A PIP-mediated osmotic stress signaling cascade plays a positive role in the salt tolerance of sugarcane

Plasma membrane intrinsic proteins (PIPs) are plant channel proteins involved in water deficit and salinity tolerance. PIPs play a major role in plant cell water balance and responses to salt stress. Although sugarcane is prone to high salt stress, there is no report on PIPs in sugarcane. In the present study, eight PIP family genes, termed ScPIP1–1, ScPIP1–2, ScPIP1–3, ScPIP1–4, ScPIP2–1, ScPIP2–2, ScPIP2–4 and ScPIP2–5, were obtained based on the sugarcane transcriptome database. Then, ScPIP2–1 in sugarcane was cloned and characterized. Confocal microscopy observation indicated that ScPIP2–1 was located in the plasma membrane and cytoplasm. A yeast two-hybridization experiment revealed that ScPIP2–1 does not have transcriptional activity. Real time quantitative PCR (RT-qPCR) analysis showed that ScPIP2–1 was mainly expressed in the leaf, root and bud, and its expression levels in both below- and aboveground tissues of ROC22 were up-regulated by abscisic acid (ABA), polyethylene glycol (PEG) 6000 and sodium chloride (NaCl) stresses. The chlorophyll content and ion leakage measurement suggested that ScPIP2–1 played a significant role in salt stress resistance in Nicotiana benthamiana through the transient expression test. Overexpression of ScPIP2–1 in Arabidopsis thaliana proved that this gene enhanced the salt tolerance of transgenic plants at the phenotypic (healthier state, more stable relative water content and longer root length), physiologic (more stable ion leakage, lower malondialdehyde content, higher proline content and superoxide dismutase activity) and molecular levels (higher expression levels of AtKIN2, AtP5CS1, AtP5CS2, AtDREB2, AtRD29A, AtNHX1, AtSOS1 and AtHKT1 genes and a lower expression level of the AtTRX5 gene). This study revealed that the ScPIP2–1-mediated osmotic stress signaling cascade played a positive role in plant response to salt stress.

former two accomplish water transport by cell-to-cell pathways or through plasmodesmata and membranes, and the last one transports water by water flow in the xylem [4]. However, the water transport channels could be blocked by casparian bands and suberin lamellae [3].
Aquaporins (AQPs) belong to the major intrinsic protein (MIP) family [5]. AQPs play a core role in water transport channels and are able to guide water transport across membranes between the protoplasm [6][7][8][9][10]. In plants, the molecular weights of AQPs are 23-30 kDa [5]. There are five water rings and six transmembrane α-helices in AQPs [11,12]. According to subcellular localization and sequence similarities, AQPs in higher plants can be divided into five subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast membrane intrinsic proteins (TIPs), nodulin26-like major intrinsic proteins (NIPs), small and basic intrinsic proteins (SIPs) and X intrinsic proteins (XIPs) [13,14]. Based on the N-terminal length of the proteins, PIPs are divided into PIP1s and PIP2s, which have different roles in plants when they respond to various stresses [5,6,15].
AQP genes have been cloned and characterized in several plant species, such as Arabidopsis thaliana [11], Oryza sativa [16], Zea mays [17] and Solanum lycopersicum [18]. As reported, AQPs play a major role during plant responses to water stress [6]. The expression of AQPs maintains the balance of water when plants suffer from various abiotic stresses such as salt, drought and cold [5]. PIPs are regarded as the major water transport proteins in roots and leaves of higher plants [5]. Previous research revealed that different PIP family members had various expression patterns and functions when plants were exposed to osmotic stress [19,20]. When Arabidopsis was treated with 250 mmol·L − 1 mannitol for 2 d, the expression levels of../%25E5%258A%259E %25E5%258,525 AC/360/Youdao/Dict/8.5.2.0/resultui/ html/index.html -/javascript:; AtPIP1-3, AtPIP1-4, AtPIP2-1 and AtPIP2-5 were 5 times as high as those in control leaves, but those of AtPIP1-5, AtPIP2-2, AtPIP2-3 and AtPIP2-6 decreased by 10% compared with the control [20]. Moreover, the expression levels of the PIP2 gene in Arabidopsis roots declined in response to salt stress [21]. The association between the health status of Arabidopsis and the higher expression levels of PIPs (AtPIP1-4 and AtPIP2 -5) showed that the expression and activity of AtPIP1-4 and AtPIP2-5 could regulate water balance in plants after osmotic stress [22]. During the process of drought stress, the expressions of NtPIP1-1 and NtPIP2-1 were down-regulated in Nicotiana benthamiana [23]. Moderate expression levels of OsPIP1-1 can increase the seed yield, salt resistance, root hydraulic conductivity and seed germination rate of rice [24]. Furthermore, the expressions of OsPIP2 genes were positively correlated with the level of salt stress, and this phenomenon was similar to drought stress [25,26]. In Rosa rugosa, RhPTM (PM-tethered MYB) could interact with RhPIP2-1 to maintain the water balance in cells [27]. These reports confirmed that PIPs could aid the response to salt stress in plants such as Z. mays [17], O. sativa [16] and A. thaliana [11]. However, there is no report on the identification and characterization of PIP genes in sugarcane (Saccharum spp. hybrid, var. ROC22) until now.
Sugarcane, the most important sugar crop in the world, is often planted in tropical and subtropical regions. Salt stress hinders sugarcane growth and development and also reduces its production. In the present study, first, according to the transcriptome database, eight sugarcane PIP genes (named ScPIPs) were obtained and analyzed by the bioinformatics method. Second, a full-length cDNA sequence of ScPIP2-1, homologous to the rice salt-resistant gene OsPIP2-1 (AF062393.1) [26], was cloned from the main sugarcane variety ROC22 (Saccharum spp. hybrid) in mainland China. The amino acid sequence, subcellular location and transcriptional activity of the ScPIP2-1 protein were analyzed. Third, in order to understand the expression patterns of ScPIP2-1 in ROC22, tissue-specific expression and the expression of ScPIP2-1 under polyethylene glycol (PEG) 6000, sodium chloride (NaCl) and abscisic acid (ABA) stresses were detected. Finally, the biological functions of ScPIP2-1 under salt stress were also investigated by transient overexpression of ScPIP2-1 in N. benthamiana leaves combined with stable overexpression in A. thaliana. The above results suggested that ScPIP2-1 might enhance salt tolerance in plants mainly by maintaining cell homeostasis and by eliminating reactive oxygen species (ROS). In other words, the salt tolerance of transgenic Arabidopsis could be enhanced by the ScPIP2-1-mediated osmotic stress signaling cascade.

Identification and classification of sugarcane PIP family genes
Based on our previous sugarcane transcriptome, eight ScPIP family genes, namely, ScPIP1-1 (MZ362000), and ScPIP2-5 (MZ362007), were mined (Table S1). Bioinformatics analysis showed that these eight ScPIP proteins had a relative molecular mass of 30-31 kDa, isoelectric points (pIs) of 6.64-9.00, and all belonged to stable hydrophobins (Table S2). A phylogenetic tree showed that these ScPIP proteins and AQP proteins from other higher plants were divided into four subfamilies: PIPs, TIPs, NIPs and SIPs. All eight ScPIP proteins belonged to the PIP subfamily ( Fig. 1).

Cloning and sequence analysis of the ScPIP2-1 gene
A full-length cDNA sequence of ScPIP2-1 was cloned from the 24-h 250 mmol·L − 1 NaCl-treated ROC22 leaves by reverse transcription-polymerase chain reaction (RT-PCR). The cDNA length of ScPIP2-1 was 1145 bp, including an open reading frame (ORF) of 873 bp that encoded 290 amino acids (Fig. 2). Moreover, the ScPIP2-1 protein had the MIP conservative structure of the NPA-motif (Asn-Pro-Ala) and the PIP feature structures (P/KDYXE/DPPP/RX3-4E/DXXELXXWSFY/WR, and GGGANXXXXGY) (Jang et al., 2004). Sequence alignment indicated that ScPIP2-1 was highly homologous with PIPs in Arabidopsis, rice, tobacco, maize and sorghum. However, the N-terminal amino acid sequences of these homologous proteins are quite different (Fig. 3).

Subcellular localization analysis of the ScPIP2-1 protein
Subcellular localization analysis (Fig. 4) showed that 35S::ScPIP2-1::eGFP was expressed in the plasma membrane and cytoplasm, and 35S::eGFP was expressed in the plasma membrane, cell nucleus and cytoplasm.

Transcription activation activity of the ScPIP2-1 protein
The DUAL membrane yeast two-hybrid system was used to detect the transcriptional activity of ScPIP2-1. Compared with the positive and negative control, pBT3-N-ScPIP2-1 did not possess transcriptional activity and showed no toxicity to the yeast strain NMY51 (Fig. 5).

Tissue-specific expression of the ScPIP2-1 in different sugarcane tissues
Real time quantitative PCR (RT-qPCR) was performed to determine the expression patterns of the ScPIP2-1 gene in different sugarcane tissues, including the stem pith, epidermis, root, leaf and bud. As shown in Fig. 6, the ScPIP2-1 gene was constitutively expressed in all these sugarcane tissues but mainly in the bud, leaf and root, which was 4.71-, 2.41-and 1.55-fold higher, respectively, than that in the stem pith.

Expression of the ScPIP2-1 gene in response to various abiotic stresses
In the case of ABA, NaCl and PEG stresses, except for the downregulation in aboveground tissues at 0.5 h under ABA treatment, the expression levels of the ScPIP2-1 gene in both below-and aboveground tissues of ROC22 were up-regulated (Fig. 7). During ABA stress, the ScPIP2-1 gene exhibited a sensitive response. Its expression levels peaked in belowground tissues at 6 h and in aboveground tissues at 3 h, which were 16.36and 2.99-fold higher, respectively, than those of the control. In response to NaCl, the expression levels of the ScPIP2-1 gene were increased from 0.5 h to 3 h in belowground tissues and from 0 h to 6 h in aboveground tissues but remained unchanged at the other time points. In response to PEG 6000, the expression levels of the ScPIP2-1 gene increased at 6 h in both below-and aboveground tissues of ROC22, which were 1.59-and 2.81-fold higher, respectively, than those of the control. However, the expression levels of ScPIP2-1 remained stable at the other time points of PEG 6000 treatment. These results suggested that ScPIP2-1 had a positive response to salt and PEG 6000 stresses, most probably mediated by the ABA signaling pathway.

Transient overexpression of ScPIP2-1 gene regulated the responses of N. benthamiana leaves to salt stress
After transient overexpression of ScPIP2-1 in N. benthamiana leaves for 2 days, the overexpression tobacco plants were treated with 250 mmol·L − 1 NaCl for 24 h. Then, the salt tolerance of tobacco leaves was determined at the physiologic, phenotypic and molecular levels ( Fig. 8). As shown in Fig. 8a, a darker DAB staining color was observed in the control (pCAMBIA 1301) than in ScPIP2-1 leaves. Moreover, the phenomenon of stronger salt tolerance was demonstrated by a higher SPAD index and more stable ion leakage in ScPIP2-1 overexpression plants after 24-h salt treatment (Figs. 8b, c). According to the results of Fig. 8d, the expression levels of salt-associated marker genes (NtESI3, NtDREB2 and NtP5CS) in ScPIP2-1 leaves were increased, which were 4.32-, 1.95-and 8.30-fold higher, respectively, than those of the control. These results laid the foundation for genetic transformation experiments and provided evidence for the ScPIP2-1 gene to respond to salt stress.

Overexpression of ScPIP2-1 gene improved the salt stress tolerance in transgenic Arabidopsis
Here, a functional analysis of Arabidopsis overexpression ScPIP2-1 for salt tolerance was conducted, and the indexes were measured at phenotypic, physiological and molecular levels. Three lines of homozygous transgenic Arabidopsis (OE1, OE2 and OE3) were selected in 1/2 MS medium containing hygromycin (HYG). At the phenotypic level, the transgenic Arabidopsis seedling had longer root length than wild-type Arabidopsis (WT) under 0-250 mmol·L − 1 NaCl (Fig. 9). In addition, compared to the 5-week-old transgenic Arabidopsis, the leaves of WT turned yellow and its growth was inhibited under 250 mmol·L − 1 NaCl stress (Fig. 10a). These results indicated that transgenic Arabidopsis might have better water absorption ability. As shown in Fig. 10c, the transgenic plantlets had a significantly higher relative water content (RWC) than WT plantlets after 250 mmol·L − 1 NaCl treatment.
As Figs. 10b-e and Fig. 11 show, the transgenic Arabidopsis exhibited a stronger salt tolerance at the physiological and molecular levels. It was noted that the transgenic Arabidopsis had a higher proline (Pro) content, and the expression levels of Pro synthesis-related genes AtP5CS1 and AtP5CS2 were up-regulated by salt stress (Fig. 10e and Figs. 11c, d). According to the results of Fig. 10b and Figs. 11a, e and f, the transgenic Arabidopsis had a more stable intracellular environment than WT, which was exhibited by the insignificantly increased ion leakage (3-7 days) and the higher expression levels of osmotic   Fig. 11b).

Discussion
AQPs play important roles as water channels in regulating water balance [6][7][8][9][10]. At present, many AQPs from various plant species have been used in transgenic  Test of the ScPIP2-1 trans-activation assay. The media were added with X-Gal (5-bromo-4-chloro-3-indoxyl-D-galactopyranoside). 10 − 1 , 10 − 2 , 10 − 3 and 10 − 4 represent the sample concentrations diluted ten-fold, hundred-fold, thousand-fold and ten thousand-fold, respectively. SD/− Leu, the synthetic dropout medium without leucine; SD/−Leu/−Trp, the synthetic dropout medium without leucine and tryptophan; SD/−Ade/− His/−Leu/−Trp, the synthetic dropout medium without adenine, histidine, leucine and tryptophan  research to improve salt stress tolerance in plants [28][29][30][31]. In this study, eight AQPs, all of which belong to the PIP subfamily, were mined from the sugarcane transcriptome database (Fig. 1). Johanson et al. suggested that the naming of AQPs should refer to the protein subcellular localization and sequence homology [11]. In subsequent research, the AQP proteins located in the plasma membrane and cytoplasm were renamed as PIPs in higher plants [5,11]. Our research revealed that ScPIP2-1 was localized in the plasma membrane and cytoplasm (Fig. 4), which is in accordance with previous studies [27,29]. Protein-protein interactions play an important role in plant development and growth [32]. Based on previous research in roses, RhPTM could interact with RhPIP2-1, indicating its important role in maintaining water balance [27]. In the present study, pBT3-N-ScPIP2-1 did not possess transcriptional activity and showed no toxicity to the yeast strain NMY51 (Fig. 5), suggesting that it could be used for yeast two-hybrid screening in future research.
Temporal and spatial expression of AQPs often represents their diverse functions [9]. SbAQPs were mainly expressed in roots and played major roles in sorghum under waterlogging [33]. AtPIP1-4 and AtPIP2-5 were mainly expressed in the leaf and root when Arabidopsis was exposed to salt and drought stresses [22]. Likewise, the tissue-specific expression of ScPIP2-1 (Fig. 6) proved that it may play an important role in benthamiana leaves after 24-h salt stress. The NtEF1-α gene was used for data normalization. All data points are presented as means ± standard error (n = 3). Different lowercase letters indicate a significant difference, as determined with Duncan's test (P < 0.05) maintaining water transport in leaf and root tissues. Accordingly, we speculated that ScPIP2-1 also participated in the sugarcane cell development due to the fact that ScPIP2-1 was mainly expressed in bud tissues of sugarcane (Fig. 6), which is similar to the phenomenon that RhPIP2-1 participated in rose petal cell swelling with higher expression in the meristem [34]. ABA is a major phytohormone that can enhance the tolerance of osmotic stress in plants [35]. In this study, the expression levels of the ScPIP2-1 gene in both belowand aboveground tissues of ROC22 were increased by ABA treatment (Fig. 7). The transcripts of the ScPIP2-1 gene were also up-regulated under NaCl and PEG 6000 stresses (Fig. 7). The above phenomena were the same as in many previous studies, for example, the expression levels of ClPIP were up-regulated under ABA treatment and had a positive response to osmotic stresses in Citrullus lanatus [36], and PvPIP2-9 withstood salt and drought stresses through ABA signal regulation in Panicum virgatum [37]. Water deficit can cause difficulty in root water absorption, mainly due to a lower osmotic potential. The research demonstrates that ABA can regulate stomatal closing to reduce the rate of water loss [38]. Taking all these findings into consideration, we believe that ScPIP2-1 may have a positive response to PEG 6000 and NaCl stresses through the ABA signal in sugarcane.
What should be stressed here is that we found the overexpression of ScPIP2-1 in Arabidopsis improved its tolerance to salt stress, which was correlated with the higher expression levels of salt-associated marker genes (Fig. 11), lower levels of stress-induced ROS  (Fig. 10c), higher activity of ROS scavenging enzyme (Fig. 10d) and higher expression levels of Pro biosynthetic genes and a higher Pro content ( Fig. 10e and Figs. 11c, d). In particular, under salt stress, ScPIP2-1 overexpression Arabidopsis displayed healthier physiological states (Fig. 10a), longer length of roots (Fig. 9) and more stable cell ion leakage (Fig. 10b) compared with WT, which suggested that the transgenic Arabidopsis had more stable cell pressure. Although the ScPIP2-1 overexpression improved salt tolerance in Arabidopsis in our study, AtPIP2-1 overexpression resulted in reduced salt stress tolerance in Arabidopsis and tobacco [39]. These seemingly conflicting results are not uncommon. In fact, many AQP overexpression studies have produced contrasting results, for example, AQP overexpression plants showed either positive or negative effects due to stress tolerance [9,40]. There are differences in gene regulation, even among highly homologous AQPs, indicating their divergent functions [39]. Another explanation for the contrasting results might lie in the differences in protein sequences between ScPIP2-1 and other plant PIP proteins. Amino acid sequence differences were mainly found in the N-terminus, which was expected to be exposed on the cytosol side [41]. From all the above, it was tempting to infer that contrasting stress phenotypes may be due to differences in the N-termini of the PIP protein (Fig. 3), which contains important motifs such as those for activity regulation, protein stability, protein interaction or even subcellular localization.
In the salt tolerance analysis of transgenic Arabidopsis, we found up-regulation of AtP5CS1 and AtP5CS2 (Figs. 11c, d), higher Pro content (Fig. 10e) and increased activity of SOD ( Fig. 10d) with reduced levels of MDA (Fig. 10c) in ScPIP2-1 overexpression Arabidopsis under salt stress. Pro mainly functions in defense and turgor pressure maintenance against water-deprived conditions [42]. The expression levels of three ion exchange-correlated genes (AtNHX1, AtSOS1 and AtHKT1) were up-regulated, which may improve the salt tolerance of ScPIP2-1 overexpression plants, which is in accordance with a previous report that AtNHX1, AtSOS1 and AtHKT1 played important roles in salt stress resistance [43]. Hence, the enhanced salt stress tolerance in ScPIP2-1 overexpression Arabidopsis seemed to result in, at least in part, the increased expression of Pro biosynthetic genes and elevated activities of ROS scavenging enzymes, as well as the downregulation of AtTRX5 (Fig. 11b). Similar to our findings, the enhanced stress tolerance in MsPIP2-2 overexpression transgenic Arabidopsis was correlated with decreased levels of MDA and increased Pro contents and activity of the SOD enzyme [29]. In the present study, salt tolerance improvement by up-regulation of salt stress-regulated genes and an increase in ROS scavenging enzyme activity suggested that ScPIP2-1 overexpression might sensitize transgenic Arabidopsis, causing the overexpression Arabidopsis react faster to salt stress signals and eventually enhance stress defense. According to the results of Figs. 11e and f, which indicated the up-regulation of AtDREB2 and AtRD29A genes under salt stress, we speculate that ScPIP2-1 in transgenic Arabidopsis could activate salt stress sensing or the upstream steps in signaling pathways to induce a better stress-tolerance mechanism than in WT. Consistent with our speculation, it has been suggested that AQPs may be part of an osmotic stress signaling cascade [9]. Additionally, it has even been proposed that AQPs may act as osmosensors [44]. Further study will be required to investigate this possibility.

Conclusions
In the present study, eight ScPIP genes that belong to the PIP subfamily of AQPs were mined from our previous sugarcane transcriptome database. A full-length cDNA of ScPIP2-1 was then cloned from ROC22. ScPIP2-1 was localized in the plasma membrane and cytoplasm. The ScPIP2-1 gene was constitutively expressed in sugarcane tissues, and its transcript levels were increased by ABA, NaCl and PEG 6000 treatments. Yeast two-hybrid screening demonstrated that pBT3-N-ScPIP2-1 did not possess transcriptional activity and had no toxicity to the yeast strain NMY51. The transient overexpression of ScPIP2-1 in N. benthamiana leaves could enhance the salt tolerance. Moreover, overexpression ScPIP2-1 Fig. 11 Expression of salt stress-related genes in ScPIP2-1-transgenic Arabidopsis based on RT-qPCR. AtKIN2, AtDREB2 and AtRD29A represent the osmotic stress-defense genes. AtTRX5 represents the reactive oxygen species (ROS) elimination gene. AtP5CS1 and AtP5CS2 represent the proline (Pro) synthesis-related genes. AtNHX1, AtSOS1 and AtHKT1 represent the ion exchange-correlated genes. Arabidopsis was grown in soil and was treated with 250 mmol·L − 1 sodium chloride (NaCl). Bars with different superscripts differ significantly (P < 0.05), and error bars represent the standard error of each treatment group (n = 3). WT, wild-type Arabidopsis; OE1, OE2 and OE3, three different lines of transgenic Arabidopsis in Arabidopsis could enhance the salt tolerance of transgenic plants at the phenotypic, physiological and molecular levels. These results would be helpful in mining and functional identification of ScPIP family genes in sugarcane in future.

Plant materials and treatments
One panel of sugarcane material was used in this study. The main sugarcane variety ROC22 (Saccharum spp. hybrid) in mainland China was provided by the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs (Fuzhou, China). For tissue-specific expression analysis, 10-month-old ROC22 plants were selected. The samples included the root, bud, + 1 leaf (the youngest fully expanded leaf with a visible dewlap), stem pith and epidermis [45]. For osmotic stress and exogenous hormone treatments, 4-month-old ROC22 plants were cultivated hydroponically for 7 days. The cultivation conditions were 28 °C with 16-h light and 8-h darkness. Then, 25 PEG 6000, 250 mmol·L − 1 NaCl and 0.1 mmol·L − 1 ABA were used as simulated environmental stress conditions [46][47][48]. The leaf and root of each treatment were collected at 0, 0.5, 3, 6 and 24 h. Three biological replicates of each sample were prepared, and all the above-mentioned samples were fixed in liquid nitrogen and stored at − 80 °C until total RNA extraction.

Total RNA extraction and the first strand of cDNA synthesis
Total RNA was extracted using TRIeasy ™ Total RNA Extraction Reagent (YEASEN, Shanghai, China), and 1.0 μg total RNA was used to synthesize first-strand cDNA using the Hifair ® 1st Strand cDNA Synthesis Kit (gDNA digester plus) (YEASEN, Shanghai, China). The cDNA was detected by 1% gel electrophoresis [49]. The cDNA of 24-h 250 mmol·L − 1 NaCl-treated ROC22 leaves was used for gene cloning.

Identification and bioinformatics analysis of ScPIP family genes
Eight ScPIP genes were minded from our previous sugarcane transcriptome database [50,51]. The sequences of these genes were translated and analyzed using the Open Reading Frame Finder (https:// www. ncbi. nlm. nih. gov/ orffi nder/) and DNAMAN V6.0.3.99 software. The signal peptides and pIs were predicted using ExPASy (http:// us. expas y. org/ tools). A phylogenetic tree was constructed with the maximum likelihood (ML) method (1000 bootstrap replicates) using the MEGA X 10.0.5 program [16,52].
The full-length cDNA sequence of ScPIP2-1, which was minded above, was cloned by RT-PCR from 24-h 250 mmol·L − 1 NaCl-treated leaf tissue of ROC22. The amplification procedure was set as 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s and 72 °C for 1 min, and then 72 °C for 10 min in an elongation step. The PCR products were purified by a gelpurification kit (Magen, Guangzhou, China), cloned into the pMD 19-T vector (Takara, Dalian, China) and sent for sequencing.

Self-activation of the ScPIP2-1 in yeast
To analyze the transcriptional activity of ScPIP2-1, the DUAL membrane yeast two-hybrid system (including three reporter genes, HIS3, ADE2 and lacZ) was used following the manufacturer's directions for the DUAL yeast two-hybrid system and according to the protocol established in our group [48]. The ScPIP2-1 gene was PCR-amplified from pMD19-T-ScPIP2-1 using primers ScPIP2-1 pBT3-F and ScPIP2-1 pBT3-R (Table S3). Then, Sfi I endonuclease was used to construct the bait vector pBT3-N-ScPIP2-1. NMY51 strains carrying the treatment (positive plasmid pBT3-N-ScPIP2-1), the negative control (empty vector pBT3-N and empty vector pRR3-N) and the positive control (pNubG-Fe65 and pTSU2-App) vectors were cultured in synthetic dropout medium without leucine and tryptophan (SD/−Leu/−Trp) for 2 days at 30 °C at 150 rpm [48].

Expression of the ScPIP2-1 gene in different sugarcane tissues and in response to various abiotic stresses
RT-qPCR analysis was conducted to reveal the expression patterns of ScPIP2-1 in sugarcane tissues (root, bud, leaf, stem pith and stem epidermis) and in response to various abiotic stresses (NaCl, PEG 6000 and ABA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank Accession Number: CA254672) was used as a reference gene (Table S3) [56]. The specific RT-qPCR primers of ScPIP2-1 (Table S3)  The standard curve plot and amplification efficiency of ScPIP2-1 and GAPDH genes in RT-qPCR analysis were showed in Fig. S1 and Table S4. The 2 −∆∆CT method [54], SPSS 9 software and Origin 8 software were used to calculate the relative expression of the target gene, to analyze the significance level of the experimental data and to structure the histogram.

Transient expression of ScPIP2-1 in N. benthamiana
To investigate the function of ScPIP2-1 under high salt treatment in N. benthamiana, the overexpression vector pCAMBIA 1301-ScPIP2-1 was constructed using T4 ligase (YEASEN, Shanghai, China). The recombinant vector of pCAMBIA 1301-ScPIP2-1 and the empty vector of pCAMBIA 1301 were separately introduced into Agrobacterium GV3101. Agrobacterium-mediated transient expression in the N. benthamiana leaf was performed [52][53][54]. The six-leaf stage N. benthamiana plants were irrigated with 50 mL of 250 mmol·L − 1 NaCl every day. All materials were cultivated at 24 °C with an illumination intensity of 15,000 μmol·m − 2 ·s − 1 , a photoperiod of 16-h light and 8-h darkness and a relative humidity of 65%. The histocytological analysis was processed by 3, 3′-diaminobenzidine (DAB) staining at 0 h and 24 h on leaves under NaCl stress. At the same time, the chlorophylls and ion leakage of the leaves were determined using soil and plant analyzer development measurements (SPAD, KONICA MINOLTA, Japan) and the soaking method [57], respectively. The expression levels of salt-associated genes, i.e., NtESI3 (salt stress-induced hydrophobic peptide gene) [58], NtDREB2 (DREB transcription factor) [59] and NtP5CS (correlation Pro production gene) [60] (Table S3), were detected by RT-qPCR. The NtEF1-α gene (Table S3) was used as an internal reference for normalization. The standard curve plot and amplification efficiency of RT-qPCR primers were showed in Fig. S1 and Table S4. The 2 −∆∆CT method [54], SPSS 9 software and Origin 8 software were used to calculate the relative expression of the target gene, to analyze the significance level of the experimental data and to structure the histogram.

Stable expression of ScPIP2-1 in Arabidopsis and the salt tolerance analysis of transgenic plants
The ScPIP2-1 gene was genetically transformed into Arabidopsis by Agrobacterium-mediated transformation. The overexpression vector was pCAMBIA 1301, which contained a marked gene for HYG resistance. Seeds of transgenic Arabidopsis lines of T 1 , T 2 and T 3 generations were germinated and selected on 1/2 MS medium with 25 mg·L − 1 HYG. As a control, WT seeds were also placed on 1/2 MS medium with HYG. Two weeks later, the seedlings were transferred to soil pots and cultured at 22 °C with an illumination intensity of 15,000 μmol·m − 2 ·s − 1 , a photoperiod of 16-h light and 8-h darkness and a relative humidity of 65%. The seeds of T 1 , T 2 and T 3 generations were generated and collected.
To determine the root lengths, the seeds of WT and three lines of T 3 transgenic Arabidopsis (OE1, OE2 and OE3) were separately placed on 1/2 MS medium containing 0, 50, 150, 200 or 250 mmol·L − 1 NaCl. A Nikon camera was used to take the photos 7 days later, and Image J 1.8.0 was used to measure the root lengths [47,61].
In order to analyze the salt tolerance of ScPIP2-1 overexpression Arabidopsis at physiological and molecular levels, the 5-week plants of WT and three lines of T 3 transgenic Arabidopsis (OE1, OE2 and OE3) were cultivated in peat medium and treated with 50 mL of 250 mmol·L − 1 NaCl every day. The Nikon camera was used to take photos at 0, 3 and 7 days post NaCl treatment. The leaf samples for the analysis of physiological indexes were collected at 0 d and 7 d post treatment, and other leaf samples for the analysis of molecular indexes were collected at 0 d, 3 d and 7 d post treatment. Three biological replicates of each sample were prepared. Three plantlets, randomly selected, were defined as one biological replicate, and three biological replicates of each sample were prepared to measure the fresh weight (FW) and dry weight (DW). The RWC was calculated by RWC = (FW-DW) × FW − 1 [62]. The contents of MDA and Pro as well as the enzymatic activity of SOD were determined by spectrophotometry using MDA, Pro and SOD assay kits (Solarbio, Beijing, China) [47,61]. The ion leakage was determined by the soaking method [57]. The expressions of the osmotic stress-defense genes (AtKIN2,