60 µM of putrescine (Put), spermidine (Spd) or spermine (Spm), or with buffer alone (control, Ctrl), after which lipids were extracted, separated by TLC, and visualized by autoradiography. Each sample represents the extract of three seedlings. (B) 32P-labeled PA fold response to 30 min Put, Spd, Spm or diaminopropane (Dap), and thermospermine (tSpm) at the indicated concentrations.

standard growth medium consisting of 1/2 Murashige and Skoog (MS) medium with Gamborg B5 vitamins (pH 5.7; KOH), 1% (w/v) sucrose, and 1% (w/v) agar. Plates were vernalized at 4◦C for 48 h and then placed vertically under the angle of 70◦ , in a growth chamber (16/8 light/dark cycle, 110–130 µmol m−2 s −1 ) at 22◦C. Five days-old seedlings were then transferred to either 2 mL Eppendorf safe-lock tubes for 32Pi labeling O/N, or to treatment plates for phenotypic analyses.

### Chemicals

All chemicals were obtained from Sigma-Aldrich except 32Pi (orthophosphate, 32PO4 3−), which was purchased from PerkinElmer. Arabidopsis incubations with polyamines and chemicals were performed in incubation buffer, consisting of 2.5 mM MES buffer [2-(N-morpholino) ethanesulfuric acid], pH 5.7 (KOH), 1 mM KCl.

### 32Pi-Phospholipid Labeling, Extraction and Analysis

Phospholipid levels were measured as described earlier (Munnik and Zarza, 2013). Briefly, three seedlings per sample were metabolically labeled overnight by flotation in continuous light in 2 ml safe-lock Eppendorf tubes containing 200 µl incubation buffer (2.5 mM MES-KOH, pH 5.7, 1 mM KCl) and 2.5–10 µCi 32PO4 3− (stock 32Pi ; carrier-free, 2.5–10 µCi/µL). For mature plants, Arabidopsis leaf disks (∅ 5 mm) were taken from 3-week-old plants and labeled using the same conditions. Treatments were performed by adding 1:1 (v/v) of a 2× solution and incubations were stopped at indicated times by adding perchloric acid (Munnik and Zarza, 2013). Lipids were extracted and analyzed by thin-layer chromatography (TLC) using an ethyl acetate solvent system (Munnik and Laxalt, 2013). Radioactivity was visualized by autoradiography and individual spots were quantified by phosphoimaging (Typhoon FLA 7000; GE Healthcare).

For certain experiments, the protocol was slightly modified, i.e.: (1) In short-labeling experiments, 32Pi was added 30 min prior treatment. (2) In transphosphatidylation assays, treatments were performed in the presence of 0.5% n-butanol (Munnik and Laxalt, 2013). (3) For tissue-dissection experiments, seedlings were labeled, treated and fixed as described above, but then carefully cut into sections with a scalpel, and every section processed separately.

For statistical analysis, letters indicate values significantly different according to Student–Newman–Keuls test at P-value <0.05, and asterisks indicate significant differences with respect to control treatments, using the Student's t-test: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005. Data represent the mean ± SD. The results obtained were confirmed by at least 2 independent experiments.

#### Detection of ROS and NO in Arabidopsis Root

ROS production in the root tip of 5-day-old seedlings was detected by DCF fluorescence as described previously (Pei et al., 2000; Zhang et al., 2009). Briefly, seedlings were treated for the indicated times and then transferred to 10 µM H2DCFDA for 10 min followed by two washes in buffer. For ROS scavenging, seedlings were pre-treated with 5 mM N,N'-Dimethylthiourea (DMTU; Lu et al., 2009) for 60 min, before the different treatments with or without Spm 120 µM for 30 min. For NO detection, seedlings were co-incubated with the corresponding treatment and 10 µM DAR-4M for 30 min, and then washed two times with buffer. For cPTIO treatment, 0.1 mM cPTIO

test at P-value < 0.05. Asterisks indicate significant differences with respect to control treatments, using the Student's t-test: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005.

was applied for 60 min prior to treatments in order to scavenge NO. All incubations were performed in dark conditions. The localization of the DCF and DAR signal was done using the AMG Evos FL digital inverted microscope equipped with transmitted light GFP (470/22 to 510/42 nm). Images were converted to 8-bit using Image-J, and data was quantified as mean pixel intensity per region of interest (ROI).

#### Ion Flux Measurement

Net K+ fluxes were measured using MIFE technique (UTas Innovation, Hobart, TAS, Australia) (Newman, 2001; Shabala et al., 2006). Five days-old Arabidopsis seedlings were placed into a 30 mL measuring chamber, containing 0.5 mM KCl, 0.2 mM CaCl2, 5 mM MES, 2 mM Tris base; pH 6.0. Roots were immobilized in a horizontal position (Bose et al., 2014) and preincubated in the above buffer for at least 30 min. Electrodes were positioned near the root surface at the elongation zone (less than 2 mm from the root cap junction). First, steady-state ion fluxes were recorded over a period of 5 min, after which different concentrations of Spm were applied and net ion fluxes measured.

### Root Phenotyping Assay on Plates

Arabidopsis seedlings were grown on vertical plates containing standard growth medium for 5 days. Then, seedlings were transferred to plates supplemented with or without Spm. Plates were scanned at indicated days after transfer (DAT) using an Epson Perfection V700 Scanner at 300 dpi. For root measurements EZ-Rhizo software was used (Armengaud et al., 2009). Main root (MR) growth was expressed as growth ratio (MR length divided by MR length at 0 DAT). A paired t-test in SPSS was used for statistical analysis.

### RESULTS

#### Polyamines Trigger the Formation of PA in Arabidopsis Seedlings

To investigate whether polyamines could affect PA signaling, Arabidopsis seedlings were 32Pi-prelabeled O/N and treated with physiological concentrations (60 µM) of Put, Spd, or Spm for 30 min. As shown in **Figure 1A**, in particular Spm but also Spd,

∗∗P < 0.01, ∗∗∗P < 0.005.

was found to induce an increase in PA. Put did not show any effect until millimolar concentrations were used (**Figure 1B**). Spm already induced significant PA responses at ≥15 µM, while 30– 60 µM was required for Spd. Thermospermine (tSpm), a minor structural isomer of Spm also present in Arabidopsis (Kakehi et al., 2008), was found to induce PA responses at a similar concentrations as Spm (**Figure 1B**). Diaminopropane (Dap), a diamine product of polyamine oxidation, exhibited a similar low potency like Put (**Figure 1B**). Together these results show that polyamines can trigger a PA response, with its potency depending on charge, i.e., Spm4+ = tSpm4+ > Spd3+>>Put2+≈Dap2+.

To further investigate the Spm-induced PA, detailed doseresponse and time-course analyses were performed. As shown in **Figure 2**, Spm induced a clear dose-dependent Michaelis– Menten PA curve when treated for 30 min; starting at low µM levels and reaching a maximum 2.5-fold increase at ∼250 µM (**Figures 2A,B**). The response was relatively fast, starting between 8 and 15 min when using 60 µM of Spm, and PA linearly increasing (**Figures 2C,D**).

### Spm Triggers PA Formation in Roots and Requires Transport Across the Plasma Membrane via RMV1

To obtain more information as to where in the seedling the PA accumulation took place, we performed the same 32Pi-labeling and treatments, but now dissected root- and shoot tissues prior to lipid extraction. Interestingly, the Spm induced-PA increase was only found in the root, not in the shoot or hypocotyl (**Figure 3A**). Within the root, the PA accumulation was equally distributed along the different root sections (**Figure 3A**). A repetition of this experiment with 120 µM Spm gave similar results (data not

shown). In contrast to seedlings, we did observe a Spm induced-PA response in 32Pi-labeled leaf disks of mature, 3-weeks old plants (**Supplementary Figure S1**).

The non-permeant cation transport blocker, gadolinium (Gd3+) is known to inhibit the uptake of Spm across the plasma membrane (Pistocchi et al., 1988; Ditomaso et al., 1992b; Pottosin et al., 2014). Incubation of seedlings with GdCl3 prior to the application of Spm triggered a small PA response itself, but significantly reduced the Spm induced-PA response to approximately 70% of the control response (**Figure 3B**). This may indicate that most of the PA response observed is caused intracellularly.

To further characterize Spm uptake, we analyzed the Arabidopsis PUT/L-type amino acid transporter (LAT), called Resistant to Methyl Viologen 1 (RMV1, PUT3, LAT1), which is localized in the plasma membrane and responsible for the high-affinity uptake of Spm (Fujita et al., 2012). Using the knockout T-DNA insertion mutant rmv1 and two independent overexpressing Pro35S::RMV1 lines, we found a 35% decrease and ∼20–40% increase in PA, respectively (**Figures 3C,D**). These results indicate that cellular uptake of Spm is required for the PA response, and that RMV1 is one of the proteins involved in internalizing Spm across the plasma membrane.

To functionally analyze the involvement of the rest of the PUT/LAT family members, of which Arabidopsis contains five homologs (Mulangi et al., 2012), two quadruple knock-out mutants were used, i.e., lat1/2/3/5 and lat1/2/4/5, because the quintuple mutant was lethal (Fujita M., unpublished). Both lines, however, showed Spm induced-PA responses similar to wild type (**Supplementary Figure S2**). This discrepancy could be due to the fact that the single rmv1-KO allele is different from the quadruple mutants and belongs to a different ecotype (i.e., Ler vs. Col-0, respectively). While at least three LAT proteins exhibit polyamine transport activity (i.e., LAT1, LAT3, LAT4; Fujita et al., 2012; Mulangi et al., 2012), only LAT1 (RMV1, PUT3) is localized to the plasma membrane; LAT3 and LAT4 are localized to the ER and Golgi, respectively (Li et al., 2013; Fujita and Shinozaki, 2014). Hence, the results obtained may reflect distinct plasma membrane activity as well as genetic redundancy for Spm uptake, involving other members from the amino acidpolyamine-choline (APC) transporter family to which LAT/PUT transporters belong (Verrey et al., 2004; Rentsch et al., 2007).

#### Spm-Triggered PA Is Predominantly, but Not Solely Generated via PLD

A rapid PA response has traditionally been associated with increased DGK- and/or PLD activity. DGK produces PA through phosphorylation of DAG that originates from the hydrolysis of phosphoinositides or structural phospholipids by PLC or NPC, respectively (Munnik, 2014). PLD hydrolyses structural phospholipids, like PE and PC, to form PA directly. To distinguish between these two routes, a differential 32P-labeling protocol was used that highlights the DGK kinase-dependent reaction (Arisz and Munnik, 2013). This method is based on the premise that the 32Pi added to seedlings is rapidly takenup and incorporated into ATP and subsequently into lipids that

FIGURE 4 | Analysis of DGK involvement in Spm triggered-PA response. Seedlings were pulse-labeled with 32Pi for 30 min and then treated with 60 µM Spm or buffer alone (Ctrl) for the indicated times. The fold-PA response of two independent experiments is shown (squares and triangles, respectively). Values were normalized to the 32P-labeling of phosphatidylinositol and to Ctrl, without Spm.

are synthesized via kinase activity (e.g., DGK), which is in huge contrast to the relatively slow incorporation of 32P into structural phospholipids via de novo synthesis (Munnik et al., 1994, 1998; Arisz and Munnik, 2013). Under short labeling conditions, PLD would hardly generate 32P-labeled PA whereas the contribution of DGK would be augmented. As shown in **Figure 4**, Spm was still able to trigger an increase in 32P-PA when seedlings were only prelabeled for 30 min rather than 16 hrs O/N, indicating that at least part of the Spm induced-PA response is generated via DGK.

To analyze the potential involvement of PLD, its unique ability to catalyze a transphosphatidylation reaction was used, which produces phosphatidylbutanol (PBut) in vivo in the presence of a low concentration of n-butanol (Munnik et al., 1995, 1998; Arisz and Munnik, 2013; Munnik and Laxalt, 2013). To get a substantial proportion (though not all) of the structural phospholipids (i.e., PLD's substrate) 32P-labeled, seedlings were incubated with 32Pi O/N and the next day treated with Spm in the presence of 0.5% n-butanol. The subsequent formation of 32P-PBut is an in vivo marker for PLD activity that can be quantified. Again dose-response and time-course experiments were performed, but this time in the presence of n-butanol. As shown in **Figures 5A,B**, Spm clearly triggered PLD activity, with the PBut following a similar pattern as PA (**Figure 2**).

Arabidopsis contains 12 PLDs, i.e., 3 PLDαs, 2 PLDβs, 3 PLDγs, 1 PLDδ, 1 PLDε, and 2 PLDζs (Zhang et al., 2005). Validating the 32P-labeled PBut- and PA response in various T-DNA KO mutants, we identified PLDδ as the main contributor, with the pldδ-KO mutant alone or in combination with other KOs, showing a ∼55% reduction in PA and ∼70% reduction in PBut accumulation (**Figures 5C,D**). Interestingly, in Arabidopsis this isoform is located in the plasma membrane (Wang and Wang, 2001; Pinosa et al., 2013), while promoter-GUS analyses

suggests it is mainly expressed in roots (Katagiri et al., 2001), which is in agreement with the PA response observed here.

### H2O2 or NO Are Not Involved in the Spm Induced-PA Response

Spermine is known to cause an accumulation of NO and H2O2 (Cona et al., 2006; Tun, 2006; Moschou et al., 2008), which is likely mediated by polyamine oxidase (PAO) and diamine oxidase (DAO) activities (Tun, 2006; Wimalasekera et al., 2011). We confirmed that Spm was able to trigger an increase in H2O2 and NO under our conditions, as evidenced by the increase in fluorescence of their reporters, i.e., 20 ,70 -dichlorofluorescein diacetate (H2DCFDA; **Figure 6A**) and diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM; **Figure 6B**).

Since PLD activity can be activated by H2O2 (Wang and Wang, 2001; Zhang et al., 2003, 2009) or by NO (Distéfano et al., 2008; Lanteri et al., 2008; Raho et al., 2011), this could be a potential mechanism by which the production of PA was stimulated, especially since H2O2 and NO have been found to act upstream of PLDδ in response to ABA inducedstomatal closure (Distéfano et al., 2012). Similarly, H2O2 has been found to promote the binding of cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) to PLDδ and increase its activity (Guo et al., 2012).

To investigate whether H2O2 and NO were responsible for the Spm induced-PA response, the effects of ROS scavenger, DMTU and NO scavenger, carboxy-PTIO (cPTIO) were analyzed. While able to significantly reduce the accumulation of Spm-derived H2O2 and NO (**Figures 6A,B**), the scavengers had no effect on the Spm induced-PA response (**Figure 6C**), suggesting that the increase in PA was independent of these secondary metabolites. Moreover, double gapc1-1 gapc2-1 or gapc1-1 gapc2-2 knock-out mutants, revealed a PA response similar to wild type (**Figure 6D**). These results indicate that the Spm-induced PA is not caused via ROS- or NO induction.

Testing polyamines on seedling growth, we observed a significant reduction in primary root growth (**Supplementary Figure S3**). Previous reports have shown that this root growth inhibition is associated to H2O2 accumulation, derived from PAO activity (de Agazio et al., 1995; Couée et al., 2004; Tisi et al., 2011). However, loss-of function pldδ seedlings did not show any apparent root phenotype when transferred to agar

plates containing µM concentrations of Spm. Only a slight increase in root growth inhibition with respect to wt was observed at higher Spm concentrations, i.e., 150 µM (**Supplementary Figure S3**). Those results are consistent with previous reports indicating that pldδ is more sensitive to H2O2-induced stress (Zhang et al., 2003).

#### PLDδ Is Involved in the Spm-Induced K +-Efflux Response in the Root-Elongation Zone

Application of exogenous polyamines has been shown to trigger a K+ efflux in pea- (Zepeda-Jazo et al., 2011) and maize roots (Pandolfi et al., 2010), which has consequences for the membrane potential, inducing the plasma membrane to depolarize (Ozawa et al., 2010; Pottosin et al., 2014). To study this in our context, we performed MIFE ion-flux analyses at the root elongation zone of Arabidopsis seedlings using different concentrations of Spm. As shown in **Figures 7A,B**, a clear dose-dependent efflux of K+ was detected, which correlated with the response in PA (**Figure 2B**). While the Spm induced-K+ efflux slowly restored to pre-treatment values after 50 min with 60–200 µM, the efflux persisted when only 10–20 µM Spm was used.

To investigate potential involvement of PLDδ, the response of wild type was compared with that of the pldδ knock-out mutant in the same root zone. Prior to Spm application, roots showed a small net K+ efflux of 150–250 nmol m−2 s −1 , likely due to transferring seedlings from nutrient-rich MS medium (∼20 mM K +) to poorer, basic-salt medium (BSM; 0.2 mM K+; **Figure 7**). Upon 60 µM Spm application to wild-type seedlings, the K+ efflux increased gradually, reaching a peak around 15 min, and returning to basal levels after ∼50 min (**Figure 7C**). In pldδ, the response was significantly different, showing an faster K+ efflux peak at 10 min, and recovery (**Figure 7C**). Overall, pldδ showed ∼60% reduction in net K+ loss compared to wt (**Figure 7D**),

placing PLDδ and derived PA upstream of the Spm induced-K+ efflux. Since the Spm induced-K+ efflux is completely abolished by gadolinium (**Supplementary Figure S4**), these results indicate that PLDδ is likely activated by Spm from the inside of the cell, after its uptake.

### DISCUSSION

#### Polyamines Trigger a Charge-Dependent PA Response

Polyamines are naturally occurring polycationic molecules involved in a plethora of cellular events (Tiburcio et al., 2014), yet it is still largely unknown how this works at the molecular level. Here, a link to the formation of the lipid second messenger PA is reported, which is predominantly generated by PLDδ and plays a role in Spm induced-K+ efflux.

In this paper, we show that low µM concentrations of Spm trigger a rapid (minutes) PA response in the roots of Arabidopsis seedlings (**Figure 2**). In older plant material, Spm also triggered a PA response in leaves (**Supplementary Figure S1**), so multiple tissues are sensitive to Spm. Other polyamines, like Spd, Put and Dap were also able to trigger an accumulation in PA but this required higher concentrations, especially the diamines. In contrast, tSpm was as effective as Spm (**Figure 1**). These results indicate that the capacity of polyamines to activate the formation of PA is charge-dependent, with Spm4+ = tSpm4+>Spd3+>>Put2+, which may indicate an electrostatic interaction between the positive charges of the polyamine and a negatively charged target (Bertoluzza et al., 1988; Kurata et al., 2004; DeRouchey et al., 2010; Rudolphi-Skórska et al., 2014).

#### Triggering PA Response Requires Uptake of Polyamines

The saturation of the response at relatively low concentrations (**Figure 2**) may reflect a saturation of polyamine uptake (Pistocchi et al., 1987; Ditomaso et al., 1992a). In that regard, the Spm induced-PA responses were found to be strongly reduced in the plasma membrane localized PUT, RMV1-KO, also known as LAT1 or PUT3 (Fujita et al., 2012; Fujita and Shinozaki, 2014), while overexpression of RMV1 resulted in a much stronger PA response upon Spm treatment (**Figure 3**). The affinity of this transporter for Spm (Km = 0.6 µM), Spd (Km = 2.2 µM), and Put (Km = 56.5 µM), respectively (Fujita et al., 2012; Fujita and Shinozaki, 2014), is consistent with their potency to activate a

PA response and indicates, together with the inhibition of the PA response by gadolinium (**Figure 3**), that polyamines are taken up before triggering a PA increase.

### Link Between Polyamine Synthesis and PLDδ

Differential 32P-labeling experiments combined with transphosphatidylation assays revealed that part of the PA response was generated via the PLD pathway. Using T-DNA insertion PLD-KO mutants, we found that the majority of the PA is generated through PLDδ. Arabidopsis contains 3α-, 2β-, 3γ-, 1δ-, 1ε-, and 2ζ PLDs, which differ in amino acidsequence conservation and lipid-binding domains (Bargmann and Munnik, 2006; Hong et al., 2016; Hou et al., 2016). PLDδ is typically localized in plasma membrane facing the cytosol (Wang and Wang, 2001; Pinosa et al., 2013) while all others PLDs are cytosolic though can transiently bind to various microsomal membranes (Wang, 2002). PLDδ has been implicated in drought and salinity stress (Katagiri et al., 2001; Bargmann et al., 2009; Distéfano et al., 2015) and in freezing tolerance (Li et al., 2004), which are stress responses in which polyamines have also been implicated to play a role (Alcázar et al., 2010). The specific involvement of PLDδ further implies that the polyamine induced-PA response predominantly occurs at the plasma membrane.

Both Spm and tSpm activated PLDδ equally well. In Arabidopsis, Spm is synthesized by spermine synthase (SPMS) while tSpm by ACAULIS5 (Panicot et al., 2002; Kakehi et al., 2008). Both are encoded by single genes, SPMS and ACL5, which are predominantly expressed in the phloem and xylem, respectively, in both roots and leaves (**Supplementary Figure S5**; Brady et al., 2007; Winter et al., 2007; Sagor et al., 2011; Yoshimoto et al., 2016). Interestingly, their expression strongly overlaps with that of PLDδ, especially SPMS, which are both strongly induced upon salt- and osmotic stress (**Supplementary Figures S5, S6**; Katagiri et al., 2001; Brady et al., 2007; Winter et al., 2007). So potentially, the local synthesis and/or transport of spermine could activate PLDδ to generate PA. In agreement, significantly increased PA levels were found in an SPMSoverexpressor line (35S::SPMS-9; Gonzalez et al., 2011) at control conditions (**Supplementary Figure S7A**). An spms-2 KO line exhibited normal PA levels, however, in response to salt stress, a strongly reduced PA response was found while the SPMSoverexpressor line revealed a much higher PA response than wt (**Supplementary Figures 7A,B**). These results support the idea of a novel, interesting link between PA and Spm in stress responses. Alternatively, since polyamines are rich in soil and produced by various microbes (Young and Chen, 1997; Chibucos and Morris, 2006; Zandonadi et al., 2013; Zhou et al., 2016), our observation that extracellular polyamines trigger intracellular PA responses may also reflect the action of natural, exogenous polyamines.

### PA Function

Phosphatidic acid is an important plant phospholipid. Besides its role as precursor for all glycerolipids at the ER, PA has emerged as important lipid second messenger, generated through the PLC/DGK- and/or PLD pathways in response to various (a)biotic stresses, including plant defense, wounding, salt, drought, cold, and heat stress, where it is linked to various cellular processes, like vesicular trafficking, membrane fission and -fusion, and transport (Munnik et al., 2000; Testerink and Munnik, 2005, 2011; Vergnolle et al., 2005; Zhao, 2015; Hong et al., 2016; Hou et al., 2016). A local accumulation of PA in cellular membranes may affect the enzymatic- or structural properties of protein targets in that membrane or, alternatively, recruit cytosolic protein targets via PA-binding domains. PA targets include protein kinases, phosphatases, ion transporters, PEPC, GAPDH, NADPH oxidases (Rboh) (Kim et al., 2013; McLoughlin et al., 2013; Pleskot et al., 2013; Putta et al., 2016; Ufer et al., 2017). Here, evidence is provided for a role of PA in ion transport. MIFE analyses showed that Spm triggers a rapid efflux of K+ ions, which was strongly reduced in the pldδ mutant (**Figure 7**), indicating a direct or indirect role for PA in K+ gating. Associated to Spm uptake, we observed a fast net H+ influx (cytosolic acidification) followed by a gradual increase of H+ efflux (cytosolic alkalinisation), which correlated with the K + efflux peak and its gradual recovery (**Supplementary Figure S8**). The cytosolic alkalinisation is related to the opening of voltage-gated inward-rectifying K+ channels (Kin) to compensate the K+ efflux (Dreyer and Uozumi, 2011; Karnik et al., 2016). In animal cells, PA has been shown to regulate voltage-gated potassium (Kv) channels and has been proposed to stabilize K+ inward channels (Kin) in its closed conformation, thus reducing K + inward currents (Hite et al., 2014). In plants, PA has been shown to inactivate Kin channels in guard cells of Vicia faba and Arabidopsis (Jacob et al., 1999; Uraji et al., 2012), in which PLDδ has been implicated (Uraji et al., 2012). These observations are in agreement with a role for PA in regulating K+ fluxes of which the precise mechanism requires further investigation.

In summary, we provide molecular evidence that polyamines functionally require PLD and PA for their mode of action. This knowledge and the use of PLD- and polyamine synthesis mutants may shed new light on this phenomenon in other studies.

### DATA AVAILABILITY

All datasets for this study are included in the manuscript and the **Supplementary Files**.

## AUTHOR CONTRIBUTIONS

XZ, SS, and TM designed the experiments. LS performed the MIFE experiments while XZ performed the rest. MF, AT, and MH added materials, ideas and discussions. XZ and TM wrote the manuscript.

## FUNDING

This work was supported by the Spanish Ministerio de Ciencia e Innovación (BIO2011-29683 and CSD2007-00036), the Generalitat de Catalunya (SGR2009-1060 and BE DGR 2011), and the Netherlands Organisation for Scientific Research (NWO 867.15.020).

#### ACKNOWLEDGMENTS

fpls-10-00601 May 20, 2019 Time: 17:9 # 11

The authors would like to thank L. Tikovsky and H. Lemereis for the assistance in the greenhouse, Dr. T. Takahashi (Okayama University, Japan) for supplying tSpm and Dr. I. Pottosin

#### REFERENCES

(University of Colima, Mexico) for discussion and critical review of the MIFE experiments.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00601/ full#supplementary-material

and transcript profiling provide new insights on the role of the tetraamine spermine in Arabidopsis defense against Pseudomonas viridiflava. Plant Physiol. 156, 2266–2277. doi: 10.1104/pp.110.171413

thaliana spermine synthase gene promoter Gene Note. Plant Biotechnol. 28, 407–411. doi: 10.5511/plantbiotechnology.11.0704a

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Zarza, Shabala, Fujita, Shabala, Haring, Tiburcio and Munnik. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Spermine Confers Stress Resilience by Modulating Abscisic Acid Biosynthesis and Stress Responses in Arabidopsis Plants

*Francisco Marco1† , Enrique Busó2† , Teresa Lafuente3 and Pedro Carrasco1 \**

*1 Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina (ERI BIOTECMED), Universitat de València, Valencia, Spain, 2 UCIM, Universitat de València, Valencia, Spain, 3Departamento de Biotecnologia de Alimentos, Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Valencia, Spain*

#### *Edited by:*

*Ana Margarida Fortes, University of Lisbon, Portugal*

#### *Reviewed by:*

*Yi Shang, Yunnan Normal University, China Mingjun Li, Northwest A&F University, China Rongrong Guo, Guangxi Academy of Agricultural Science, China*

#### *\*Correspondence:*

*Pedro Carrasco<EMAIL_ADDRESS>
*† These authors have contributed equally to this work*

#### *Specialty section:*

*This article was submitted to Plant Metabolism and Chemodiversity, a section of the journal Frontiers in Plant Science*

*Received: 26 February 2019 Accepted: 11 July 2019 Published: 31 July 2019*

#### *Citation:*

*Marco F, Busó E, Lafuente T and Carrasco P (2019) Spermine Confers Stress Resilience by Modulating Abscisic Acid Biosynthesis and Stress Responses in Arabidopsis Plants. Front. Plant Sci. 10:972. doi: 10.3389/fpls.2019.00972*

Polyamines (PAs) constitute a group of low molecular weight aliphatic amines that have been implicated as key players in growth and development processes, as well as in the response to biotic and abiotic stresses. Transgenic plants overexpressing PA-biosynthetic genes show increased tolerance to abiotic stress. Therein, abscisic acid (ABA) is the hormone involved in plant responses to environmental stresses such as drought or high salinity. An increase in the level of free spermine (Spm) in transgenic Arabidopsis plants resulted in increased levels of endogenous ABA and promoted, in a Spm-dependent way, transcription of different ABA inducible genes. This phenotype was only partially reversed by blocking ABA biosynthesis, indicating an ABA independent response mediated by Spm. Moreover, the phenotype was reproduced by adding Spm to Col0 wild-type Arabidopsis plants. In contrast, Spm-deficient mutants showed a lower tolerance to salt stress. These results indicate that Spm plays a key role in modulating plant stress responses.

Keywords: abscisic acid, spermine, salt stress, stress response, stress tolerance

### INTRODUCTION

Polyamines (PAs) are small aliphatic amines found in all living organisms (Kusano et al., 2008). In plants, the three major PAs include putrescine (Put), spermidine (Spd), and spermine (Spm). In Arabidopsis, Put biosynthesis is done mainly by arginine decarboxylase (ADC; EC<IP_ADDRESS>) activity, which decarboxylates arginine as the first step in the PA-biosynthetic pathway. Put serves as the precursor for higher molecular weight PAs Spd and Spm. Spd synthase (SPDS;EC<IP_ADDRESS>) and Spm synthase (SPMS;EC <IP_ADDRESS>), respectively, catalyze the addition to Put of aminopropyl groups generated from S-adenosylmethionine (SAM) by SAM decarboxylase (SAMDC, EC <IP_ADDRESS>) (Tiburcio et al., 1990). Arabidopsis genome carries two genes that encode for ADC enzyme (ADC1 and ADC2; Urano et al., 2005), four genes for SAMDC (SAMDC1-4; Urano et al., 2003), two genes for SPDS (*SPDS1* and *SPDS2*; Hanzawa et al., 2002) and one single gene for SPMS (Hanzawa et al., 2002). *ACAULIS5* (*ACL5* gene), originally assigned as a putative Spm synthase (Hanzawa et al., 2000), encodes a thermospermine (tSpm) synthase (Knott et al., 2007). Free PAs levels of the plant are the result of a balance between biosynthesis and degradation, the former process done mainly through the activity of diamine oxidases (DAO, EC<IP_ADDRESS>) and polyamine oxidases (PAO, EC<IP_ADDRESS>), that exhibit different substrate preferences. Diamines like Put are a preferred substrate for DAOs, while higher molecular weight PAs, like Spm, tSpm, and Spd, are oxidized by PAOs (Cona et al., 2006). Some PAOs terminally oxidize PAs, while other isoforms are involved in a PA backconversion process of tSpm to Spm and Put, releasing H2O2 (Moschou et al., 2012). Ten genes encoding DAOs, as well as five genes that encode PAOs are present in the Arabidopsis genome (Takahashi et al., 2010; Planas-Portell et al., 2013).

As sessile organisms, plants respond to environmental stresses through a series of physiological, cellular, and molecular changes. Consequence of these changes, transcriptomic, proteomic, and metabolic modifications, which help to mitigate the effect of stress and lead to the adaptation of the plant, occur. Classically, PAs have been assigned to play an important role in modulating the response of plants to diverse environmental stresses (Bouchereau et al., 1999). Elevated PA levels are one of the most remarkable metabolic hallmarks in plants exposed to stresses such as drought, salinity, chilling, heat, hypoxia, ozone, UV, or heavy metals (Alcázar et al., 2010; Gill and Tuteja, 2010). In the last years, it has been elegantly summarized the relationship between the functional significance of PAs and their roles in tolerance and/or amelioration of stress responses in plants (Minocha et al., 2014). It has been suggested that PAs could act as stress messengers in plant responses interacting with different stress pathways (Takahashi et al., 2003; Tuteja and Sopory, 2008; Alcázar et al., 2010; Marco et al., 2011).

It is well known that abscisic acid (ABA) is involved in the response of plants to abiotic stresses (Vishwakarma et al., 2017). Abscisic acid (ABA) is a plant hormone that quickly accumulates in plants exposed to different abiotic stresses. ABA is a sesquiterpenoid that is synthetized from C40 carotenoids in three steps catalyzed by zeaxanthin epoxidase (Marin et al., 1996), 9-cis-epoxycarotenoid dioxygenase (NCED; Schwartz et al., 1997) and abscisic aldehyde oxidase (AAO; Seo et al., 2000). The key step in ABA biosynthesis is the cleavage reaction of epoxy carotenoids to produce xanthoxin catalyzed by NCED (Kende and Zeevaart, 1997). In fact, ectopic expression of NCED causes overproduction of ABA in tobacco and tomato (Thompson et al., 2000). Accumulation of ABA stimulates stomatal closure to limit water loss from leaves, as well as changes in the level of expression of genes involved in stress responses and in the biosynthesis of osmoprotectant species that help the cells to handle damage and restore cellular homeostasis (Fujita et al., 2011). Transcriptome comparison of Arabidopsis plants exposed to salt or drought stress shows that about half of the genes induced by these stresses are also induced by ABA (Seki et al., 2002). Moreover, the disruption of ABA synthesis by transgenic approaches in Arabidopsis leads to plants more sensitive to drought stress (Iuchi et al., 2001). These observations suggest the predominant role of this hormone as a signaling molecule in the response against drought and high salinity stresses (Sah et al., 2016).

Hierarchical clustering expression analyses in Arabidopsis using public microarray data indicate that some, but not all, PA biosynthesis gene paralogs share similar expression patterns, in agreement with their different implications in stress and development (Tiburcio et al., 2014). Moreover, transcriptome studies performed in Arabidopsis have revealed differential regulation of PA biosynthesis genes by abiotic stress (Alcazar et al., 2006b). Recent studies have been reported that Spm-overproducer plants overexpress a number of ABA related genes (Marco et al., 2011). On the other hand, the characterization of PA loss-of-function mutants has provided evidence for the involvement of PAs in resistance traits (Urano et al., 2003; Yamaguchi et al., 2007; Cuevas et al., 2008; Zarza et al., 2017). Besides that, although taken together these evidences suggest that PAs are involved in modulating plant responses to abiotic stress, the relationship between PA, and ABA remains to be fully understood.

In this work, we have observed that overexpression of *SAMDC1* gene in Arabidopsis produces Spm accumulation and leads to plants with an improved tolerance to salt stress. Furthermore, since overexpression of the *NCED3* gene and ABA accumulation were observed on these Spm-accumulating transgenic lines, the expression of several ABA-response related genes was checked in plants with different Spm levels, several Spm-deficient mutant lines, as well as WT plants treated with external Spm. A Spm-dependent expression pattern was observed for *NCDE3* and some ABA-responsive genes. These results suggest that Spm plays a key role in modulating ABA levels and that stress tolerance could be improved by manipulation of Spm biosynthesis, causing the accumulation of endogenous ABA and triggering the expression of a number of stress-response genes. On the other hand, expression of *NCDE3* and other ABA-responsive genes was increased in these Spm-overproducer plants even when ABA biosynthesis was inhibited, suggesting the existence of a Spm-dependent pathway response that would include some ABA-dependent factors.

### MATERIALS AND METHODS

### Plant Growth Conditions

Experiments were performed using several *Arabidopsis thaliana* (Arabidopsis) lines. Ecotype Col-0, obtained from the Nottingham Arabidopsis Stock Centre (University of Nottingham, Loughborough, UK) was used as the wild type (WT). On the other hand, different lines of Arabidopsis plants with altered Spm levels have been used: three transgenic lines overexpressing the SAMDC1 gene under the control of CaMV35S constitutive promoter (pBISDCs-S3', pBISDCs-S9', pBISDCs-S15, Marco et al., 2011), a T-DNA insertion mutant line of the *SPMS* gene (*spms-1*, Imai et al., 2004), an ethyl methane-sulfonatemutant line of the *ACL5* gene (*acl5-1*, Hanzawa et al., 1997) and a double mutant line obtained from crossing of the two lines mentioned above (*acl5-1/spms-1*, Imai et al., 2004). Both *acl5-1* and *acl5-1/spms-1* mutants showed stem-reduced growth phenotypes (Hanzawa et al., 1997; Imai et al., 2004), however, *spms-1* plants exhibited similar phenotypes to WT plants in terms of growth and development (Imai et al., 2004).

Plants were cultivated in growth chambers Sanyo MLR-350 (Sanyo Electric Co., Japan) under long day conditions, illumination at 23°C for 16 h, in darkness at 16° for 8 h. Previously, all the seeds were stratified for 2 days at 4°C. Adult plants were grown from seeds sown in pots with a 1:1:1 mixture of soil, vermiculite, and sand, and watered with mild nutrient solution [recipe from Arabidopsis Biological Resource Center (ABRC, The Ohio State University, USA) handling plants and seeds guide, http://www.biosci.ohio-state.edu/pcmb/Facilities/ abrc/handling.htm].

Five day-old seedlings were grown under the same light and temperature conditions. Seeds were surface sterilized by washing for 10 min in 30% (v/v) commercial bleach, 0.01% (v/v) Triton X-100 and rinsed three times with sterile distilled water. Sterile seeds were plated on 4% agar plates containing one half strength MS medium (Murashige and Skoog, 1962).

#### Plant Treatments

Adult plants were grown in pots, salt stress treatments were performed by adding NaCl (0–250 mM) to the watering solution. Visible damage of stress was estimated in 3-week old plants exposed for 10 days to 0–250 mM NaCl, and pictures were taken. Additionally, 4-week old plants were exposed for 6 h to 250 mM NaCl and rosette leaf samples were harvested to isolate RNA. Furthermore, a stress recovery assay was performed by exposing 2-week old plants to 250 mM NaCl for 2 days. After 12 days of recovery in control conditions, stem length and shoot fresh weight (FW) were measured.

Salt stress and inhibition of ABA biosynthesis experiments were also performed in plates by adding 250 mM NaCl to 5-day grown seedlings grown in ½ MS medium with or without 100 μM sodium tungstate dehydrate (Fluka). Entire seedling samples were taken after 6 h.

Seedlings were also grown in ½ MS plates supplemented with 0.1, 0.5, and 1 mM, Put, Spd, or Spm. Entire seedling samples were taken after 5 days of growth. All tissues were harvested and immediately frozen in liquid nitrogen and stored at −80°C until used.

Germination of seeds was also studied in ½ MS plates supplemented with 0, 50, 100, 150, or 250 mM NaCl. Fifty seeds were sowed per plate and the number of seeds that developed cotyledons until 8 days after stratification was counted. All experiments were done by triplicate.

#### Quantitative RT-PCR

Total RNA was extracted from plant tissue using Total Quick RNA Cells and Tissues Kit (Talent SRL, Italy), following protocol established by manufacturer. RNA was quantified by their absorbance al 260 nM, and its integrity checked by denaturing agarose gel electrophoresis.

RNA was treated with RNAse free-DNAse (Roche diagnostics, Spain), according to manufacturer's instructions. A total of 1 μg of DNA free-total RNA was reverse transcribed to Firststrand complementary DNA (cDNA) with random hexamers using SuperScript® III First-Strand Synthesis System 1st (Invitrogen, Spain) according to manufacturer's instructions. Quantitative real time PCR (qRT-PCR) was performed on Gene AmpR 5,700 Sequence Detection System (PE Applied Biosystems, Japan), using Power SYBR® Green PCR Master Mix (PE Applied Biosystems) first-strand cDNA as a template. Each 20 μl reaction contained 1 μl of cDNA, 100 nM of each pair of target primers (FW and REV), and 10 μl of SYBR Green PCR Master Mix. The PCR conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s and 60°C for 1 min. Three replications were performed for each sample in each experiment. Primers used for real-time PCR are described in **Table 1**. Data was analyzed according to 2−ΔΔ*C*T Method (Livak and Schmittgen, 2001). Actin-2 (AT5G09810; An et al., 1996) was used as a reference gene.

#### Abscisic Acid Quantification

ABA was measured in seedlings and 4-week old Arabidopsis plants. Plant tissue (0.2 g FW) was homogenized with 5 ml of extraction buffer (80% acetone, 100 mg L−1 butylated hydroxytoluene, 0.5 g L−1 citric acid) and centrifuged 12,000 ×*g* for 5 min. Supernatant was recovered, dried, and resuspended in 0.5 ml TBS buffer (6.05 g L−1 Tris, 0.20 mg L−1 MgCl2, and 8.8 g L−1 NaCl, pH 7.8). ABA was quantified following an indirect ELISA method (Walker-Simmons, 1987). ABA-BSA conjugates were made according to Weiler (1980) as modified by Norman et al. (1988). ABA levels are expressed as ng (g FW)−1.

#### Statistical Analyses

Data was analyzed by one-way Analysis of Variance (ANOVA) followed by *post-hoc* comparisons by Tukey's HSD *t* test. A probability level < 0.05 was considered statistically significant. Calculations were performed using IBM® SPSS® Statistics v22.0 Software.

### RESULTS

#### pBISDCs Plants Are Tolerant to Salt Stress

In a previous work, we were able to describe that overexpression of SAMDC1 gene in Arabidopsis leads to with higher Spm levels than WT plants (**Supplementary Figure S1**). Also, the transcriptome of pBISDCs transgenic lines showed an increase in the expression of a set of genes enriched in functional categories involved in defense-related processes against both biotic and abiotic stresses (Marco et al., 2011).

To assess the effects of this defense-related transcriptome expression changes in salt stress tolerance, pBISDCs and WT plants were grown in soil for 3 weeks and treated with irrigating solution with presence or absence of NaCl. Under control conditions, pBISDCs showed no phenotype in terms of growth and development. However, after 10 days of treatment, WT plants irrigated with excess NaCl showed generalized chlorosis and wilt, while pBISDCs lines appeared with a healthier aspect and diminished symptoms (**Figure 1A; Supplementary Figure S3**). A saline stress recovery assay was also performed by exposing pBISDCs and WT plants to 250 mM NaCl during 2 days and leaving them to recover for 12 days in the absence of salt. After the recovery period, salt treated plants showed a reduction in their growth compared to untreated plants, but pBISDCs lines were able to develop higher recovery percentages of stem length (46.1–48.7%) and shoot fresh weight (72.8–75.2%) than WT plants (26 and 54%, respectively) (**Supplementary Figure S4**).

Additionally, salt stress tolerance was estimated by establishing seed germination percentage after 8 days, of seeds sowed in plates with increasing concentrations of NaCl (0–250 mM). Germination percentage decreased with increasing NaCl concentration for all lines studied (transgenic and WT), dropping to 10% for the highest salt concentration (250 mM) (**Figure 1B**). However, Spm-accumulating lines maintained better germination percentages (about 15% higher than WT) at intermediate NaCl concentrations (50–150 mM) (**Figure 1B**).

#### *SAMdC1* Overexpression Alters Abscisic Acid Metabolism and Abscisic Acid-Responsive Gene Expression

Given the previous result, a more specific search of genes related to the salt stress response within the transcriptome of the pBISDCs plants was carried out, locating a set of genes that code for proteins related to the ABA synthesis and response that showed significant expression changes compared to WT plants (**Supplementary Figure S2**). The expression of some of these ABA-related genes was checked by qRT-PCR to confirm the results observed in transcriptome studies.  SAMDC1-overexpressing lines showed higher expression levels of all these ABA-related genes when compared to WT plants (**Figure 2A**). ABA-biosynthesis gene *NCED3* showed the most pronounced changes (5 to 10-fold).

Plants of same age were also exposed to salt stress. After 6 h of 250 mM NaCl exposure, the level of expression of all five ABA-related genes increased in WT plants (**Figure 2B**). Regarding pBISDCs transgenic lines response to NaCl exposure,

FIGURE 1 | Salt stress tolerance of pBISDCs lines. (A) Appearance of Arabidopsis WT and pBISDCs transgenic plants overexpressing SAMDC1 (S3′, S9 and S15) after 10 days of salt treatment. 3 week-old plants were watered with solution without (control) and with the supplementation of 250 mM NaCl. Pictures were taken after 10 days. (B) Seed germination percentage in MS plates supplemented with different concentrations of NaCl. The number of seeds that developed cotyledons until 8 days after stratification was counted. Graphs show the mean ± standard deviation from MS plate triplicates. Significant changes from control treatment are highlighted (\*) (ANOVA, Tukey HSD test, *p* < 0.05).

*RD26* (all three lines) and *NCED3* (two lines) maintained expression levels similar to control conditions, while *COR15A* (two lines) raised to similar levels observed in WT plants exposed to stress. Also, *RD29A* (all lines) increased its expression in pBISDCs lines to higher levels than WT plants in salt stress conditions (**Figure 2B**).

ABA levels were also determined in both pBISDCs and WT lines in control and salt stress conditions. *SAMDC1* overexpressing lines showed also higher ABA levels (about 3-fold) than WT lines on non-stressed 4-week old plants (**Figure 2C**). After 6 h of salt exposure, ABA levels raised in WT in greater extent than transgenic lines, reaching to a slightly higher level than in Spm-accumulating transgenic lines (**Figure 2D**). Similar results in gene expression and ABA levels were found for 5 day-old plate-grown seedlings and did not changed when photoperiod was set to short day (data not shown).

#### Spm Treatment Raises ABA Levels and Induces ABA-Related Gene Expression

Results described above suggested a possible link between the observed differences in ABA and high Spm levels. To further test this possibility, PAs were exogenously supplied to plates where WT seeds were sown and let grow for 5 days. ABA levels increased in Spm-supplemented seedlings, with a positive correlation between ABA levels and external Spm concentration (**Figure 3A**). This positive correlation affected also to expression of the chosen set of ABA-related genes, with *NCED3* showing the most important induction in response to external Spm (**Figure 3B**). However, the growth of seedlings in the presence of external Put or Spd did not produce significant changes in ABA levels or in the expression of ABA-related genes (**Figure 3**).

#### Spm Deficiency Affects Expression of Abscisic Acid-Induced Genes

Moreover, the possible relationship between ABA-related stress response and Spm levels was also tested in different Spm-deficiency scenarios. Therefore, ABA levels were determined in 5-day old plate-grown seedlings of tSpm synthase mutants (*acl5-1*), that have similar free Spm levels than WT plants (Hanzawa et al., 2000; Imai et al., 2004), as well as in *spms-1* mutants and double mutant *acl5-1/spms-1*, with no detectable levels of Spm (**Figure 4A**; Imai et al., 2004). In control conditions, all mutants showed similar ABA levels to those of WT seedlings. On the other hand, when seedlings were exposed to 250 mM NaCl for 6 h (salt stress conditions), ABA on *acl5-1* mutant rose to levels similar to those observed stressed WT seedlings. On the contrary, *spm-1* and *acl5-1/spm-1* mutants showed slightly lower ABA levels than WT stressed seedlings (**Figure 4A**). In the same conditions, Spm-accumulating line pBISDCs-S15 had the highest levels of ABA for both control and salt stress conditions (**Figure 4A**).

Expression of the ABA-responsive genes previously analyzed was also determined by qRT-PCR in Spm-deficient mutants (**Figures 4B–E**). Under control conditions, expression levels in the mutants were similar to those found in WT plants for all genes, but their expression pattern changed in salt exposed seedlings, depending on the mutant considered. Thus, *spms-1* and *acl5-1/spms-1* mutants were not able to raise *NCED3* and *RD26* expression when exposed to salt to the levels observed

in salt-stressed WT and *acl5-1* seedlings (**Figures 4C,E**, respectively). Similar observations were made for *COR15A*, although the expression levels were more close to WT stressed seedlings (**Figure 4B**). On the other side, *RD22BP1* gene showed expression fold-change levels closer to the observed in WT salt-stressed seedlings for both *spms-1* mutants assayed (**Figure 4D**).

#### Spm Is Able to Modulate Plant Responses to Salt Stress in an Abscisic Acid-Independent Way

ABA synthesis was blocked by addition of 0.1 mM tungstate in plates. As expected, ABA levels dropped to levels <1 ng ABA g FW−1 in 5 days seedlings of WT, Spm-treated WT or pBISDCs plants (**Figure 5A**). Expression levels of *COR15A*

and *RD22BP1* were higher in Spm-treated WT and pBISDCs lines than in WT seedlings (**Figures 5B,D**). In the presence of tungstate, expression of those genes was similar to the levels observed in the WT seedlings. (Fold changes ≤1) (**Figures 5B,D**), suggesting that their induction by Spm is determined by ABA-dependent pathways. On the contrary, *NCED3* expression levels in presence of tungstate remained higher in Spm-treated plants and pBISDCs lines than in WT plants (**Figure 5C**). *RD26* also showed maintenance of their expression after tungstate treatment combined with Spm or in pBISDCs seedlings (**Figure 5E**) but with fold change differences relative to WT control less pronounced than those observed for *NCDE3* (**Figure 5E**).

Once determined that higher Spm levels affect expression of ABA-related genes, we also tested the effect of blocking ABA biosynthesis in the different Spm-deficient situations

determine the expression levels of salt stress-related response genes in all conditions assayed (B–G). For each gene, data are expressed as fold change relative to the level measured in WT plants in control conditions (2−ΔΔCT). Graph show the mean of three biological replicates ± standard deviation. Significant differences between treatments are indicated with letters (ANOVA, Tukey HSD test, *p* < 0.05).

described previously. Expression of the ABA dependent genes *COR15A*, *RD22BP1, NCED3*, and *RD26* was analyzed under control and salt stress conditio*ns* in the presence or absence of tungstate. Expression levels of *COR15A* and *RD22BP1* did not respond to salt stress in the presence of tungstate either in WT type, Spm-deficient mutants or the pBISDCs line S15 (**Figures 4B,D**). However, after salt stress, *NCDE3* rises to levels higher than control conditions in WT, the pBISDCs line S15 and *acl5-1* seedlings, and only dropped to similar levels observed in WT in Spm-deficient *spm-1* and *acl5-1/ spm-1* mutants (**Figure 4C**). *RD26* also showed maintenance of their expression with tungstate treatment combined with salt stress, but with fold change differences relative to WT control less pronounced than those observed for *NCDE3* (**Figure 4E**). Similar results were obtained when ABA biosynthesis was blocked with Fluoridone (data not shown).

The specificity of Spm in relation with ABA-dependent stress responses was checked by analyzing the expression levels

of *SOS1* (AT2G01980) and *SOS3* (AT5G24270), two members of the ABA-independent SOS (Salt Overly Sensitive) saltsignaling response pathway (Ji et al., 2013). *SOS1* and *SOS3*·expression was similar in all seedlings of the lines assayed, and salt stress induced their expression at similar levels independently of tungstate presence or the level of Spm in the seedlings (**Figures 4F,G**).

deviation. Significant differences between treatments are indicated with letters (ANOVA, Tukey HSD test, *p* < 0.05).

#### DISCUSSION

During the last years, the manipulation of polyamine levels by transgenic approaches or use of loss or gain mutants has proved a useful tool to gain knowledge about possible polyamine roles in plant processes (Alcazar et al., 2006b). Transgenic approaches include heterologous overexpression studies of *ODC*, *ADC*, *SAMDC*, and *SPDS* from different animal and plant sources in rice, tobacco, tomato, and Arabidopsis (Alcázar et al., 2010).

Likewise, Spm accumulation has been previously observed in transgenic rice plants constitutively overexpressing the *Datura stramonium* SAMDC cDNA in transgenic rice (Thu-Hang et al., 2002) or the yeast SAMDC in tobacco (Cheng et al., 2009), as well as using ABA inducible expression of Tritordeum SAMDC in rice (Roy and Wu, 2002) or ripening-induced expression of yeast SAMDC in tomato (Mehta et al., 2002), where accumulation of Spd and Spm was observed. High levels of Put, Spd, and Spm were also observed in tobacco plants that overexpress constitutively carnation SAMDC (Wi et al., 2006). In a previous work, we were able to describe that overexpression of *SAMDC1* gene in Arabidopsis leads to plants with higher Spm content than WT plants. Also, the transcriptome

of pBISDCs transgenic lines showed an increase in the expression of a set of genes enriched in functional categories involved in defense-related processes against both biotic and abiotic stresses (Marco et al., 2011). In line with these results, Arabidopsis Spm-accumulating lines obtained by overexpression of SPMS share a common set of 234 genes that includes genes related with the response to water deprivation or cold acclimation (Gonzalez et al., 2011; Marco et al., 2011). In addition, it was previously observed that external Spm treatment modulated the expression of a large number of defense-related genes (Mitsuya et al., 2009). In fact, 28 genes induced from that study are also overexpressed in the pBISDCs transgenic lines, including the transcription factor AtbZIP60 (Iwata and Koizumi, 2005) and the mitogen-activated protein kinase AtMAPK3 (Takahashi et al., 2003). Moreover, Arabidopsis AtPO4-deficient plants show increased Spm levels in the roots and up-regulation of several genes encoding drought stress response proteins (Kamada-Nobusada et al., 2008).

The activation of this set of stress response genes due to Spm accumulation could be the factor that would explain the tolerance of those plants to salt stress (**Figure 1**). Absence of Spm on mutant line *acl5-1/spm-1* causes a defect of Ca2+ homeostasis and resulted in hypersensitivity to salt stress, being this phenotype mitigated only by exogenously applied Spm, but not by Spd or Put (Yamaguchi et al., 2006). On the other hand, increased tolerance to abiotic stress by increasing Spm levels had been previously observed in other transgenic plant systems. Rice plants over-expressing *Tritordeum* SAMDC under the control of an ABA inducible promoter accumulate Spd and Spm and are less sensitive to salt stress (Roy and Wu, 2002). Also, rice plants with constitutive expression of *D. stramonium SAMDC* showed increased levels of Spm and an improved recovery after exposure to drought conditions (Peremarti et al., 2009). In the same trend, over-expression of carnation SAMDC produced accumulation of total PAs in tobacco, and generated a broad-spectrum tolerance to abiotic stresses (Wi et al., 2006). More recently, is has been described that constitutive overexpression of *Capsicum annuum S*AMDC in Arabidopsis increases Spd and Spm levels and leads to an increased drought tolerance of the transgenic plants compared to WT (Wi et al., 2014). Raise of Spm and Spd levels was also obtained in Arabidopsis plants by overexpression of cucurbita *SPDS* gene, leading to an enhanced tolerance to multiple environmental stresses (Kasukabe et al., 2004). Conversely, alterations of Spm levels have also been reported by downregulation of SAMDC gene by RNA interference strategies in tobacco (Moschou et al., 2008) and rice (Chen et al., 2014), leading to plants with reduced Spd and Spm levels and an enhanced salinity-induced programmed cell death in tobacco (Moschou et al., 2008) or with a reduced tolerance to stress by drought, salinity or chilling in rice (Chen et al., 2014).

The relationship among PA metabolism, abiotic stress, and ABA has been previously reported in several studies. Expression of *ADC2*, *SPMS*, and *SAMDC2* genes is induced by the exogenous application of ABA in Arabidopsis (Urano et al., 2003). Also, induction of *ADC2*, *SPDS1*, and *SPMS* by drought stress is Arabidopsis is an ABA-dependent response, since up-regulation is not observed in ABA deficient (*aba2*) and insensitive (*abi1*) mutants (Alcazar et al., 2006a). Moreover, maize *ADC2*, *ZmSPDS1* and *ZmSPDS2* genes are also induced by NaCl and ABA treatments (Jiménez-Bremont et al., 2007).

Based on these results, the effect of Spm accumulation on the expression of ABA-related genes in pBISDCs plants was examined more in depth with the MAPMAN tool (**Supplementary Figure S2**; Usadel et al., 2005). We found three upregulated genes (*NCED3*, *NCED4*, and *ABA2*) that code for ABA biosynthesis enzymes (González-Guzmán et al., 2002; Tan et al., 2003). In addition, other genes coding for ABA-induced proteins like ATHVA22A, ATHVA22B (Chen et al., 2002), KIN1 (Wang et al., 1995) or the gram-domain containing protein GER5 (AT5G13200; Baron et al., 2014) were also up-regulated (**Supplementary Figure S2**, overexpressed). The set of under-expressed genes also includes some ABA-responsive genes as AAO2 (Seo et al., 2000), ABF4 (Kang et al., 2002) and a couple more of ABA-responsive proteins. (**Supplementary Figure S2**, under-expressed). These observations pointed us to carry out a more detailed study of the ABA levels and ABA response in pBISDCs plants. In order to confirm the expression data observed in the transcriptome study, expression levels of several ABA-related genes were checked by qRT-PCR. pBISDCs lines have higher levels of expression of genes that code for ABA biosynthesis genes (**Supplementary Figure S2**), including *NCED3* (**Figure 2A**), and consequently higher ABA levels than WT lines in control conditions (**Figure 2C**).

Expression levels of a set of ABA-responsive genes were also checked by qRT-PCR. Cold-Regulated 15A gene (*COR15A*) codes for a member of the Late Embryogenesis Abundant protein family with a role in the protection of the chloroplast structures during freeze-induced dehydration (Steponkus et al., 1998). Responsive to Desiccation 26 gene (*RD26*) is induced in response to desiccation, and encodes a transcriptional activator that acts in ABA-mediated dehydration response (Yamaguchi-Shinozaki et al., 1992; Song et al., 2016). *RD22BP1* gene also encodes a MYC-related transcriptional activator that is induced by dehydration stress and ABA treatment (Abe et al., 2003; Liu et al., 2018). *RD29A* encodes a hydrophilic protein of unknown function that is induced by salt and drought stresses (Yamaguchi-Shinozaki and Shinozaki, 1993). Expression levels of all these ABA-dependent genes were increased in the pBISDCs lines (**Figure 2A**). Induction of *RD26* and *COR15A* has been previously observed in Arabidopsis plants overexpressing cucurbita *SPDS*, with increased levels of Spd and Spm (Kasukabe et al., 2004). Higher ABA levels on pBISDCs lines could also explain the activation of at least part of the stress-related genes observed in these Spm-accumulating lines (**Supplementary Figure S2**).

Additionally, external application of Spm to WT plants lead to similar changes in *NCDE3* expression, ABA levels, and expression levels of the other ABA-responsive genes (**Figure 5**). Moreover, Spm-deficient mutants (*spm-1*, *acl5-1/ spm-1*) were not able to raise ABA to similar levels than WT plants when exposed to salt stress (**Figure 4A**). Same observation was done for the expression levels of genes *NCED3*

(**Figure 4C**) and *RD26* (**Figure 4E**). An increase in ABA levels has been also reported in soybean seeds treated with Spm (Radhakrishnan and Lee, 2013).

Finally, when ABA synthesis was blocked with tungstate, *NCED3* maintained a certain level of induction in plants with normal (WT, *acl5-1*) or high (Spm-treated WT and pBISDCs lines) levels of Spm, whereas this was not observed in Spm-deficient mutants *spm-1* and *acl5-1/spm-1* (**Figure 5**, tungstate and **Figure 4**, saline + tungstate). The existence of an ABA-independent pathway responsible for part of the induction of ABA biosynthesis genes by salt stress was proposed previously (Barrero et al., 2006). Those authors pointed out that severe ABA-deficient mutants still showed a NaCl-dependent induction of *NCED3*, *AAO3*, and *ABA1*, being *NCED3* the gene that showed a stronger induction with NaCl in ABA absence. Taken together, our results suggest that Spm could have a possible role in the induction of the expression of *NCED3* by this ABA-independent pathway salt stress response.

On the other hand, expression levels of two members of the SOS signaling pathway, involved in the maintenance of ion homeostasis during salt stress (Ji et al., 2013) are not altered in Spm-accumulating lines in response to NaCl stress (**Figures 4F,G**). This observation was previously reported in Spm-deficient mutants (Yamaguchi et al., 2006).

In summary, the results obtained in this study add more evidences to the involvement of Spm in plant stress responses, for which various protective roles have been proposed. Our results suggest that one of these mechanisms could involve the modulation of ABA levels in salt stress response through modulation of ABA biosynthesis by affecting *NCED3* gene expression. At the same time, Spm could be involved in an ABA-independent stress response pathway, as suggested by the results observed when ABA biosynthesis is blocked with tungstate.

#### REFERENCES

It remains to be determined which other changes in gene expression observed in Spm-accumulating plants results from the direct action of Spm or which gene expression changes are the consequence of cross-talking between Spm and other stress-response signaling pathways, including ABA.

### DATA AVAILABILITY

All datasets generated for this study are included in the manuscript and/or the **Supplementary Files**.

### AUTHOR CONTRIBUTIONS

FM, EB and PC conceived the experimental design. FM and EB conducted the experiments. EB and TL conducted ABA measurements FM, EB and PC conducted gene selection and primer design. FM and PC wrote the manuscript.

#### ACKNOWLEDGMENTS

We are grateful to Drs Oliver Laule and Andreas Fürholz (RIP) for their assessment in the Arabidopsis genomic experiments carried out in ETH Zürich.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00972/ full#supplementary-material

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2019 Marco, Busó, Lafuente and Carrasco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Increasing Polyamine Contents Enhances the Stress Tolerance *via*  Reinforcement of Antioxidative Properties

*So Yeon Seo, Yu Jung Kim and Ky Young Park\**

*Department of Biology, Sunchon National University, Suncheon, South Korea*

The diamine putrescine and the polyamines (PAs), spermidine (Spd) and spermine (Spm), are ubiquitously occurring polycations associated with several important cellular functions, especially antisenescence. Numerous studies have reported increased levels of PA in plant cells under conditions of abiotic and biotic stress such as drought, high salt concentrations, and pathogen attack. However, the physiological mechanism of elevated PA levels in response to abiotic and biotic stresses remains undetermined. Transgenic plants having overexpression of *SAMDC* complementary DNA and increased levels of putrescine (1.4-fold), Spd (2.3-fold), and Spm (1.8-fold) under unstressed conditions were compared to wild-type (WT) plants in the current study. The most abundant PA in transgenic plants was Spd. Under salt stress conditions, enhancement of endogenous PAs due to overexpression of the *SAMDC* gene and exogenous treatment with Spd considerably reduces the reactive oxygen species (ROS) accumulation in intra- and extracellular compartments. Conversely, as compared to the WT, PA oxidase transcription rapidly increases in the *S16-S-4* transgenic strain subsequent to salt stress. Furthermore, transcription levels of ROS detoxifying enzymes are elevated in transgenic plants as compared to the WT. Our findings with OxyBlot analysis indicate that upregulated amounts of endogenous PAs in transgenic tobacco plants show antioxidative effects for protein homeostasis against stress-induced protein oxidation. These results imply that the increased PAs induce transcription of PA oxidases, which oxidize PAs, which in turn trigger signal antioxidative responses resulting to lower the ROS load. Furthermore, total proteins from leaves with exogenously supplemented Spd and Spm upregulate the chaperone activity. These effects of PAs for antioxidative properties and antiaggregation of proteins contribute towards maintaining the physiological cellular functions against abiotic stresses. It is suggested that these functions of PAs are beneficial for protein homeostasis during abiotic stresses. Taken together, these results indicate that PA molecules function as antisenescence regulators through inducing ROS detoxification, antioxidative properties, and molecular chaperone activity under stress conditions, thereby providing broad-spectrum tolerance against a variety of stresses.

Keywords: polyamines, spermidine, reactive oxygen species, chaperone activity, S-adenosylmethionine decarboxylase

#### *Edited by:*

*Ana Margarida Fortes, University of Lisbon, Portugal*

#### *Reviewed by:*

*Autar Krishen Mattoo, Agricultural Research Service (USDA), United States Shaohua Zeng, Chinese Academy of Sciences, China*

> *\*Correspondence: Ky Young Park<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Plant Metabolism and Chemodiversity, a section of the journal Frontiers in Plant Science*

*Received: 12 March 2019 Accepted: 25 September 2019 Published: 31 October 2019*

#### *Citation:*

*Seo SY, Kim YJ and Park KY (2019) Increasing Polyamine Contents Enhances the Stress Tolerance via Reinforcement of Antioxidative Properties. Front. Plant Sci. 10:1331. doi: 10.3389/fpls.2019.01331*

#### INTRODUCTION

Polyamines (PAs) are naturally occurring polycations ubiquitous to all living cells, which are essential for development, growth, and survival (Tabor and Tabor, 1985; Tiburcio et al., 2014). The diamine putrescine (Put) and the higher PAs [spermidine (Spd; triamine) and spermine (Spm; tetraamine)] contribute to biochemical and physiological cellular processes such as ion channel regulation and the maintenance of chromatin structure and cell membranes. They also modulate enzyme functions and are required for the regulation of DNA replication, transcription, and translation (Madeo et al., 2018), many of which are related to the positive charge at physiological pH for strong binding capacity to the negatively charged ions of DNA, RNA, and protein molecules (Murray-Stewart et al., 2018). Especially in plants, PAs and their metabolic products act as cell signaling molecules in enhancing tolerance to pathogen attacks and numerous abiotic stresses, including antisenescence in fruits (Wi et al., 2006; Mattoo et al., 2007; Moschou et al., 2008; Pathak et al., 2014; Jiménez-Bremont et al., 2014; Tiburcio et al., 2014; Handa et al., 2018). Attenuation of whole-plant senescence in transgenic plants with overexpression of yeast Spd synthase provides evidence for the role of PAs, particularly Spd, in increasing fruit shelf life, probably by reducing the postharvest senescence (Nambeesan et al., 2010).

The biosynthesis and degradation pathway of PAs in plants is well studied (Bagni and Tassoni, 2001; Moschou et al., 2008). The starting points for PA biosynthesis are the basic amino acids ornithine and arginine, which are decarboxylated by ornithine decarboxylase and arginine decarboxylase, respectively, to yield Put, which serves as the substrate for biosynthesis of Spd and Spm *via* the activities of S-adenosylmethionine decarboxylase (SAMDC) and Spd synthase and Spm synthase (Walters, 2003; Tiburcio et al., 2014). The oxidation of PAs is catalyzed by amine oxidases (AOs) including diamine oxidases (DAOs) and PA oxidases (PAOs), localized either intercellularly (i.e., apoplast) or intracellularly (i.e., cytoplasm and peroxisomes) (Tiburcio et al., 2014; Gémes et al., 2016). The activities of these two enzymes produce hydrogen peroxide (H2O2), which acts as a signal molecule or an antimicrobial compound involved in the resistance to pathogen attack (Walters, 2003; Moschou et al., 2008).

PAs have been linked to ROS homeostasis, in which PAs act as scavengers of reactive oxygen species (ROS) and activate the antioxidant enzyme machinery (Pottosin et al., 2014). An important rate-limiting step in PA biosynthesis is catalyzed by SAMDC. Cellular accumulation of ROS significantly reduces under drought stress in transgenic *Arabidopsis SAMDC* overexpressor plants exhibiting higher endogenous PAs (Wi et al., 2014). On the other hand, PAs exhibit an inverse relationship with PAOs, which correlate with developmental and stress responses (Paschalidis and Roubelakis-Angelakis, 2005). Furthermore, the respiratory burst oxidase homologs [nicotinamide adenine dinucleotide phosphate (NADPH) oxidase] and the apoplastic PAO form a feedforward ROS amplification loop, which impinges on oxidative state and culminates in the execution of cell damages. This loop is a central hub in the plethora of responses controlling

salt stress tolerance, with potential functions extending beyond stress tolerance (Gémes et al., 2016). Therefore, both functions of PAs are proposed to augment antioxidants for protection against oxygen-radical-mediated damages and are substrates for oxidation reactions that produce H2O2 (Murray-Stewart et al., 2018).

Under physiological or stress conditions, superoxide anions (O2 •−) are generated mainly by NADPH oxidase. Superoxide dismutation by superoxide dismutase is considered one of the major routes for subsequent H2O2 production (Gémes et al., 2016). At low/moderate concentrations, ROS are implicated as second messengers in intracellular signaling cascades that mediate several plant responses in plant cells, including stomatal closure, programmed cell death (PCD), gravitropism, and acquisition of tolerance to both biotic and abiotic stresses such as systemic acquired resistance (Sharma et al., 2012). However, it remains unknown whether the major ROS generators, namely, PAOs and NADPH oxidase, are functionally inked or interplayed. In addition, it remains ambiguous as to which enzyme is more effective in generating ROS under abiotic and biotic stress (Tiburcio et al., 2014).

An imbalance between ROS generation and scavenging often results in oxidative stress, which is a common phenomenon for stress-induced detrimental effects in abiotic stress, which sometimes induces a hypersensitive response against incompatible pathogens (Pottosin et al., 2014). In plants, the relative abundance of the PAs depends on the species, the developmental stages, and environmental conditions (Tiburcio et al., 2014). PAs are detected at relatively high concentrations in actively growing tissues and under conditions of biotic or abiotic stress (Jiménez-Bremont et al., 2014).

Theoretically, under stressed conditions, both phenomena of increased accumulations of PAs and ROS can occur independently in plants. Even if PAs are increased in response to a variety of stresses, the stress-induced ROS generation is more rapid and effectively increased than the antioxidative function of PAs. Although the exact mechanism of action of PAs remains elusive in plants under stressed conditions, their indispensable roles are getting recognized as beneficial SAMDC activity in plants and as differential roles in human health and disease (Handa et al., 2018). It has recently been proposed that high doses of PAs are detrimental to disease conditions involving higher cellular proliferation, although PAs are involved in increased longevity and reducing some age-related diseases. Despite the increasing trend in our understanding regarding effects on health and diagnostic use of PAs in humans, the physiological functions of PAs remain ambiguous, since the functions are often opposed to each other depending on the conditions. In fact, this phenomenon is associated to the strict homeostasis of PAs in living organisms.

Recently, there is an increased focus on the link between metabolism and defense response of major PAs during plant– stress interactions (Seifi and Shelp, 2019). We therefore undertook to investigate the physiological mechanism of PAs on cellular homeostasis in response to salt stress, using transgenic tobacco plants with overexpression of *SAMDC* complementary DNA (cDNA) from the carnation (*Dianthus caryophyllus* L.) flower. Our results provide evidence for the physiological function of PAs required for stress tolerance under high salt condition, *via*  signaling through the intracellular and intercellular ROS levels.

### MATERIALS AND METHODS

#### Chemicals and Reagents

All chemicals procured were either analytical or laboratory grade and were used as received without further purification. Reagent and stock/standard solutions were prepared in deionized water (pH 7.0) purified by Barnstead Water Purification Systems (Thermo Scientific, Waltham, MA, USA). NaCl and sodium carbonate were purchased from Junsei (Japan). Standard PAs, Put, Spd, and Spm were obtained from Sigma-Aldrich, Saint Louis, MO, USA. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid, phenol, chloroform, trimethylamine, perchloric acid, and most other chemicals were obtained from Sigma-Aldrich, Saint Louis, MO, USA. Glycerol and lactic acid were purchased from Biobasic (Canada).

### Plant Materials and Growth Conditions

In our previous study, transgenic tobacco (*Nicotiana tabacum* Wisconsin 38) plants were generated with overexpression construct of the *CaMV 35S* promoter-driven *SAMDC* gene *via Agrobacterium tumefaciens*-mediated gene transfer (Wi et al., 2006). A DNA fragment corresponding to nucleotides 39-1794 of the full-length *SAMDC* cDNA (GenBank accession no. U38527) from carnation (*D. caryophyllus* L.) flowers was included in the 465 bp of 5′-untranslated leader sequence, which included 52 amino acids of the upstream open reading frame (uORF) and 377 amino acids of SAMDC ORF. Although several independent transgenic tobacco lines were obtained from the transgenic experiment, we used the *S16-S-4* transgenic plant (T3 homo line) in this study since the expression trait of the SAMDC gene was characterized to the maximum in our previous studies (Wi et al., 2006). Surface-sterilized seeds were cultured on solid Murashige and Skoog medium (pH 5.8) (Duchefa Biochemi, Netherlands) under light (16L/8D, 100 μmol photons m−2 s−1) at room temperature (25°C). Wild-type (WT) tobacco plants were used as the controls. Fully expanded, green, healthy leaves were plucked, and whole leaves were treated with 200 mM NaCl solution for inducing salt stress. For mock treatment, whole tobacco leaves were floated on 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.1) devoid of any other chemicals.

### RNA Isolation and Real-Time qPCR

Using a High-Fidelity PrimeScript RT-PCR kit (Takara, Japan), total RNA was extracted from whole leaves that had been subjected to salt stress (Wi et al., 2006). Gene-specific PCR primers, sequence information of which was obtained from the GenBank database, were designed using a stringent set of criteria (**Supplemental Table S1**). Real-time quantitative PCR (qPCR) was performed in optical 96-well plates using a Chromo 4 continuous fluorescence detector (Bio-Rad, USA). Reaction mixtures (20 ml) comprised of 10 ml 23 SYBR Green master mix, 0.5 mM of each primer, and 10 ng cDNA. PCR conditions were as follows: 95°C for 15 min; 45 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 30 s; followed by 72°C for 10 min. Fluorescence threshold data were analyzed using the MJ Opticon monitor software version 3.1 (Bio-Rad, USA) and subsequently exported to Microsoft Excel for further analysis. Relative expression levels in each cDNA sample were normalized to the reference gene β-actin. PCR efficiencies (90–95%) for all primers were determined by serial dilution of cDNA from RNA samples.

### Determination of PA Contents by High-Performance Thin-Layer Chromatography

PA levels were assessed as described by Pedrol and Tiburcio (2001). Leaves (0.3 g) were homogenized in 1 ml of 5% (*v*/*v*) perchloric acid and centrifuged at 12,000×*g* for 15 min. To 0.2 ml of the isolated supernatant, we added 0.2 ml of saturated sodium carbonate and 0.4 ml of dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride; Sigma-Aldrich, Saint Louis, MO, USA) (1 mg ml−1 stock solution prepared in acetone), and incubated the mixture overnight at room temperature*.* The dansylated product was extracted with toluene and separated on thin layer chromatography in chloroform/trimethylamine (4:1, *v*/*v*) using a silica gel 60 Å high-performance thin-layer chromatography plate (Merck, USA). The separated PAs were scraped off and quantified against commercial standards using a spectrophotofluorometer (RF-1501, Shimadzu, Japan), where emission was recorded at 495 nm after excitation at 350 nm.

#### Trypan Blue Staining

To monitor plant cell death, tobacco leaves were stained as described previously (Wi et al., 2012). Whole leaves were immersed for 1 min in a boiling solution comprising 10 ml lactic acid, 10 ml glycerol, 10 g phenol, and 0.4% (*w*/*v*) trypan blue (Sigma, USA). After cooling to room temperature for 1 h, the solution was replaced with 70% (*w*/*v*) chloral hydrate. Stained plants were decolorized overnight and photographed using a digital camera.

#### Histochemical Detection of Hydrogen Peroxide and Superoxide Anion

The accumulation of superoxide (O2 •**−**) anion and hydrogen peroxide (H2O2) was histochemically assessed by nitroblue tetrazolium (NBT) (Biobasic, Canada) and 3,3′-diaminobenzidine (DAB) (Sigma-Aldrich, Saint Louis, MO, USA) staining (Kumar et al., 2013). For detection of superoxide, the leaves were floated in 50 mM potassium phosphate (pH 7.8) containing 0.2% NBT for 2 h at 25°C, leading to the formation of the dark blue insoluble formazan compound. For the detection of hydrogen peroxide, the leaves were immersed for 2 h in a solution of DAB (1 mg ml−1, pH3.8) at 25°C. Thereafter, chlorophyll was removed by boiling in 96% (*v*/*v*) ethanol for 10 min. After complete removal of chlorophyll, the stained leaves were photographed using a digital camera. H2O2 was visualized as a reddish-brown stain formed by the reaction of DAB with endogenous H2O2. The O2 •**−** content was detected as dark blue stain of formazan compound formed as a result of NBT reacting with the endogenous O2 •**−**.

#### Measurement of ROS With 2**′**,7**′**-Dichlorodihydrofluorescein Diacetate

For histochemical staining of total ROS level, leaf epidermal strips were peeled from tobacco leaves subjected to stress treatment for the indicated time. Leaf epidermal strips were floated for 10 min on a solution of 50 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Invitrogen, USA) prepared in 20 mM potassium phosphate buffer (pH 6.0). Mechanistically, DCFH-DA can be transported across the cell membrane and deacetylated by esterase to form nonfluorescent DCFH. This compound is then trapped inside the cells and is subsequently converted into the highly fluorescent compound (DCF) through the action of ROS and peroxidase, which can be detected and quantified based on fluorescence intensity. ROS was observed by fluorescence microscopy (excitation: 450 ± 490 nm; barrier 520 ± 560 nm) equipped with a cooled charge-coupled device camera (Olympus, FV300, Japan). Fluorescence intensity of histochemical staining from each photograph was quantified by densitometry in ImageJ.

#### Detection of Intra- and Extracellular ROS in Guard Cells

For fluorescent detection of ROS, we used the lower leaf epidermal strips. For detecting the intra- and extracellular superoxide anions, benzene sulfonyl (BES)-So-Am and BES-So (WAKO Chemicals, Japan), respectively, were used at a concentration of 20 mM, prepared in potassium phosphate buffer (pH 6). For detecting intra- and extracellular hydrogen peroxide, BES-H2O2-Ac and BES-H2O2 (WAKO Chemicals, Japan), respectively, were used at a concentration of 50 mM in 20 mM potassium phosphate buffer (pH 6). After incubation of epidermal tissues with ROS/superoxide detection solution at room temperature in the dark for 1 h, fluorescence was observed using the confocal laser scanning microscope FluoView 300 (FV 300; Olympus, Japan). Fluorescence intensity for superoxide detection was observed in the dark with excitation at 485 nm and emission at 530 nm. Fluorescence intensity for hydrogen peroxide was observed with excitation at 505 nm and emission at 544 nm.

#### Oxidized Protein Analysis

Oxidized proteins were detected using an OxyBlot protein oxidation detection kit (Merck Millipore, USA), according to the manufacturer's instructions. Dinitrophenyl hydrazine was added to crude total proteins (10 μg) extracted from tobacco leaves post-NaCl treatment to derive carbonyl groups from the protein side chains. Carbonylated proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis was performed using the provided 2,4-DNP antibody (1:150). The DNP signals in integrated intensity of each fraction were quantified by densitometry in ImageJ and normalized to the total protein value of the WT 0 h control, which was set as 1.

#### Analysis of Chaperone Activity

The chaperone activity of Spd was assayed by measuring its capacity to suppress thermal aggregation of malate dehydrogenase (MDH) (Sigma-Aldrich, Saint Louis, MO, USA) from malic dehydrogenase, using porcine heart (Sigma-Aldrich, Saint Louis, MO, USA) as a model substrate (Jung et al., 2015). MDH (1 μM) was incubated in 50 mM 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid-KOH (pH 8.0) buffer with total proteins extracted from salt-stressed plants after addition of 0.2 mM Spd. Aggregation of the substrate was monitored under heat denaturation at 45°C for 30min, by measuring the turbidity at 340nm using a Shimadzu UV-1601 spectrophotometer (Shimadzu, Japan).

#### Quantitation and Statistical Analysis

All experiments were repeated at least three times with three independent biological replicates. The photographs represented are from one representative experiment in more than three independent biological experiments with more than three replicates after verifying the reproducibility of the results (**Figures 2A**, **3A**–**C**, **4A**, **B**, **5A**, **B**, **7A**, and **8A**, **B**). Statistically significant differences using Student's *t* test (Microsoft Excel) between transgenic lines and respective controls at each time point are indicated with one asterisk (\*) (*P* < 0.05) or two asterisks (\*\*) (*P* < 0.01). A two-way analysis of variance (ANOVA) is performed to determine significant differences between NaCl and PAs treatment (*P* < 0.001) using GraphPad Prism (GraphPad, San Diego, CA, USA). Evaluation of differences between two groups are evaluated using Student's *t* test of GraphPad Prism.

#### RESULTS

#### Enhancement of PA Biosynthesis Is Induced in Transgenic Tobacco Plants

The full-length of tobacco *SAMDC16* cDNA clone (GenBank accession no. U38527) was overexpressed with the *35S CaMV* promoter-driven construct. We originally isolated two *SAMDC* cDNA clones, *SAMDC9* and *SAMDC16*, from the petals of carnation flowers (Lee et al., 1997). Since the *SAMDC16* clone is more effectively expressed in the leaves of carnation plants, we used *SAMDC16* cDNA for making the transgenic plants (Wi et al., 2006).

Transgenic plants overexpress the full-length *SAMDC16* cDNA fragment that contains an uORF of 52 amino acids located at 5′-untranslated region. It is previously reported that the *SAMDC uORF* sequence or SAMDC uORF protein is a translational inhibitor of its own downstream ORF, which is responsible for the homeostatic regulation of PAs even under stressed conditions (Hanfrey et al., 2003; Hu et al., 2005; Choi and Park, 2011). Therefore, we used the *SAMDC* gene with *uORF* region for developing the transgenic plant in our attempt to overexpress the *SAMDC* gene without it being affected by the removal of *SAMDC* uORF.

After confirming *SAMDC* gene integration in kanamycinresistant transgenic lines by Southern blot analysis and Northern blot analysis, four transgenic lines were finally selected. SAMDC activity in three transgenic lines (*S16-S-1*, *S16-S-2*, and *S16- S-4*) was significantly increased as compared to the WT (**Supplementary Material Figure S1**); the *S16-S-4* transgenic line had the highest SAMDC activity (increased by 70%), compared to the WT control.

Within 3–4 months, the four transgenic plants produced flowers and seeds from self-fertilization. The seeds produced from each flower of transgenic plants were counted. Seed mass was increased by 1.6 times in all transgenic lines, as compared to the nontransformed WT plants (Wi et al., 2006). However, the transgenic plants with empty vector control (*pBI121*) produced almost the same number of seeds as the WT tobacco. Based on these results, the *S16-S-4* transgenic line was selected for further studies.

In our experiments, the major PAs such as Put, Spd, and Spm were significantly increased in the leaves of transgenic *S16-S-4* under unstressed condition, having the most effective increase in Spd levels (2.3-fold), as compared to the WT (**Figure 1A**). In addition, the most abundant PA in transgenic plants was Spd. After being subjected to salt stress, we observed a rapid increase in the amount of *SAMDC* transcript, which in turn may result in increased PA biosynthesis. Transcript levels of *SAMDC16* were

significantly increased in WT and transgenic tobacco leaves in response to salt stress with 200 mM NaCl, resulting in elevated *SAMDC* transcription. This was more significant in stressed tobacco T3 transgenic homozygous lines (*S16-S-4*) as compared to stressed WT plants (**Figure 1B**). Although *SAMDC16* was driven by constitutive *35S CaMV* promoter in these transgenic plants, the transcripts of *SAMDC16* increased by only ~1.5 fold transcripts when compared to WT plants during the entire period of high salinity.

In addition, induction of transcription of *PAO* rapidly increased from 1 h after salt stress (**Figure 1C**), which contributed to the degradation of the PAs. The induction of *PAO* transcripts was much higher in transgenic plants as compared to WT. Stress-induced increase in transcriptions of PA biosynthetic gene and catalytic gene were transient in both the WT and transgenic plants, which peaked at 12 and 1 h, respectively, after salt stress (**Figures 1B**, **C**).

Therefore, we next examined for alterations of endogenous PA levels of tobacco leaves in transgenic plants under salt stress. Following salt stress, levels of the major PAs (Put, Spd, and Spm) were found to be considerably higher in *SAMDC*overexpressing transgenic plants as compared to WT plants (**Figure 1D**). These increases in Spd and Spm levels were mainly due to overexpression of the *SAMDC* transgene. Furthermore, the levels of Put, a precursor of these PAs, were also increased and were found to be higher in transgenic plants than in WT during the entire period of salt stress. Increase in the amount of PAs due to high salinity stress was highest at 12 h after salt stress but decreased to almost initial levels after 24 h. These phenomena indicate the tight regulation of PA biosynthesis for homeostasis, even under abiotic stress conditions.

#### Enhanced Expression of *SAMDC16* Induces a Tolerance in Response to Salt Stress

To elucidate the physiological functions of PAs in response to salt stress, we first compared stress-induced damages between WT and PA-overproducing transgenic plants. We observed that leaves of *S16-S-4* transgenic plants showed greater tolerance to salt stress when compared to WT and vector control transgenic plants, as determined by trypan blue staining for cell death (**Figure 2A**) and attenuation in stress-induced chlorophyll degradation (**Figure 2B**). Furthermore, the maximal protective effects of *SAMDC* overexpression appeared after 2 days of salt stress, as determined by chlorophyll degradation.

The effects of increased PAs in the *S16-S-4* line were examined on the expression of metacaspase type II gene (*MCP2*), which plays a positive role in biotic and abiotic-induced PCD (Watanabe and Lam, 2011). Upon salt stress, the transcription levels of *MCP2* rapidly increased in both WT and *S16-S-4* transgenic plants, showing lower levels in *S16-S-4* as compared to WT (**Figure 2C**). Our findings show that upregulation of *MCP2* expression under salt stress is significantly attenuated in transgenic plants. Taken together, we believe that enhancement of endogenous PAs by overexpression of *SAMDC* gene induces stress tolerance based on cell damages under conditions of high salinity.

#### Downregulation of ROS Accumulation by PAs Under High Salinity

To investigate ROS generation in whole leaves of WT and transgenic tobacco plants following induction of high salinity with 200 mM NaCl, we histochemically monitored two important ROS, superoxide anion and hydrogen peroxide, using NBT and DAB as chromogenic substrates, respectively. NBT reacts with O2 •**−** to form a dark blue insoluble formazan compound, whereas DAB is oxidized by hydrogen peroxide to produce a reddishbrown precipitate (Kumar et al., 2013). Both ROS were detected in the leaves of WT from 1 h after salt stress, following which the levels were significantly increased until 24 h after salt stress (**Figures 3A**, **B**). However, both the stress-induced ROS were significantly downregulated in transgenic plants as compared to WT, suggesting that the upregulated PAs in transgenic tobacco plants inhibit ROS accumulation under salt stress. These results indicate that PAs have significant roles as antioxidative agents rather than acting as a source of hydrogen peroxide through PA degradation.

Next, by applying the DCFHDA histochemical assay, we analyzed ROS accumulation at the cellular level in response to salt stress. Since guard cells have been used as a well-developed single-cell model system and are particularly useful for the study of ROS signaling (Qi et al., 2017), we examined the histochemical pattern of ROS produced by the guard cells. Our data reveal that ROS are rapidly and transiently generated in guard cells in response to salt stress and significantly downregulated in transgenic plants of *S16-S-4* as compared to WT (**Figure 3C**). No differences were observed in rapid ROS accumulation in the WT and vector control transgenic plants under salt stress (**Supplementary Material Figure S2**). Surprisingly, only very low levels of ROS were produced in guard cells of *S16-S-4*, compared to WT. These results imply that stress-induced ROS generation is effectively inhibited by upregulated PA levels by overexpression of the *SAMDC* gene.

In aerobic organisms, ROS are produced during normal cellular metabolism as by-products of metabolic pathways and electron flows in both mitochondria and chloroplasts. RboH, called neutrophil NADPH oxidase, is a transmembrane protein that generates superoxide radicals in plant cells. Its isoforms, NtRbohD and NtRbohF, are expressed in all tobacco plants and produce the superoxide anion, which is unable to permeate cell membranes under ambient pH conditions due to the presence of a negative charge. The highly reactive superoxide anion is catalyzed to O2 and H2O2 either spontaneously or by superoxide dismutase (SOD) (Libik-Konieczny et al., 2015). H2O2 is less reactive than O2 •**−** but is more stable and can diffuse through membranes *via*  aquaporins (Bienert et al., 2007). Thus, H2O2 is recognized as the most potent signaling ROS in plants. We therefore determined gene expression profiles of tobacco plants in the context of ROS, including H2O2, following salt stress.

Transcription levels of the two *NtRboh* genes were evaluated in tobacco leaves using real-time qPCR analysis. Transcription of *NtRbohD* and *NtRbohF* occurred in a time-dependent manner, which were significantly downregulated in leaves of transgenic plants during the entire period of stress treatment, as compared

were generated from one representative experiment with three independent biological replicates after verifying the reproducibility of the results in three experiments. An asterisk indicates a significant difference between WT and transgenic plants (\*\**P* < 0.01).

to WT (**Figures 3E**, **F**). These results suggest that PAs inhibit ROS generation by downregulating the expression of both *NtRbohD* and *NtRbohF* genes during stress responses. Therefore, our findings confirm that increased PAs inhibit the generation and accumulation of total ROS.

We further determined the accumulation of each superoxide anion and hydrogen peroxide in the subcellular regions using specific fluorescent dyes. Intracellular generation of O2 •− was detected using BES-So-AM, a highly specific fluorescent probe (Park and Roubelakis-Angelakis, 2018). Under control conditions, no significant accumulation of O2 •− was detected in the guard cells of WT and *S16-S-4* (**Figure 4A**, 0 h). After 1 and 6 h of salt stress, the levels of intracellular O2 •− were increased significantly in guard cells of WT, with ROS accumulation in the chloroplasts of guard cells, and a very large amount of ROS accumulation especially in the nucleus. At 6 h after stress treatment, the accumulated ROS in the chloroplast were considerably small but still very large in the guard cell nuclei of WT tobacco leaves. However, the accumulation of intracellular O2 •− was dramatically reduced in the chloroplasts and nucleus of guard cells of the *S16-S-4* transgenic plants (**Figure 4A**).

Next, extracellular O2 •− levels were detected using BES-So. Similar to the intracellular O2 •−, no significant accumulation of fluorescent BES-So was detected under control conditions in the guard cells of WT and *S16-S-4* transgenic plants (**Figures 4A, C**, 0h). Although extracellular ROS levels were observed somewhat after 1 h, increased levels were observed after 6 h on the exterior of guard cells in WT under salt stress condition (**Figures 4B**, **D**, 1 and 6 h). In addition, a higher amount of BES-So fluorescence was observed in the stomatal pores of WT tobacco leaves under salt stress, indicating that stress-induced superoxide anions were localized outside the guard cells, since its anionic property prevented entry

FIGURE 3 | Kinetics of ROS production in response to salt stress. (A) Superoxide radical (sporadic O2 •− accumulation in leaves was detected after NBT staining in WT and transgenic plants with or without 200 mM NaCl. (B) Sporadic accumulation of H2O2 was determined by 3,3′-diaminobenzidine (DAB) staining in leaves from WT and transgenic plants after administering 200 mM NaCl. (C, D) Histochemical analysis of cellular reactive oxygen species (ROS) accumulation in response to salt stress. ROS accumulation was determined by incubation with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 10 min. Staining images of leaves were obtained by confocal microscopy (C) and quantified by ImageJ software (D). (E, F) Relative mRNA levels of *NtRbohD* and *NtRbohF* genes after salt stress. Transcription levels of *NtRbohD* or *NtRbohF* are expressed as means ± SD. Transcription levels are expressed relative to the reference gene β*-actin* after qPCR. The photographs represented (A–C) are from one representative experiment after verifying the reproducibility of the results in three independent experiments with three leaves. Data were generated from one representative experiment with ten guard cells (D) and three independent biological replicates (E, F). An asterisk indicates a significant difference between WT and transgenic plants (\**P* < 0.05; \*\**P* < 0.01).

•− (C) and extracellular O2 by ImageJ. The photographs represented (A, B) are from one representative experiment with three leaves after verifying the reproducibility of the results in three experiments. Data (C, D) were generated from 10 cells in one representative experiment after verifying the reproducibility of the results in three experiments. An asterisk indicates a significant difference between WT and transgenic plants (\*\**P* < 0.01).

into the cells. These results suggest that increased levels of PAs are responsible for significant reduction in intra- and extracellular accumulation of superoxide anion in response to salt stress. Differences between WT and transgenic plants were significantly greater in the late phase under salt stress, implying that endogenous PAs might have more important effects on the later stage in response to salt stress.

We then determined the intra- and extracellular H2O2 accumulations after salt stress. Since DCF and DAB are highly sensitive to peroxidase (Noctor et al., 2016), we employed a peroxidase-independent method for the estimation of H2O2 levels. We used the highly specific BES-H2O2 and BES-H2O2-Ac probes to estimate intra- and extracellular H2O2 levels, respectively (**Figure 5**). Higher levels of intra- and extracellular H2O2 accumulation were observed in the guard cells of WT than in corresponding *S16-S-4* plant cells under salt stress (**Figure 5A**). Intra- and extracellular H2O2 remained at significant levels after 6 h of salt stress in leaf cells of WT. However, H2O2 accumulation was only marginally detected by BES-H2O2 and BES-H2O2-Ac in the *S16-S-4* transgenic plants under salt stress (**Figures 5A**, **B**). Our results additionally imply that BES-H2O2 and BES-H2O2-Ac enter the guard cells under salt stress conditions.

#### Downregulation in the Expression of ROS-Detoxifying Enzymes by PAs Under Salt Stress

We next investigated whether PAs contribute to the expression of ROS-detoxifying enzymes in stress-induced ROS accumulation. An enzymatic dismutation reaction converts superoxide into a more stable, membrane-permeable H2O2 derivative, which is required for cell-to-cell signaling. ROS-scavenging enzymes such as SOD, ascorbate peroxide (APX), and catalase (CAT) provide the cells with a highly efficient machinery for detoxifying superoxide and H2O2 (Foyer and Noctor, 2005).

The level of ROS is tightly regulated by enzymes involved in the ROS-detoxifying pathways, including mitochondrial manganese-SOD (MnSODmi), cytosolic copper/zinc SOD (CuZnSODc), cytosolic APX (APXc), CAT (CAT1 and CAT2), and phi glutathione-S-transferase (GSTF). In this study, *CAT1*

determined using confocal scanning microscopy followed by incubation with BES-H2O2-Ac. Images of H2O2 stained with BES-So-Am (green) and chlorophyll autofluorescence (red). (B) Extracellular hydrogen peroxide was determined using confocal scanning microscopy by incubation with BES-H2O2. Images of the H2O2 stained with BES-H2O2 (green) and chlorophyll autofluorescence (red). (C, D) Green fluorescence signals for intracellular H2O2 (C) and extracellular H2O2 (D) were quantified by ImageJ. The photographs represented (A, B) are from one representative experiment with three leaves after verifying the reproducibility of the results in three experiments. Data (C, D) were generated from 10 cells in one representative experiment after verifying the reproducibility of the results in three experiments. An asterisk indicates a significant difference between WT and transgenic plants (\*\**P* < 0.01).

and *CAT2* expressions were induced biphasically, which peaked at 6 h, after which they decreased, but again showed a high level at 24 h (**Figure 6**, *CAT1* and *CAT2*). On the other hand, expressions of other ROS-detoxifying enzymes in response to salt stress were significantly induced monophasically. Salt stress upregulated the transcription of *SODs*, *MnSOMmi*, and *CuZnSODc*, reaching maximum peaks at 24 and 12 h, respectively, with accompanying elevation of *APXc* transcription peaking at 6 h for detoxification of H2O2. Plant GSTs are known to induce tolerance to abiotic stresses due to their ability to regulate specific redox signaling pathways responsible for activating defense gene transcription (Cummins et al., 2011). Expression of GSTF, a plant-specific phi class of stress-induced GST, reached a peaked at 12 h under salt stress. Taken together, these results indicate that PAs activate the ROS detoxification pathway, thereby lowering ROS accumulation and resulting in the prevention of severe stressinduced cell damages.

### Downregulation of Oxidized Proteins by PAs Under Salt Stress

Oxidized proteins were detected using an OxyBlot protein oxidation detection kit (Merck Millipore, USA), according to the manufacturer's instructions. After dinitrophenyl hydrazine was added to crude total proteins to derive carbonyl groups from the protein side chains, Western blot analysis was performed using the provided 2,4-DNP antibody (1:150). DNP signals in integrated intensity of each fraction were quantified by densitometry in ImageJ and normalized to the total protein value of the WT 0 h control, which was set as 1.

We observed that, under unstressed condition, the amount of oxidized proteins was much higher in WT than in transgenic plants. In addition, salt stress caused a rapid increase in the amount of oxidized protein, which was higher in leaves of WT as compared to transgenic plants (**Figure 7**). These results indicate that the upregulation of endogenous PAs in transgenic tobacco

the results in three experiments. An asterisk indicates a significant difference between WT and transgenic plants (\**P* < 0.05; \*\**P* < 0.01).

plants might show antioxidative effects for protein homeostasis against stress-induced protein oxidation.

#### Exogenous Addition of PA Downregulates ROS Accumulation and Upregulates the Chaperone Activity Induced by Salt Stress

Abiotic stresses such as salt stress usually cause the accumulation of not only oxidized proteins but also protein aggregation (Luo et al., 2017). Molecular chaperones are key components contributing to the homeostasis and the quality control of proteins under stress conditions (Wang et al., 2004). In the current study, MDH protein alone as the target protein showed substantial aggregation after 6 min at 45°C, whereas proteins purified from the leaves after cotreatment with salt stress and PAs for 6 h induced a marked reduction in light scattering, indicating that PAs prevent the heat-induced aggregation of cellular target proteins and enhance the chaperone activity of cellular proteins under salt stress (**Figure 9**). Two-way ANOVA found statistically significant effects of PAs for chaperone activity [*F*(60, 156) = 2.13, *P* < 0.0001] compared to mock- and NaCl-treated conditions. Spd and Spm show similar induction of chaperone activity, and their effect was significantly higher than that exerted by Put.

#### DISCUSSION

PAs are small ubiquitous polycations involved in numerous processes of plant growth and development and are well known for their antisenescence and antistress effects due to their acid neutralizing and antioxidant properties, as well as for their membrane and cell wall stabilizing abilities (Gill and Tuteja, 2010; Tiburcio et al., 2014). Engineered PA accumulation by the expression of the yeast SAMdc enhances phytonutrient content, juice quality, and vine life in tomato, which supplied the direct evidence for a physiological role of PAs and demonstrates an approach to improving nutritional quality (Mehta et al., 2002). Mattoo et al. (2007) suggested that higher levels of Spd and Spm in the tomato fruit causes a marked shift for antisenescence in gene expression, especially for genes involved in metabolism, signal transduction, and defense/stress responses. It has been suggested that Put plays more active roles against pathogen attacks (Rodrıguez-Kessler et al., 2008), whereas Spd and Spm modulate the defense response of plants to numerous environmental stresses including metal toxicity, oxidative stress, drought, salinity, and chilling stress (Liu et al., 2000). Handa and Mattoo (2010) proposed that due to the chemical attributes of PAs and their derivatives/conjugates, they interact differentially with cellular components including chromatin, transcriptional

expressed as means ± SD. The photographs represented (A) are from one representative experiment in four independent biological experiments after verifying the reproducibility of the results. Data (B) were generated from four independent biological replicates. An asterisk indicates a significant difference between WT and transgenic plants (\*\**P* < 0.01).

machinery, translational machinery, and macromolecules, and result in modified metabolic profiles. However, little is understood regarding the detailed physiological mechanism of elevated PA levels in response to abiotic and biotic stresses.

The PA-derived H2O2 by PAO triggers signal transduction pathways causing defense gene expression and stress tolerance in plants, particularly due to the potential of H2O2 to act as a secondary messenger and a signaling molecule (Tavladoraki et al., 2012; Handa et al., 2018). However, it is possible that PAO is involved in the back-conversion reactions during PA catabolism, in which Spm is oxidized to Spd, and Spd is further oxidized to Put (Cona et al., 2006; Tavladoraki et al., 2012).

As previously reported, there exists a feedforward amplification loop between the apoplastic PAO and the plasma membrane NADPH oxidase for controlling ROS accumulation in response to abiotic stress (Gémes et al., 2016). This model suggests that the

(D) Oxidation index (integrated density/area) was quantified with Oxyblot by ImageJ. The photographs represented (A) and data (B) were generated from 10 cells in one representative experiment after verifying the reproducibility of the results at three experiments. The photograph (C) are from one representative experiment after verifying the reproducibility of the results in four experiments. Data (D) were expressed as means ± SD from four independent experiments. An asterisk indicates a significant difference between WT and transgenic plants (\**P* < 0.05; \*\**P* < 0.01).

apoplastic PAO feeds a stress-inducible ROS amplification loop that can lead to tolerance responses during stresses. Although it is hypothesized that PAO-related ROS triggers further cascade of ROS signaling for stress tolerance, apoplastic ROS was not detected in our histochemical experiments in *S16-S-4* transgenic plants. The temporal differences between transcript levels of *PAO* and *SAMDC16* indicate that the early increase in *PAO* is not induced due to the catabolism of stress-induced PAs. We therefore hypothesize that the catabolism of PA occurs independently from stress-induced PA accumulation.

The proposed role of PAs is for mediating stress responses through redox homeostasis as antioxidative molecules (Saha

treatment under salt stress. Light scattering of total proteins was determined using a model substrate MDH (1 μM) under thermal denaturing conditions (45°C) for 30 min. Data were expressed as means ± SD from three independent biological replicates. The statistical analysis was performed with two-way ANOVA using GraphPad Prism, comparing NaCl vs. NaCl + Put, NaCl vs. NaCl vs. NaCl + Spd, or NaCl vs. NaCl + Spm.  One possible conjecture is that the PA molecules themselves induce stress-induced ROS detoxification and/or encompass an antioxidative property. The remnant extracellular superoxide anion is thought to cause serious damage by salt stress. However, the accumulation of extracellular superoxide anion is dramatically inhibited in *S16-S-4* under conditions of salt stress. These data indicate that under salt stress, PAs are bestowed with tolerance by the removal of extracellular superoxide anions.

Although NADPH oxidase plays a role in the production of extracellular superoxide anions, various mechanisms are involved in the production of different ROS, including H2O2. Transcript accumulation of *NtRbohD* and *NtRbohF* was significantly inhibited in *S16-S-4* transgenic plants and by exogenously added PAs, indicating that ROS production is specifically inhibited by the increase in cellular PAs in transgenic plants. Even 1 h after salt stress treatment, in which *PAO* transcripts exhibited a maximum value, only a low level of apoplastic H2O2 was observed in *S16-S-4*. Therefore, stress-induced *PAO* transcripts do not seem to contribute significantly to induce extracellular H2O2 accumulation in transgenic plants.

We also observed that superoxide anions and hydrogen peroxide prominently accumulate in the nucleus after salt stress. Intracellular ROS are mainly produced in chloroplasts, endoplasmic reticulum, and peroxisomes and, to a lesser extent, in mitochondria in plants (Sharma et al., 2012; Qi et al., 2017). It is yet unknown how ROS accumulates in the nucleus of plant cells. Recently, however, the isoform of NADPH oxidase, which produces ROS in the nucleus, has been reported in animal cells including human vascular epithelial cells (Kuroda et al., 2005; Weyemi et al., 2012). In plant cells, it is suggested that ROS produced in the chloroplast or peroxisome may be transferred into the nucleus (Shapiguzov et al., 2012). On the other hand, it is impossible to rule out the possibility that ROS are produced directly in the nucleus. The fact that nuclear ROS is significantly downregulated by PAs implies that PAs are effective on the nuclear gene expression for stress tolerance in a redoxdependent manner.

Stress-induced ROS burst and oxidative stress drive the protein oxidation and result in cellular toxicity that culminates in cell death (Sharma et al., 2012). The effects of PAs imparting antioxidative properties and antiaggregation of proteins contribute towards maintaining physiological cellular functions against abiotic stresses. It is suggested that these functions of PA are advantageous for protein homeostasis during abiotic stresses. Transcriptome analysis in transgenic fruit that accumulates higher PAs revealed upregulation of chaperones transcripts as compared to WT control (Mattoo et al., 2007). Interestingly, recent reports indicate that high concentrations of Spm (2.5 mM) and Spd (5 mM) exhibit chaperone-like activity against thermal and oxidative stress when incubated with bovine seminal plasma proteins (Singh et al., 2017).

The cytotoxicity of PAs through their oxidation resulted in PCD, which triggers the caspase-3 activity in leukemia cells (Stefanelli et al., 1999). In some cases, analogues of Spm are known to induce PCD in animal systems by activation of PA degradation and subsequent generation of H2O2 (Walters, 2003). Only transient enhancement of PA levels was observed in both WT and transgenic plants, which induce tolerant responses under salt stress condition. Taken together, these results indicate that the amounts of increased PA under salt stress were below the threshold levels of toxicity.

Increased resistance to stress correlates to extended leaf longevity and the overall life span (Sharabi-Schwager et al., 2009). They reported that older leaves had lower levels of antioxidants and persistently lower activities of the antioxidative enzymes in tobacco plants. These changes resulted in lower stress tolerance, with consequent acceleration of leaf senescence. Our current research supports the inference that increased resistance to salt stress results in attenuation of leaf senescence in transgenic plants. Taken together, our findings are consistent with the known role of Spd and Spm as antisenescence effectors (Mattoo et al., 2007). The next study will be towards understanding the roles of PAs and ROS signals with ROS generation and detoxification,

### REFERENCES

antioxidative machinery, and molecular chaperone activity for the fine orchestration of stress tolerance.

### DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

### AUTHOR CONTRIBUTIONS

KYP designed the experiments, analyzed the data, and wrote the manuscript. SYS and YJK executed all experiments and data analysis.

### FUNDING

This research was supported by funding from National Research Foundation of Korea (Project No. NRF-2017R1D1A3B03034134) to KYP.

### ACKNOWLEDGMENTS

The authors thank Prof. K.A. Roubelakis-Angelakis for giving helpful advice on interpreting the data.

### SUPPLEMENTARY MATERIALS

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.01331/ full#supplementary-material

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2019 Seo, Kim and Park. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Differential Nitrogen Nutrition Modifies Polyamines and the Amino-Acid Profile of Sweet Pepper Under Salinity Stress

M. C. Piñero1 , Manuel E. Porras2 , Josefa López-Marín1 , Mari C. Sánchez-Guerrero2 , Evangelina Medrano2 , Pilar Lorenzo2 and Francisco M. del Amor1 \*

1 Department of Crop Production and Agri-Tecnology, Murcia Institute of Agri-Food Research and Development, Murcia, Spain, 2 Agricultural Research and Development Centre of Almería (IFAPA), Almería, Spain

#### Edited by:

Ana Margarida Fortes, Universidade de Lisboa, Portugal

#### Reviewed by:

Christine Becker, Hochschule Geisenheim University, Germany Oscar A. Ruiz, CONICET Institute of Biotechnological Research (IIB-INTECH), Argentina

#### \*Correspondence:

Francisco M. del Amor<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Metabolism and Chemodiversity, a section of the journal Frontiers in Plant Science

Received: 29 October 2018 Accepted: 25 February 2019 Published: 02 April 2019

Citation:

Piñero MC, Porras ME, López-Marín J, Sánchez-Guerrero MC, Medrano E, Lorenzo P and del Amor FM (2019) Differential Nitrogen Nutrition Modifies Polyamines and the Amino-Acid Profile of Sweet Pepper Under Salinity Stress. Front. Plant Sci. 10:301. doi: 10.3389/fpls.2019.00301 The horticultural industry demands high-quality resources to achieve excellence in yield and optimal revenues. Nitrogen is a pivotal nutrient to accomplish these goals for plant growth and product quality. However, competition for water in semi-arid regions can force the use of brackish waters, which can impair N uptake. The lower N uptake can be due to several reasons, such as an antagonism between ions, an absence of ATP, and/or alteration of N metabolism. The effect of supplying N as NO− 3 alone or in combination with NH+ 4 , coupled with low or high salinity (8 or 20 mM NaCl), has been studied in sweet pepper fruits (Capsicum annuum L. cv. Melchor). The application of NH+ 4 at high salinity affected chromatic parameters (a∗ , b∗ , and C∗ ), while chlorophyll a and b levels declined and β-carotene increased. The concentrations of P, K, Ca, Mg, and Cu were reduced in the fruits of plants irrigated with NH+ 4 . The concentration of Na was only reduced when NH+ 4 was supplied. Likewise, the concentration of total phenolics was also reduced at high salinity. However, total protein was unaffected. The amino acid profile was altered by the supply of NH+ 4 , which reduced the concentrations of histidine and phenylalanine. Moreover, the concentrations of putrescine and cadaverine were increased by NH+ 4 at high salinity, whereas that of cadaverine was reduced by NH+ 4 at low salinity. The observed changes in fruit quality triggered by salinity, under the conditions of this study, should be borne in mind for this crop with regard to the envisaged palliative effect of the supply of N-NH+ 4 .

Keywords: nitrogen, salinity, Capsicum annuum L., nutrients, free amino acids, polyamines

## INTRODUCTION

Consumers are increasingly demanding high-quality products with benefits for human health (Bortolin et al., 2016). However, the agricultural industry usually prioritizes the maximization of yield and quality without investing in the improvement of plant tolerance of potentially harmful abiotic stress factors, which frequently impair crop yields. The current climate drift that presumably will increase the frequency and severity of dry heatwaves is now a source of concern to many growers worldwide, but especially in Mediterranean-climate areas (Surasinghe, 2011).

Therefore, new, efficient, and effective ameliorative tools are necessary, to reduce the impacts and to maintain ecosystem biodiversity, sustainability, economic growth, food security, and social equity (Dhankher and Foyer, 2018). The competition for good-quality water, which is forcing farmers to use brackish waters for irrigation, could be alleviated by the use of salt-tolerant crops or appropriate management (fertirrigation) practices (Piñero et al., 2018). Salinity is one of the main stressors limiting plant development and crop productivity. Munns and Tester (2008) observed that under salinity stress the plant N content declines while the leaf Cl− content increases. However, the negative effects of salinity could, in part, be compensated by an appropriate, balanced N supply. Some studies indicated that the supply of NO− 3 as the sole N source may be detrimental under salt stress, since salinity reduces its uptake rate (Kant et al., 2007) and decreases the plant N content (Piñero et al., 2016). Several reasons have been put forward for the decrease in leaf N content under salinity: (i) an antagonism between Cl− and NO− 3 transport and/or inactivation of NO− 3 transporters by the toxic effects of salinity; (ii) an absence of ATP, which is required for active NO− 3 transport (Piñero et al., 2016); and (iii) an alteration in the activities of enzymes involved in N metabolism induced by salt (Srivastava and Mishra, 2014). The supply of NH+ 4 has an advantage, related to the lower energy cost of its uptake, compared with NO− 3 (Fernández-Crespo et al., 2012), but it can induce cell acidification, nutrient deficiencies, and inhibition of root growth (Sarasketa et al., 2016). Moreover, NH+ 4 has also been associated with amino acids that can act as signaling molecules to trigger metabolic pathways that could limit oxidative damage (Hessini et al., 2013), such as that provoked by salinity stress. Consequently, the growth response of plants to N fertilization under salinity stress varies depending on whether N is supplied as NO− 3 or NH+ 4 , as well as on the species considered (Misra and Dwivedi, 1990).

Sweet pepper (Capsicum annuum L.) is one of the most valuable crops in the Mediterranean area and is usually cultivated in greenhouses, which allow higher yield and exceptional fruit quality in comparison with open field (conventional cultivation) conditions (Serret et al., 2018). The fruit contains a large number of health-promoting compounds such as vitamins, carotenoids, lycopene, capsaicinoids, phenolic compounds, and amino acids, all of which have antioxidant properties and provide protection against cancer (Bramley, 2000; Navarro et al., 2006; González-Chavira et al., 2018). Moreover, the synthesis of phenolic compounds, vitamin C, and carotenoids in pepper and other vegetables depends on several factors, such as the cultivar, agricultural practices, maturity, and storage conditions. It has been observed that the availability of N has the ability to modify the synthesis of phenolic compounds and soluble solids (Doll et al., 1994; González-Chavira et al., 2018).

Polyamines, also known as biogenic amines, are low molecular weight compounds involved in plant responses to abiotic and biotic stresses (Hussain et al., 2011), as well as in the processes of cell growth and differentiation (Regla-Márquez et al., 2016). They are also linked to molecular plant defense mechanisms involved in the scavenging and/or generation of free radicals, regulation of gene expression, and formation of toxic defense products (Cai et al., 2015). Moreover, polyamines are also related to the hormonal balance (Houdusse et al., 2008) and the direct effect of climate change, as influenced by CO2 concentrations (Piñero et al., 2017).

In this study, we hypothesized that the additional supply of NH+ 4 to the nutrient solution (instead of using NO− 3 as the sole N source) might partially or totally overcome the predicted deleterious effect of salinity on the fruit quality of sweet pepper, as this crop is considered salt sensitive (del Amor and Cuadra-Crespo, 2011; Piñero et al., 2014). Most plants show a strong preference for NO− 3 over NH+ 4 ions, and others grow best if they have access to both NO− 3 and NH+ 4 (Zhou et al., 2011). However, the optimum NO− 3 /NH+ 4 ratio changes depending on the plant species, the stage of development, and the environmental conditions (Hu et al., 2015). Since the reported studies concerning the effect of different N forms and salinity on sweet pepper quality are very few, this study offers a new insight into the impact of the interaction between the NO− 3 /NH+ 4 ratio and salinity, in this important crop. Therefore, our objective was to determine the influence of the NO− 3 /NH+ 4 nutritional regime, under salt stress, on the fruit colorimetric properties, pigments (chlorophylls, lycopene, and β-carotene), mineral composition, total protein and total phenolics contents, polyamines, and amino-acids profile.

### MATERIALS AND METHODS

#### Plant Material and Growth Conditions

The experiments took place in a greenhouse located in the IFAPA center "La Mojonera" (Almería, Spain, latitude 36◦ 480 N, longitude 2◦ 410 W). It was of the multi-tunnel type (3 spans), oriented east–west, and had an area of 720 m2 . The greenhouse height was 4.7 m to the ridge and 3 m to the gutter of each span. The greenhouse cover was thermal polyethylene (PE) (0.2 mm thick). Ventilation was provided by two roof vents (opening area: 1 m × 30 m) and two side vents (opening area: 1.5 m × 26 m) set in both the south and north arches. The greenhouse was equipped with a commercial climate control system (CDC, INTA S.A.). A continuous register of the temperature and humidity (HMP45C sensors, Campbell Sci.) was maintained. In the greenhouse, pepper seedlings (cv. Melchor) were transplanted on 19 August 2013, two plants into each 27-L container filled with perlite, with a density of 2.5 plants per m2 . The cultivation was carried out with a type of Dutch pruning; two stalks were left on each plant.