvPIP2;5 was able to grow until 10 −6 dilution ( Figure 1B). Similarly, empty vector containing cells grew only to 10 −3 dilution on the YPD medium supplemented with 4% polyethylene glycol (PEG), while yeast cells with HvPIP2;5 displayed growth until 10 −6 dilution under the same condition ( Figure 1C). Taken together, these results showed that HvPIP2;5 expression in yeast resulted in increased stress tolerance under high salt and high osmotic conditions.

Enhanced Stress Tolerance of HvPIP2;5-Overexpressing Arabidopsis
To investigate the functional roles of HvPIP2;5 in planta, we expressed HvPIP2;5 in Arabidopsis under the control of CaMV 35S promoter (Supplementary Figure 2A). Two independent homozygous HvPIP2;5 overexpressing (OE) lines were selected. PCR analysis confirmed the presence of the HvPIP2;5 gene in transgenic plants (data not shown) and semi quantitative RT-PCR for HvPIP2;5 expression analysis demonstrated that transgenic lines were overexpressing HvPIP2;5 gene (Supplementary Figure 2B).
Germination under 200 mM NaCl (high salt stress) and 20% PEG (high osmotic stress) was first tested for two independent HvPIP2;5 OE lines along with WT. On the control medium, all lines germinated successfully after 7 days of planting. On the medium supplemented with 200 mM NaCl, WT germination ratios were decreased to about 50%, whereas HvPIP2;5 OE lines displayed a 59-60% germination ratio on salt medium (Figures 2A,D). Also, HvPIP2;5 OE lines displayed a 41-44% germination ratio in MS medium with 20% PEG whereas WT germination was reduced to 18% (Figures 2A,D).
Root growth under salt and osmotic stress conditions was also compared. In root growth assays, we observed increased tolerance of HvPIP2;5 OE lines under salinity and osmotic stresses compared to WT. The relative root length of HvPIP2;5 OE lines was significantly higher in the presence of 200 mM NaCl (59-62% vs. 34%) or 20% PEG (47-50% vs. 28%) compared to that of WT (Figures 2B,E).
We further evaluated growth performance of WT and HvPIP2;5 OE lines during drought stress. WT and HvPIP2;5 OE plants were grown in well-watered conditions for 4 weeks and then subjected to drought conditions. After 21 days of water withdrawal, WT lines became wilted with retarded growth, whereas HvPIP2;5 OE lines did not wilt as severely as WT. Upon re-watering, WT plants were so severely damaged that they were unable to resume growth. In contrast, the transgenic lines were able to recover and retained survival upon rehydration ( Figure 2C).
Chlorophyll degradation is among the changes caused by salt and osmotic stresses. No remarkable differences were observed between WT and HvPIP2;5 OE lines grown under control conditions; however, after stress treatments with 200 mM NaCl or 20% PEG, HvPIP2;5 OE lines showed less chlorophyll degradation when compared with WT. Measurement of chlorophyll contents of stress-treated plants confirmed the better retention of chlorophyll A in OE lines than in WT ( Figure 2F). WT under salinity stress with 200 mM NaCl contained only 23% of chlorophyll A levels under normal conditions, while OE lines under salt stress had largely higher levels of chlorophyll A retention (27-43%). Additionally, osmotic stress with 20% PEG caused 51 and 25-31% chlorophyll A loss in WT and HvPIP2;5 OE, respectively ( Figure 2F).
Low Water Loss and High Water Retention in HvPIP2;5 Overexpressing Lines under Stresses Dehydration tolerance in HvPIP2;5 lines was assessed by measuring the water loss percentage. For this, rosette leaves from WT and HvPIP2;5 OE lines were detached, placed on paper for dehydration and fresh weight measured. After 5 h of dehydration, the leaves of WT plants lost ∼45% of their original fresh weight, whereas leaves of the HvPIP2;5 OE lines lost only about 30-35% from their initial fresh weight ( Figure 3A).
In addition, relative water content (RWC) was measured from the root tissues of WT and HvPIP2;5 OE lines under salt or osmotic stress. Under salt stress with 200 mM NaCl, WT lines held 68.1% RWC, whereas HvPIP2;5 OE lines kept 75-77% RWC ( Figure 3B). Likewise, OE lines contained largely higher RWC than did WT under osmotic stress with 20% PEG (Figure 3B).

Reduction of Oxidative Stress in HvPIP2;5 Overexpressing Lines under stresses
Reactive oxygen species (ROS) are generated in plants under drought, salt, and temperature stresses. Using nitro blue tetrazolium (NBT) staining for superoxide and diaminobenzidine (DAB) staining for hydrogen peroxide, we measured the levels of ROS in WT and HvPIP2;5 OE lines under high salt (200 mM NaCl) and high osmotic stress (20% PEG). Compared to WT, the HvPIP2;5 OE lines showed significantly weaker NBT staining and less O − 2 amount under osmotic or salt stress conditions (Figures 4A,B). Similarly, DAB staining and quantification of H 2 O 2 content revealed lower amounts of H 2 O 2 in the OE lines than in WT after NaCl treatment (Figures 4C,D). However, HvPIP2;5 OE lines did not seem to display very significant reduction in H 2 O 2 levels after PEG treatment when compared to WT.
Malondialdehyde (MDA) is an end product of lipid peroxidation of cell membrane lipids and a good indicator of oxidative damage (Diao et al., 2011). After salt stress (200 mM NaCl) and osmotic stress (20% PEG), MDA content was significantly lower in HvPIP2;5 OE lines than in WT, which implicates lesser membrane damage in HvPIP2;5 OE lines ( Figure 4E). These results suggested that enhanced stress tolerance in HvPIP2;5 OE lines might be due, at least in part, to reduced levels of oxidative stress caused by salt and osmotic stresses.

Increased Activities of ROS Scavenging Enzymes in HvPIP2;5 Overexpressing Lines
To assess the contribution of ROS scavenging enzymes in reduction of oxidative stress caused by salt and osmotic stresses,  we measured the activities of catalase (CAT) and superoxide dismutase (SOD) in WT and HvPIP2;5 OE lines under salt and osmotic stress conditions. Although, CAT activity was elevated in both WT and HvPIP2;5 OE lines, consistent with increase of osmotic and saline stresses (Figure 5A), the level of increase of CAT activity was significantly higher in HvPIP2;5 OE lines than in WT. Patterns largely similar to those found in the CAT activity assay were observed in SOD activity assay. SOD activity in HvPIP2;5 OE lines were higher than those in WT under salt and osmotic stresses ( Figure 5B). Interestingly, both CAT and SOD activities in HvPIP2;5 OE lines were generally higher than those of WT even under normal conditions.
In addition, we measured the activities of glutathione reductase (GR) and ascorbate peroxidase (APX) in HvPIP2;5 OE lines under 100 and 200 mM NaCl conditions. GR and APX are major enzymes for the ascorbate-glutathione cycle which is an important component of the ROS scavenging system in plants (Pang and Wang, 2010). Generally, elevated activities of APX and GR have been shown to correlate with increased salt tolerance in plants (Pang and Wang, 2010). The activities of GR were higher in HvPIP2;5 OE lines than in WT under both normal and salt conditions ( Figure 5C). The activities of APX were also largely higher in HvPIP2;5 OE lines than in WT under normal and salt conditions ( Figure 5D).
Taken together, these results indicated that reduction of oxidative stress in HvPIP2;5 OE lines under salt and osmotic stresses is possibly related to enhanced activities of ROS scavenging enzymes.

Increased Levels of Proline in HvPIP2;5 Overexpressing Lines
The amino acid proline acts as an osmolytes and an antioxidant, and high levels of proline enhance stress adaptation under unfavorable conditions (Bates et al., 1973;Hayat et al., 2012). Thus, we investigated expression patterns of the Arabidopsis proline biosynthesis genes, 1 -Pyrroline-5-Carboxylate Synthase 1 and 2 (P5CS1 and P5CS2) in WT and HvPIP2;5 OE lines. Real time PCR analysis revealed that gene expression levels of P5CS1 and P5CS2 were higher in HvPIP2;5 OE lines than in WT under salt and osmotic stresses (Figures 6A,B). We also measured proline levels in WT and HvPIP2;5 OE lines and found that even under normal conditions, HvPIP2;5 OE lines demonstrated slightly higher proline levels than WT (Figure 6C). After salt treatment, proline levels were increased in both WT and HvPIP2;5 OE lines with much higher increase in OE lines than in WT ( Figure 6C). Interestingly, our PEG treatment did not seem to induce proline accumulation. Still, proline levels remained higher in the HvPIP2;5 OE lines than in WT ( Figure 6C). This salt-induced proline accumulation correlated with the up-regulation of P5CS1 and P5CS2 expression in HvPIP2;5 OE lines, suggesting a possible molecular mechanism behind the enhanced stress tolerance in HvPIP2;5 OE lines.

DISCUSSION
Water-deficit stress such as salt and osmotic stress and impede plant growth and development by affecting plant water balance. Aquaporins play important roles as water channels in regulating FIGURE 6 | Analysis of P5CS1 and P5CS2 gene expression and proline levels in HvPIP2;5 overexpressing lines under salt and osmotic stresses. The expression levels of (A,B) P5CS1 and P5CS2 in WT and OE lines were estimated by quantitative real time PCR. Arabidopsis clatharin gene was used as an internal control for normalization. The expression levels of each gene in WT control (calibrator) were assumed as 1. Three biological replicates were averaged and bars indicate standard error. (C) Proline contents in WT and OE were quantified with three biological replicates. Bars indicate standard error and significant differences between WT and OE lines were marked with asterisks (P < 0.05). plant water status; thus, many aquaporin genes from diverse plant species have been used in transgenic research to improve waterdeficit stress tolerance in plants (Martre et al., 2002;Cui et al., 2008;Khan et al., 2015).
We found that overexpression of barley PIP2;5 (HvPIP2;5) in both yeast and Arabidopsis improved tolerance to high salt and high osmotic stresses (Figures 1-3). These improved tolerances in HvPIP2;5 overexpressing Arabidopsis were also observed in milder stress conditions (100 mM NaCl and 10% PEG) (Supplementary Figures 3, 4). Increased stress tolerance in HvPIP2;5 overexpressing Arabidopsis was correlated with lower levels of stress-induced ROS (Figure 4 and Supplementary Figure 5), high activity of ROS scavenging enzymes (Figure 5), higher induction of proline biosynthetic gene expression and high levels of osmoprotectant proline (Figure 6). In particular, HvPIP2;5 overexpressing Arabidopsis displayed lower water loss in shoots and higher relative water contents in roots than did WT under salt and osmotic stresses. While HvPIP2;5 overexpression improved water-stress tolerance in both yeast and Arabidopsis, Arabidopsis PIP2;5 (AtPIP2;5) overexpression brought about reduced osmotic stress tolerance in Arabidopsis and tobacco (Jang et al., 2007). These seemingly contradicting results are not uncommon. In fact, many aquaporin overexpression studies have produced contrasting results-aquaporin-overexpressing plants have shown either positive or negative effects on stress tolerance (Maurel et al., 2008;Martinez-Ballesta and Carvajal, 2014;Zhou et al., 2014). Even overexpression of aquaporins with high homology has resulted in different sensitivities to dehydration stress. For example, Arabidopsis AtPIP1;2, rice RWC3, and tobacco NtAQP1 share approximately 80% sequence identity. Despite this, overexpression of AtPIP1;2 in tobacco has caused reduced stress tolerance (Aharon et al., 2003) while RWC3 overexpression in rice and NtAQP1 overexpression in tomato have shown enhanced tolerance under drought and salt stress, respectively (Lian et al., 2004;Sade et al., 2010). In addition, Arabidopsis pip2;2 mutants display defects in hydraulic conductivity despite the expression of a very close homolog AtPIP2;3 which shares >96% homology, demonstrating that close aquaporin homologs could not function redundantly even within the same plant (Javot et al., 2003).
Although, HvPIP2;5 and AtPIP2;5 share high sequence homology, there are differences in gene regulation. AtPIP2;5 expression levels remain low in Arabidopsis and are only upregulated by drought and cold (Jang et al., 2007). However, HvPIP2;5 is one of the highly expressed PIPs in barley which is down-regulated by osmotic stress (Katsuhara et al., 2014). These different patterns of gene expression might indicate the divergent functions of PIP2;5 in Arabidopsis and barley, and may be attributed to contrasting stress responses in HvPIP2;5 and AtPIP2;5 overexpressors.
Another explanation for the contrasting results might lie in the difference in protein sequence between HvPIP2;5 and AtPIP2;5 proteins. Amino acid sequence differences are mainly found in the N-terminus which is expected to be exposed on the cytosol side (Walz et al., 1997; Supplementary Figures 1A, 6). Thus, it is tempting to speculate that contrasting stress phenotypes may be due to differences in the N-termini of PIP2;5 proteins which may contain important motifs such as for activity regulation, protein stability, protein interaction, or even subcellular localization. Although transcriptional control of aquaporins appeared to be important for physiological functions (Alexandersson et al., 2005;Guo et al., 2006;Jiang et al., 2016), aquaporin activity is also post-translationally regulated by protein modification including phosphorylation (Johansson et al., 1998;Santoni et al., 2003;Daniels and Yeager, 2005). We have found some differences in phosphorylation sites at the N-terminus of two PIP2;5 proteins (Supplementary Figure 6) which might be important for PIP2;5 function. This differential protein modification might cause variable functional activities of PIP2;5 in plant tissue, particularly where proteins are ectopically expressed due to constant promoter activity. It is interesting to note that Arabidopsis PIP2;1, an AtPIP2;5 close homolog was shown to be a drought response-negative regulator which is a target of ubiquitination and degradation by a RING membrane-anchor 1 E3 ubiquitin ligase (Lee et al., 2009). Thus, it might be possible that this kind of Arabidopsis regulation system might function differently on the endogenous PIP2;5 (AtPIP2;5) and the heterologous PIP2;5 (HvPIP2;5) due to the different amino acid residues in the N-termini.
We found, in HvPIP2;5 overexpressing Arabidopsis, upregulation of P5CS2, high levels of proline, and increased activities of SOD, CAT, GR, and APX with reduced levels of ROS under drought and salt stress conditions. Particularly, up-regulation of the key proline biosynthetic P5CS genes coincided with increased levels of osmoprotectant proline under salt stress (Figure 6). Proline mainly functions in defense and turgor pressure maintenance against water-deprived conditions (Oregan et al., 1993;Kishor et al., 1995;Khedr et al., 2003). Thus, enhanced stress tolerance in HvPIP2;5 overexpressing plants seemed to result, at least in part, from increased expression of proline biosynthetic genes and elevated activities of ROS scavenging enzymes. Similar to our findings, the overexpression of wheat TaAQP7, one of the closest homologs of HvPIP2;5, in tobacco enhanced drought tolerance in correlation with decreased levels of MDA and H 2 O 2 and increased activities of SOD and CAT enzymes (Zhou et al., 2012). Improved osmotic stress tolerance by upregulation of stress-induced genes and an increase of ROS scavenging enzyme activity suggest that HvPIP2;5 overexpression might sensitize transgenic plants, making the overexpressors react faster to osmotic stress signals and eventually induce enhanced stress defense. Thus, one might speculate that HvPIP2;5 aquaporins in transgenic plants might activate osmotic and salt stress sensing or upstream steps in signaling pathways to induce better stress-tolerance mechanism than WT under stress conditions. Consistent with our speculation, it has been suggested that aquaporins may be part of an osmotic stress signaling cascade (Maurel et al., 2008); additionally, it has even been proposed that aquaporins may act as osmosensors (Hill et al., 2004). Further, study will be required to investigate this possibility.
In conclusion, we have shown that HvPIP2;5 can improve tolerance to salt and osmotic stresses when overexpressed in yeast and Arabidopsis. Our results contrast with a previous AtPIP2;5 overexpression study where osmotic stress sensitive phenotypes in AtPIP2;5 overexpressing plants were reported (Jang et al., 2007). These results suggest the diversity of PIP regulation and function in acquiring stress tolerance in plants. Further studies should be conducted to understand the functional differences among aquaporins for crop improvement under abiotic stress.

AUTHOR CONTRIBUTIONS
HA, BhL, and SKP designed the experiments; HA, JPA performed the experiments; GR, LS, BhL, and SKP advised the research; HA, BhL, and SKP discussed the results and wrote the paper.

ACKNOWLEDGMENTS
Authors thank the lab members for advice and help. The research was supported by Next-Generation BioGreen21 Program (PJ011006), Rural Development Administration, Republic of Korea.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 01566 Supplementary Table 1 | Sequences of primers used in the study.