Populus euphratica Phospholipase Dδ Increases Salt Tolerance by Regulating K+/Na+ and ROS Homeostasis in Arabidopsis

Phospholipase Dα (PLDα), which produces signaling molecules phosphatidic acid (PA), has been shown to play a critical role in plants adapting to salt environments. However, it is unclear whether phospholipase Dδ (PLDδ) can mediate the salt response in higher plants. PePLDδ was isolated from salt-resistant Populus euphratica and transferred to Arabidopsis thaliana to testify the salt tolerance of transgenic plants. The NaCl treatment (130 mM) reduced the root growth and whole-plant fresh weight of wild-type (WT) A. thaliana, vector controls (VC) and PePLDδ-overexpressed lines, although a less pronounced effect was observed in transgenic plants. Under salt treatment, PePLDδ-transgenic Arabidopsis exhibited lower electrolyte leakage, malondialdehyde content and H2O2 levels than WT and VC, resulting from the activated antioxidant enzymes and upregulated transcripts of genes encoding superoxide dismutase, ascorbic acid peroxidase and peroxidase. In addition, PePLDδ-overexpressed plants increased the transcription of genes encoding the plasma membrane Na+/H+ antiporter (AtSOS1) and H+-ATPase (AtAHA2), which enabled transgenic plants to proceed with Na+ extrusion and reduce K+ loss under salinity. The capacity to regulate reactive oxygen species (ROS) and K+/Na+ homeostasis was associated with the abundance of specific PA species in plants overexpressing PePLDδ. PePLDδ-transgenic plants retained a typically higher abundance of PA species, 34:2 (16:0–18:2), 34:3 (16:0–18:3), 36:4 (18:2–18:2), 36:5 (18:2–18:3) and 36:6 (18:3–18:3), under control and saline conditions. It is noteworthy that PA species 34:2 (16:0–18:2), 34:3 (16:0–18:3), 36:4 (18:2–18:2) and 36:5 (18:2–18:3) markedly increased in response to NaCl in transgenic plants. In conclusion, we suppose that PePLDδ-derived PA enhanced the salinity tolerance by regulating ROS and K+/Na+ homeostasis in Arabidopsis.


PePLDδ Gene Cloning and Sequence Analyses
In this study, a phospholipase Dδ gene, PLDδ, was cloned from P. euphratica leaves. The amino acid sequences were compared with PLDδ proteins from different plant species. The PePLDδ sequence displayed a high similarity to P. tricocarpa PLDδ ( Figure 1A). The comparative phylogenetic analysis revealed that PePLDδ displayed a homology to StPLDδ and PtPLDδ, but was distinct from AtPLDδ ( Figure 1B). , which contributed to the maintenance of ionic and ROS homeostasis in NaCl-stressed transgenic plants.

PePLDδ Gene Cloning and Sequence Analyses
In this study, a phospholipase Dδ gene, PLDδ, was cloned from P. euphratica leaves.
The amino acid sequences were compared with PLDδ proteins from different plant species. The PePLDδ sequence displayed a high similarity to P. tricocarpa PLDδ ( Figure 1A).
The comparative phylogenetic analysis revealed that PePLDδ displayed a homology to StPLDδ and PtPLDδ, but was distinct from AtPLDδ ( Figure 1B).

Transformation of PePLDδ Gene
Seven transgenic lines, OE1, OE2, OE3, OE4, OE5, OE6 and OE7, were obtained by transferring the PePLDδ gene into Arabidopsis thaliana. The transgenic lines were identified with semi-quantitative reverse transcription PCR and real-time quantitative PCR ( Figure 2). The wild-type (WT) Arabidopsis, empty vector control (VC) and two transgenic lines (OE6 and OE7) that showed the highest abundance of PePLDδ were used for salt treatments.

Transformation of PePLDδ Gene
Seven transgenic lines, OE1, OE2, OE3, OE4, OE5, OE6 and OE7, were obtain transferring the PePLDδ gene into Arabidopsis thaliana. The transgenic lines were fied with semi-quantitative reverse transcription PCR and real-time quantitativ ( Figure 2). The wild-type (WT) Arabidopsis, empty vector control (VC) and two genic lines (OE6 and OE7) that showed the highest abundance of PePLDδ were us salt treatments. Semi-quantitative reverse transcription PCR assay. Ten-day-ol lings of wild-type (WT) Arabidopsis, empty vector control (VC) and the transgenic lines (OE were sampled for total RNA extraction, semi-quantitative PCR and real-time quantitative PC yses. Arabidopsis β-actin 2 (AtACTIN2) was used as an internal reference gene. The prim signed to target PePLDδ and internal control gene, AtACTIN2, are shown in Supplementar S2. In (A), data are presented as the mean of three independent experiments, and error bars sent SE. Columns with different letters, a, b, c and d show significant differences, with p < 0. (A) RT-qRCR assay. (B) Semi-quantitative reverse transcription PCR assay. Ten-day-old seedlings of wild-type (WT) Arabidopsis, empty vector control (VC) and the transgenic lines (OE1-OE7) were sampled for total RNA extraction, semi-quantitative PCR and real-time quantitative PCR analyses. Arabidopsis β-actin 2 (AtACTIN2) was used as an internal reference gene. The primers designed to target PePLDδ and internal control gene, AtACTIN2, are shown in Supplementary Table S2. In (A), data are presented as the mean of three independent experiments, and error bars represent SE. Columns with different letters, a, b, c and d show significant differences, with p < 0.05.

Salt Tolerance Tests of PePLDδ-Transgenic Arabidopsis
The change in growth can sensitively reflect the plant's ability to adapt to a salt environment [33]. The root length and whole plant weight of all tested genotypes were compared. The root length of WT, VC and PePLDδ-transgenic lines, OE6 and OE7, signifi-cantly decreased upon seven days of 130 mM NaCl treatment ( Figure 3A,B). The restriction of NaCl was more pronounced in WT and VC than in transgenic plants ( Figure 3A,B). Moreover, the whole-plant fresh weight of salt-stressed WT and VC showed a 10-31% higher reduction than transgenic lines ( Figure 3C). Our results showed that there was no significant difference between the tested lines, WT, VC and transgenic Arabidopsis, in root growth and fresh weight under no-salt conditions (Figure 3).

Salt Tolerance Tests of PePLDδ-Transgenic Arabidopsis
The change in growth can sensitively reflect the plant's ability to adapt to a salt environment [33]. The root length and whole plant weight of all tested genotypes were compared. The root length of WT, VC and PePLDδ-transgenic lines, OE6 and OE7, significantly decreased upon seven days of 130 mM NaCl treatment ( Figure 3A,B). The restriction of NaCl was more pronounced in WT and VC than in transgenic plants ( Figure  3A,B). Moreover, the whole-plant fresh weight of salt-stressed WT and VC showed a 10-31% higher reduction than transgenic lines ( Figure 3C). Our results showed that there was no significant difference between the tested lines, WT, VC and transgenic Arabidopsis, in root growth and fresh weight under no-salt conditions ( Figure 3).

Salt-Stress-Induced Electrolyte Leakage and Membrane Peroxidation
To test the salt damage effect on cell membrane integrity, the relative electrolyte leakage (EL) of control and salinized plants was examined [33]. Compared with WT and VC, PePLDδ-overexpressed Arabidopsis exhibited a 12-27% lower relative EL after 12 h of salt treatment ( Figure 4A). Therefore, PePLDδ could alleviate the damage of high NaCl on the cell membrane and consequently improved the salt tolerance of transgenic Arabidopsis.
The electrolyte leakage usually results from membrane peroxidation under salt stress [33]. Here, the content of malondialdehyde (MDA), which is the end-product of membrane lipid peroxidation, was examined. The results showed that the MDA content of WT and VC significantly increased upon the salt treatment, whereas MDA remained unchanged in PePLDδ-overexpressed lines ( Figure 4B). This indicates that PePLDδ reduced the oxidative damage caused by NaCl in transgenic A. thaliana.

Salt-Stress-Induced Electrolyte Leakage and Membrane Peroxidation
To test the salt damage effect on cell membrane integrity, the relative electrolyte leakage (EL) of control and salinized plants was examined [33]. Compared with WT and VC, PePLDδ-overexpressed Arabidopsis exhibited a 12-27% lower relative EL after 12 h of salt treatment ( Figure 4A). Therefore, PePLDδ could alleviate the damage of high NaCl on the cell membrane and consequently improved the salt tolerance of transgenic Arabidopsis.
The electrolyte leakage usually results from membrane peroxidation under salt stress [33]. Here, the content of malondialdehyde (MDA), which is the end-product of membrane lipid peroxidation, was examined. The results showed that the MDA content of WT and VC significantly increased upon the salt treatment, whereas MDA remained unchanged in PePLDδ-overexpressed lines ( Figure 4B). This indicates that PePLDδ reduced the oxidative damage caused by NaCl in transgenic A. thaliana.

H2O2 Content in Root Cells under Salt Stress
Reactive oxygen species (ROS) cause membrane peroxidation and electrolyte leakage under salt stress [31][32][33]. We used the fluorescent probe, H2DCFDA, to detect salt-elicited H2O2, as the fluorescence intensity of intracellular DCF is positively correlated with the level of intracellular H2O2 [34]. The intensity of H2DCFDA showed that H2O2 levels were almost undetectable in the no-salt controls of all tested genotypes. After high salt treatment, the H2O2 level in WT and VC markedly increased in root cells, which was significantly higher than transgenic lines, OE6 and OE7 ( Figure 5).

H 2 O 2 Content in Root Cells under Salt Stress
Reactive oxygen species (ROS) cause membrane peroxidation and electrolyte leakage under salt stress [31][32][33]. We used the fluorescent probe, H 2 DCFDA, to detect salt-elicited H 2 O 2 , as the fluorescence intensity of intracellular DCF is positively correlated with the level of intracellular H 2 O 2 [34]. The intensity of H 2 DCFDA showed that H 2 O 2 levels were almost undetectable in the no-salt controls of all tested genotypes. After high salt treatment, the H 2 O 2 level in WT and VC markedly increased in root cells, which was significantly higher than transgenic lines, OE6 and OE7 ( Figure 5).

H2O2 Content in Root Cells under Salt Stress
Reactive oxygen species (ROS) cause membrane peroxidation and electrolyte leakage under salt stress [31][32][33]. We used the fluorescent probe, H2DCFDA, to detect salt-elicited H2O2, as the fluorescence intensity of intracellular DCF is positively correlated with the level of intracellular H2O2 [34]. The intensity of H2DCFDA showed that H2O2 levels were almost undetectable in the no-salt controls of all tested genotypes. After high salt treatment, the H2O2 level in WT and VC markedly increased in root cells, which was significantly higher than transgenic lines, OE6 and OE7 ( Figure 5).  and transgenic lines overexpressing PePLDδ under NaCl stress. Seeds from WT, VC and PePLDδoverexpressed lines (OE6 and OE7) were allowed to germinate on 1/2 MS medium and grown for seven days. The seedlings were transferred to liquid medium containing 0 or 130 mM NaCl for 12 h. Then, Arabidopsis roots were incubated with 10 µM H 2 DCFDA for 15 min, followed by washing 4-5 times. Green fluorescence within cells was detected with a laser confocal microscope, and the relative H 2 O 2 concentrations were calculated according to the fluorescence intensity. Data are presented as the mean of 6-9 individual plants, and error bars represent SE. Values with different letters, a, b, and c show significant differences, with p < 0.05. Scale bar = 250 µm.

Activity and Transcription of Antioxidant Enzymes under Salt Stress
The high levels of H 2 O 2 in WT and VC mainly result from the decreased ability to scavenge ROS in salt-stressed plants [31][32][33]. To confirm whether the PePLDδ-transgenic lines had the capacity to maintain ROS homeostasis, the activity and transcription of antioxidant enzymes, including ascorbic acid peroxidase (APX), peroxidase (POD) and superoxide dismutase (SOD), were testified in this study. After NaCl treatment (130 mM, 7 d), the total activities of tested antioxidant enzymes, SOD, POD and APX, increased by approximately 50% in OE6 and OE7, whereas the salt stimulation was less pronounced in WT and VC ( Figure 6A-C). In accordance, the expression of AtSOD, AtPOD and AtAPX genes showed a higher increase in PePLDδ-overexpressed plants compared to WT and VC, similar to the trend of enzymic activity ( Figure 6D-F).   Supplementary Table S2. Data are presented as the mean of three repeated experiments, and error bars represent SE. Columns with different letters, a, b, c and d show significant differences, with p < 0.05.

Na + Concentration within Root Cells under Salinity Stress
The Na + accumulation in cells results in an increase in ROS production under NaCl stress [31,32]. The content of Na + in root cells was detected by a Na + -specific probe, CoroNa TM Green. Under control conditions, the fluorescence intensity of the Na + probe was very low in the roots of all tested genotypes (Figure 7). After the short-term salt treatment (NaCl 130 mM, 12 h), the fluorescence intensity significantly increased in root cells (Figure 7). It is worth noting that WT and VC displayed a 2.1-3.9-fold higher CoroNa TM intensity than that of transgenic plants (Figure 7), indicating the higher buildup of Na + in the roots of WT and VC.

Na + Concentration within Root Cells under Salinity Stress
The Na + accumulation in cells results in an increase in ROS production under NaCl stress [31,32]. The content of Na + in root cells was detected by a Na + -specific probe, Coro-Na TM Green. Under control conditions, the fluorescence intensity of the Na + probe was very low in the roots of all tested genotypes (Figure 7). After the short-term salt treatment (NaCl 130 mM, 12 h), the fluorescence intensity significantly increased in root cells ( Figure  7). It is worth noting that WT and VC displayed a 2.1-3.9-fold higher CoroNa TM intensity than that of transgenic plants (Figure 7), indicating the higher buildup of Na + in the roots of WT and VC. Figure 7. Na + concentrations in root cells of wild-type (WT) Arabidopsis, empty vector control (VC) and transgenic lines overexpressing PePLDδ under NaCl stress. Seeds from WT, VC and PePLDδoverexpressed lines (OE6 and OE7) were allowed to germinate on 1/2 MS medium and grown for seven days. The seedlings were transferred to liquid medium containing 0 or 130 mM NaCl for 12 h. Then, Arabidopsis roots were incubated with 20 M CoroNa™ Green for 1 h, followed by washing 4-5 times. Green fluorescence within cells was detected with a laser confocal microscope, and the relative Na + concentrations were calculated according to the fluorescence intensity. Data are presented as the mean of 6-9 individual plants, and error bars represent SE. Values with different letters, a, b, and c show significant differences, with p < 0.05. Scale bar = 250 μm.

Na + and K + Fluxes under Salt Stress
Salt-resistant species retain low Na + levels by an active salt extrusion across the PM [24,25]. To confirm whether PePLDδ-transgenic plants could maintain Na + extrusion under salinity, the Na + flow in root tips was recorded with a non-invasive micro-test technique (NMT). Upon short-term exposure to NaCl (130 mM 12 h), the Na + efflux increased significantly in all tested genotypes, and a higher flux rate was found in the PePLDδ-transgenic plants ( Figure 8A). However, the Na + /H + antiporter inhibitor, amiloride (AMI), drastically reduced the salt-elicited efflux of Na + in WT, VC and PePLDδ-overexpressed plants ( Figure 8A). The pharmacological data indicate that the Na + efflux resulted from an active Na + /H + exchange across the PM [24,25]. and transgenic lines overexpressing PePLDδ under NaCl stress. Seeds from WT, VC and PePLDδoverexpressed lines (OE6 and OE7) were allowed to germinate on 1/2 MS medium and grown for seven days. The seedlings were transferred to liquid medium containing 0 or 130 mM NaCl for 12 h. Then, Arabidopsis roots were incubated with 20 µM CoroNa™ Green for 1 h, followed by washing 4-5 times. Green fluorescence within cells was detected with a laser confocal microscope, and the relative Na + concentrations were calculated according to the fluorescence intensity. Data are presented as the mean of 6-9 individual plants, and error bars represent SE. Values with different letters, a, b, and c show significant differences, with p < 0.05. Scale bar = 250 µm.

Na + and K + Fluxes under Salt Stress
Salt-resistant species retain low Na + levels by an active salt extrusion across the PM [24,25]. To confirm whether PePLDδ-transgenic plants could maintain Na + extrusion under salinity, the Na + flow in root tips was recorded with a non-invasive micro-test technique (NMT). Upon short-term exposure to NaCl (130 mM 12 h), the Na + efflux increased significantly in all tested genotypes, and a higher flux rate was found in the PePLDδtransgenic plants ( Figure 8A). However, the Na + /H + antiporter inhibitor, amiloride (AMI), drastically reduced the salt-elicited efflux of Na + in WT, VC and PePLDδ-overexpressed plants ( Figure 8A). The pharmacological data indicate that the Na + efflux resulted from an active Na + /H + exchange across the PM [24,25].  The H + flux recordings showed that NaCl decreased the net influx of H + in WT and VC, but the salt effects were less pronounced in the two transgenic lines ( Figure 8B). When the specific inhibitor of PM H + -ATPase, vanadate, was applied, the net H + influx markedly increased in Arabidopsis roots irrespective of the control and NaCl treatment ( Figure 8B). Therefore, the increased net H + influx was due to a decreased efflux of H + , which was pumped by H + -ATPases in the PM ( Figure 8B) [26,27,29]. In comparison, the vanadateincreased H + influx was lower in OE6 and OE7 than in the WT and VC in the absence and presence of NaCl ( Figure 8B). This suggest that H + -ATPases in the PM were severely inhibited by vanadate in the root cells of WT and VC [27,29].
NaCl leads to a more pronounced K + loss in salt-sensitive species than in salt-resistant species [24,25]. In this study, NaCl treatment increased K + efflux in the roots of all tested lines ( Figure 8C). WT and VC exhibited a 0.2-2.0-fold higher K + loss than OE6 and OE7, although there is no significant difference between VC plants and the OE6 transgenic line in the mean K + fluxes ( Figure 8C). This shows that PePLDδ-transgenic Arabidopsis, and, in particular, OE7, had a greater capacity to retain K + under NaCl salinity.

Transcription of SOS1 and AHA2 under NaCl Stress
It has been shown that the activated plasmalemma H + -ATPase and Na + /H + antiporter result, at least in part, from the upregulated transcription of encoding genes [26,29,35,36]. Here, we examined the abundance of a typical Na + /H + antiporter gene, SOS1, and a H + -ATPase gene, AHA2, in Arabidopsis plants. We observed that the transcription of AtSOS1 and AtAHA2 was significantly up-regulated in all tested lines under a high salt treatment (with the exception of AtSOS1 in WT) ( Figure 9A,B). It is worth noting that the salt-enhanced gene expression was more pronounced in the PePLDδ-overexpressed plants than in the WT and VC ( Figure 9A,B). The results support the finding of a high Na + /H + exchange in the roots of PePLDδ-transgenic plants ( Figure 8A).

Phosphatidic Acid Content of PePLDδ-Transgenic Plants
The capacity of PePLDδ-transgenic plants in maintaining K + /Na + and H2O2 homeostasis might be related to phosphatidic acids (PA), the key signaling molecule in plant abiotic stress responses [6][7][8]. We measured the content of phosphatidic acids, since phospholipase D can hydrolyze membrane phospholipids to produce PA. We found that PePLDδtransgenic plants retained a higher content of total PA than WT and VC ( Figure 10A). It is interesting that PA significantly increased in OE6 and OE7 under salt stress, which was nearly twice that of WT and VC ( Figure 10A) Supplementary Table S2. Data are presented as the mean of three repeated experiments, and error bars represent SE. Columns with different letters, a, b, c, d, and e show significant differences, with p < 0.05.

Phosphatidic Acid Content of PePLDδ-Transgenic Plants
The capacity of PePLDδ-transgenic plants in maintaining K + /Na + and H 2 O 2 homeostasis might be related to phosphatidic acids (PA), the key signaling molecule in plant abiotic stress responses [6][7][8]. We measured the content of phosphatidic acids, since phospholipase D can hydrolyze membrane phospholipids to produce PA. We found that PePLDδtransgenic plants retained a higher content of total PA than WT and VC ( Figure 10A). It is interesting that PA significantly increased in OE6 and OE7 under salt stress, which was nearly twice that of WT and VC ( Figure 10A)

PePLDδ Enhances Salt Tolerance in Transgenic Plants
In this study, PePLDδ increased the plant's ability to tolerate salinity stress in terms of the growth response to NaCl (Figure 3). This is consistent with the reports overexpressing PLDα in herbaceous and woody species [14][15][16][17]. For example, the heterologous expression of the Ammopiptanthus nanus PLDα gene in Arabidopsis PLDα1 knockout mutants enhanced the salinity tolerance of transgenic plants. Similar findings were observed in Populus tomentosa and tobacco overexpressing phospholipase Dα genes from Arabidopsis (AtPLDα) [15] and cucumber (CsPLDα) [16,17]. In this study, the PePLDδ-enhanced salt tolerance was associated with the increased phosphatidic acids in transgenic plants. Being the lipid products of phospholipase D, phosphatidic acids mediate salt stress signaling in higher plants [6][7][8].
Our data showed that PePLDδ-overexpressed plants retained a typically higher abundance of PA species  [23]. Among these species, the specific PA 34:2 (a 16:0-18:2 PA) was suggested to play a crucial role in mediating the salt stress signal transduction [23]. Accordingly, the increased PA species in PePLDδ-transgenic plants could mediate the plant salt stress response. Therefore, PePLDδ overexpression positively regulates the plant's tolerance to NaCl salinity. Our data showed that Populus euphratica phospholipase Dδ increases salt tolerance by regulating K + /Na + and ROS homeostasis in Arabidopsis.

PePLDδ Mediates ROS Homeostasis under Salt Stress
Salinity increased the production of reactive oxygen species, e.g., H 2 O 2 ( Figure 5), which destroyed the integrity of the cell membrane, leading to solute leakage (Figure 4, [31][32][33] (Figures 4 and 5). Similarly, the MDA content and ROS (O 2 − · and H 2 O 2 ) production are much lower in tobacco overexpressing cucumber CsPLDα under NaCl stress compared to WT plants [17]. In addition, the AtPLDα-transgenic P. tomentosa plants displayed a greater capacity in scavenging ROS than the wild-type [15]. The overexpression of PePLDδ upregulated activities of antioxidant enzymes, such as APX, SOD and POD (Figure 6), which scavenged the salt-elicited excessive ROS, thus reducing the ROS-induced membrane oxidation (Figures 4 and 5). Therefore, PePLDδ constitutes a signaling cascade controlling ROS and improving the salinity tolerance in transgenic plants. Similarly, in Arabidopsis, PLD and PA decrease the cell death induced by H 2 O 2 [37]. We suppose that the increase in PA in PePLDδ-overexpressed plants might initiate the salt signaling cascade in retaining the ROS homeostasis. It is suggested that PLD and PA mediate the generation of superoxide in Arabidopsis [38,39]. Moreover, Arabidopsis PLDδ was shown to transduce H 2 O 2 signaling by interacting with glyceraldehyde-3-phosphate dehydrogenases under stress conditions [40]. The PePLDδ-and PA-stimulated ROS might activate the antioxidant enzymes, i.e., SOD, APX and POD, in salt-stressed plants overexpressing PePLDδ, since reactive oxygen species have been implicated as second messengers to induce antioxidant defenses [41,42]. We have previously shown that the activated SOD, APX and GR in salt-resistant P. euphratica was associated the rapid increase in ROS (O 2 − · and H 2 O 2 ) after the onset of salt treatment [31,32]. In accordance, CsPLDα-overexpressed tobacco plants displayed much higher activities of SOD, POD, CAT and APX than those of the wild-type [17]. The activated SOD, CAT and POD were also observed in leaf discs of AtPLD-overexpressed poplars during NaCl treatment [15]. Under high salinity, the up-regulated expression of SOD, APX and POD also contributed to the enzyme activity in plants overexpressing PePLDδ ( Figure 6). The interaction between PePLDδ, PA and these antioxidant enzymes needs to be further investigated.

PePLDδ Mediates K + /Na + Homeostasis under Salt Stress
PePLDδ-overexpressed plants retained Na + homoeostasis under salt stress (Figure 7) as a result of the greater Na + extrusion from the root cells ( Figure 8). The increased gene expression of AtSOS1 and AtAHA2 in transgenic lines suggests that the Na + efflux resulted from an active Na + /H + exchange promoted by the PM H + -ATPase ( Figure 9) [35,36]. It is suggested that the NaCl-increased specific PA species, i.e., 16:0-18:2, were able to interact with mitogen-activated protein kinase 6 (MPK6), which directly phosphorylates the downstream Na + /H + exchanger SOS1 under a high salt condition [23]. Accordingly, PePLDδ-derived PA species, 34:2 (16:0-18:2), in transgenic plants could also activate At-SOS1 through a MAPK signaling pathway, thus promoting the Na + extrusion via a Na + /H + exchanger across the plasma membrane. Moreover, it has been proposed that the PLDproduced PA activated the H + -ATPase and Na + /H + exchanger in the vacuolar membrane to increase the salinity tolerance [43]. We suggest that PePLDδ, similar to PLDα, enhanced the PM H + -ATPase and Na + /H + antiporter to extrude Na + from salt-stressed roots. However, how PePLDδ and PA activate the Na + /H + antiport system in the PM needs to be further clarified.
The lower K + loss in PePLDδ-overexpressed plants, particularly OE7, was presumably related to the PM H + -ATPase (Figures 8 and 9). The upregulation of AtAHA2 resulted in an increased activity of H + -pumps, as PM H + -ATPases are transcriptionally regulated in transgenic lines [26,29], in addition to post-translational modulation [27]. The activated H + -pumps not only promoted Na + /H + exchange but also hyperpolarized the membrane potential, thus reducing the K + loss through depolarization-activated channels, e.g., outward rectifying potassium channels and non-selective cation channels [24,25]. Consequently, PePLDδ-overexpressed plants increased their ability to retain K + /Na + homeostasis under NaCl stress. Similarly, Ji et al. suggest that the proton-pumps activated by cucumber CsPLDα and CsPLDα-produced PA that enabled transgenic tobacco plants to maintain K + /Na + homeostasis under salinity stress [17]. We noticed that the transgenic line OE7 exhibited a greater capacity than OE6 to retain K + under salt stress. This might be related to the higher level of specific PA 34:2 in PePLDδ-OE7 (Figure 10), which serves as a critical molecule that mediates salt stress signaling [23]. Nevertheless, long-term experiments are needed to evaluate the transgenic lines' capability to tolerate long-term salt stress conditions, produce flowers and set fruits.

Culture of Populus euphratica and Arabidopsis thaliana
P. euphratica seedlings (1-year-old) from Xinjiang Uygur Autonomous Region, China were raised at a greenhouse of Beijing Forestry University (BFU). The plants were wellirrigated and fertilized during three months of culture [24,44]. Upper mature leaves were sampled for total RNA isolation and PePLDδ gene cloning.
Arabidopsis thaliana were seeded in 1/2 MS agar medium and cultured in climate chamber after 3 d of low-temperature stratification treatment. The temperature was 22 ± 1 • C and humidity was maintained at 50-60%. Photosynthetically active radiation was 150 µmol m −2 s −1 during a long-day photoperiod (16 h). After 10 days of culture in plates, the A. thaliana seedlings with 4 cotyledons were planted in 200 mL pots containing nursery soil and vermiculite in a ratio of 1:1, and placed in the culture room at BFU.

Cloning of PePLDδ Gene
Total RNA for first-strand cDNA synthesis was extracted from P. euphratica leaves using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. RNA (1 µg) was then reverse transcribed using Oligo dT adaptor primers (Promega, Madison, WI, USA). The gene sequence of P. euphratica phospholipase Dδ (Pe; reference sequence number XM_011023928.1 on NCBI) was used to design primers. The primer sequences (5 -to-3 ) were as follows: forward, ATG GCT GAG CTC CAG TCA AC; reverse, TTA TGT TAA CAT CGG GAA G. The PCR processes for full-length gene cloning were followed as previously described [45,46]. The PCR products were purified and cloned into the pMD18-T vector (Takara, Kusatsu, Japan), and then the recombinant plasmid was transformed into Escherichia coli Top10 competent cells (Invitrogen, Carlsbad, CA, USA). E. coli bacterial cells were grown on LB sterile agar medium, and the ampicillin-resistant single colonies were cultured in liquid medium to obtain the full-length of PePLDδ.

Sequence and Phylogenetic Analyses
We performed PLDδ protein multiple-sequence alignments using ClustalW (http: //www.genome.jp/tools/clustalw/, accessed on 18 August 2020, EMBL-EBI, Hinxton, Cambridgeshire, UK). The phylogenetic tree was determined with MEGA 5.2 software (http: //www.megasoftware.net/index.php, accessed on 18 August 2020, Center for Evolutionary Medicine and Informatics, Tempe, AZ, USA). Accession numbers of PLDδ orthologs used in multiple-sequence alignment and phylogenetic analysis are shown in Supplementary  Table S1.

Construction and Screening of PePLDδ-Transgenic Lines
Transformation of Arabidopsis thaliana was performed by flower dipping method. The full-length PePLDδ gene was inserted into the expression vector pMDC85, containing cauliflower mosaic virus 35S (CaMV 35S) promoter to obtain recombinant plasmids pMDC85-PePLDδ. The cloned plasmid with the target gene was transformed into Agrobacterium tumefaciens. The flower buds of four-week-old soil-cultivated Arabidopsis thaliana were immersed in the bacterial solution for 5-10 s, then cultured at 22 ± 1 • C for 16-24 h in darkness. Thereafter, the infected plants were transferred to Arabidopsis culture room and well-watered until mature seeds were collected. The seeds were dried for 1 week and used for antibiotic screening. The antibiotic-resistant plants were grown in nutrient soil to obtain homozygous T3 generation seeds. The transgene expression levels of seven T3 homozygous lines, OE1, OE2, OE3, OE4, OE5, OE6 and OE7, were quantified with semi-quantitative reverse transcription PCR and RT-qPCR.

Phenotype Tests of Transgenic Plants
Phenotypic screening of PePLDδ-transgenic Arabidopsis thaliana under NaCl salinity was performed in plates with 1/2 MS medium. Seeds of wild-type (WT) Arabidopsis, empty vector control (VC) and transgenic lines overexpressing PePLDδ, OE6 and OE7 were sterilized with 1% sodium hypochlorite for 10 min, followed by washing 5-6 times, and sown in 1/2 MS medium. After vernalization treatment for 3-5 days, the A. thaliana seedlings were transferred to 1/2 MS medium supplemented with 0 or 130 mM NaCl. The petri dishes were vertically placed, and photos were taken for root length measurement after seven days of salinity treatment. The root length was measured using image processing software ImageJ pro6 (http://rsb.info.nih.gov/ij/, accessed on 8 October 2021). The fresh weight of A. thaliana seedlings was obtained immediately after the plants were harvested. Salt tolerance tests for transgenic lines were repeated three times.

Electrolyte Leakage and MDA Measurement
Mature leaves were sampled from seedlings of WT Arabidopsis, VC and transgenic lines overexpressing PePLDδ after treatment without or with 130 mM NaCl. The initial conductivity and final conductivity of leaf samples were examined to determine the relative electrolyte leakage (EL) as previously described [33,45].
To measure MDA content, 1 g (fresh weight) of leaves was sampled from control and NaCl-treated plants of WT, VC and PePLDδ-overexpressed lines. Samples were immersed in 10 mL 10% trichloroacetic acid (TCA) for full grinding, then centrifuged at 4000 rpm for 10 min. Two milliliters of the supernatant was mixed with the same volume of 0.6% 2-thiobarbituric acid (TBA) solution and boiled for 20 min. For blank controls, 2 mL distilled water was mixed with TBA solution. After cooling to room temperature, the absorbance was measure at 450, 532 and 600 nm, respectively. The MDA concentration (µmol L −1 ) was calculated as: 6.45 × (D532-D600) − 0.56 × D450 [33,45].

Real-Time Quantitative PCR
Seedlings of WT Arabidopsis, VC and PePLDδ-overexpressed lines treated without or with 130 mM NaCl were harvested for RT-qPCR analysis. The Trizol reagent (Invitrogen, Carlsbad, CA, USA) and EASYspin Plus Plant RNA Kit (Aidlab Biotech, Beijing, China) were used to isolate total RNA from Arabidopsis leaves. Based on the manufacturer's recommended protocol, RNA (1 µg) was used for reverse transcription with Moloney murine leukemia virus (M-MLV) reverse transcriptase and an oligo (dT) primer (Promega, Madison, WI, USA). The resulting cDNA products were used as templates for RT-qPCR. We used Arabidopsis β-actin 2 (AtACTIN2) as the internal reference gene. Forward and reverse primers designed to target AtSOS1, AtAHA2, AtSOD, AtPOD, AtAPX and internal control gene, AtACTIN2, are listed in Supplementary Table S2. The composition of reaction mixture and running conditions for RT-qPCR have been described elsewhere [33,45,46]. Each sample was repeated at least three times. The relative target gene expression level was normalized to the reference gene AtACTIN2 using the cycle threshold (Ct) values [47].

Na + and H 2 O 2 Contents in Root Cells
Na + specific probe, CoroNa TM Green AM (Invitrogen, Carlsbad, CA, USA), was used to measure Na + concentrations within Arabidopsis root cells. Seven-day-old seedlings of WT, VC and PePLDδ-overexpressed lines (OE6 and OE7) grown on 1/2 MS medium were exposed to 0 or 130 mM NaCl for 12 h. The control and NaCl-stressed roots were incubated in CoroNa TM Green (20 µM) in 5 mM MES/KCl loading buffer (pH 5.7) [27,33]. After 2 h incubation in darkness, roots were rinsed with 1/2 MS solution 4-5 times. For H 2 O 2 assay, Arabidopsis roots were incubated in 10 µM H 2 DCFDA (Molecular Probe, Eugene, OR, USA) for 15 min and washed 4-5 times before confocal analysis [25,33,35]. Leica SP8 confocal microscope was used to detect the fluorescence intensity, with excitation wavelength 488 nm and emission wavelength 510-530 nm [25,33,35]. The relative fluorescence intensity of Na + and H 2 O 2 was analyzed quantitatively in Image Pro Plus 6.0 (Media cybernetics, silver spring, Rockville, MD, USA).

Determination of Antioxidant Enzyme Activity
The seeds from WT, VC and PePLDδ-overexpressed lines (OE6 and OE7) were germinated and cultured on 1/2 MS medium for seven days. These seedlings were then treated with 0 or 130 mM NaCl for another seven days. Control and salt-stressed seedlings were harvested and ground in liquid-nitrogen-precooled mortars. The samples (0.1 g fresh weight) were mixed with 1 mL precooled extraction buffer containing 1mM EDTA, 1% PVP, 1mM ASA and 50 mM potassium phosphate buffer (pH 7.0). Through centrifugation (12,000 g) at 4 • C for 10 min, the supernatant solution was obtained to determine enzyme activities of superoxide dismutase (SOD), peroxidase (POD) and ascorbic acid peroxidase (APX). Antioxidant enzyme activity assay kits, such as A001-3-2 (total SOD determination kit), A084-3-1 (POD assay kit) and A123-1-1 (APX test box) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), were used to detect enzymatic activity according to the manufacturer instructions. The total protein in crude enzyme extract was assayed with A045-2-2 (total protein determination kit) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Phosphatidic Acid Species Analysis
Seeds from WT, VC and PePLDδ-overexpressed lines (OE6 and OE7) were allowed to germinate on 1/2 MS medium and grown for seven days. Then, seedlings were transferred to liquid medium containing 0 or 130 mM NaCl for 24 h. Control and salinized Arabidopsis plants were harvested, frozen in liquid nitrogen and used to measure phosphatidic acids. The lipid was extracted and phosphatidic acids were analyzed and quantified by means of electrospray ionization-tandem mass spectrometry (ESI-MS/MS) as previously described [48].

Data Analysis
Na + , K + and H + fluxes were calculated using the program JCal V3.2.1, a free MS Excel spreadsheet developed by Yue Xu (http://www.xuyue.net/, accessed on 5 May 2021). All of the experimental data were subjected to SPSS version 19.0 (IBM Corporation, Armonk, NY, USA) for tests of normality and homogeneity of variances. One-way ANOVA was applied to compare the means between different treatments. Post hoc test was performed using S-N-K method. p < 0.05 was considered significant unless otherwise noted.

Conclusions
We propose that PePLDδ overexpression positively regulates plant tolerance to NaCl salinity. The PePLDδ-enhanced salt tolerance was associated with an increased PA in transgenic plants. PePLDδ-overexpressed plants retained a typically higher abundance of PA species, 34