Superoxides (O•− 2 ) and H2O2 are the most well studied ROS; they are important players in physiological and pathological processes (Pitzschke et al., 2006; Dikalov et al., 2011; Suzuki et al., 2013). NADPH-oxidase catalyzes the conversion of molecular oxygen to O•− 2 , and its activation accounts mostly for the large consumption of oxygen that characterizes the respiratory burst in mammalian phagocytic cells (Vignais, 2002). In mammals NADPH-oxidase is composed of membrane-bound and cytosolic proteins. In the center of the NADPH oxidase complex lies the heterodimeric NADPH-binding flavocytochrome b558, consisting of the glycosylated transmembrane protein gp91phox and the non-glycosylated p22phox subunit. Upon activation, the cytosolic proteins p47phox and p67phox become phosphorylated and translocate, together with p40phox and p21rac, to the membrane components, to form the active NADPH-oxidase complex (Segal and Abo, 1993). Plants deficient in gp91phox homologs have compromised responses to stress and have a reduced ability to accumulate ROS. Antisense tomato lines (*Lerboh1*) show reduced ROS accumulation and compromise wound response (Sagi et al., 2004). *Arabidopsis* plants disrupted in the *gp91phox* homologs, the respiratory burst oxidase homolog D (*AtrbohD*) and *AtrbohF*, exhibit reduced ROS production and treatment with the avirulent bacterium *Pseudomonas syringae* pv tomato DC3000 results to cell death (Torres et al., 2005), whereas they have diminished stomatal closure in response to abscissic acid (ABA; Kwak et al., 2003). These data suggest that NADPH-oxidase homologs in plants are important for ROS accumulation.

Another important source of ROS is the mitochondrial electron transport chain (ETC; Muller et al., 2004; Vacca et al., 2004). It consists of four complexes, tightly bound to the intermembrane space of mitochondria. Electrons derived from the tricarboxylic acid (TCA) cycle in the matrix move toward the ETC and in turn pass through the four complexes. Transfer of electrons between complex 3 and 4 of the ETC is facilitated *via* the electron carrier cytochrome-c (cyt-c pathway). The electron motion generates a proton gradient which in turn drives an ATPase. Dysfunction of the mitochondrial ETC leads to the leakage of electrons toward oxygen resulting in the generation of O•− 2 (Muller et al., 2004). In order to dissipate the excess electrons, the mitochondria possess another pathway, the alternative pathway, which depends on an alternative terminal oxidase (AOX; Atkin et al., 2002). AOX alleviates mitochondrial ETC from the excess electron load (Yip and Vanlerberghe, 2001).

Previous workfrom our lab suggested that a regulatory crosstalk between PAs and NADPH-oxidase takes place during tobacco protoplast regeneration (Papadakis and Roubelakis-Angelakis, 2005). PAs seem to be necessary for protoplasts to retain their totipotent state, and prevention of PCD. The interaction between main cellular sources of ROS, such as mitochondria and NADPH-oxidases, however, remains obscure. More importantly, a feed-forward regulation of different ROS sources has been reported (Dikalov et al., 2011). Therefore, the regulatory crosstalk between ROS sources merits further examination.

Here, we report that exogenous PAs stimulate oxygen consumption in *Arabidopsis* in an NADPH-oxidase dependent manner. Plants overexpressing the peroxisomal AtPAO3 show decreased oxygen consumption rate, in strict contrast to lossof-function *Atpao3* plants which show increased consumption through the AOX pathway. Surprisingly, this increase is attenuated by diphenyleneiodonium iodide (DPI) but not by ascorbate (ASA), suggesting that NADPH-oxidase is upstream of a respiratory increase mediated by AOX. By delving the regulatory function of O•− 2 in oxygen consumption rate, we found that *AtPAO3* overexpressing plants show a balanced production of both O•− 2 and H2O2, while *Atpao3* loss-of-function plants show a high ratio of O•− 2 *versus* H2O2 production. These data suggest that NADPH-oxidase and AtPAO3 cross-talk for balancing intracellular O•− 2 /H2O2 which in turn affect the cyt-c/AOX pathways.

## **MATERIALS AND METHODS**

## **PLANT MATERIAL AND GROWTH CONDITIONS**

*Arabidopsis thaliana* wild type (WT) plants of the ecotype Columbia (Col-0) were used along with transgenic plants overexpressing the peroxisomal *AtPAO3* (S-*AtPAO3*) and *Atpao3* T-DNA loss-of-function insertional mutants, previously described (Moschou et al., 2008c; Wu et al., 2010; Fincato et al., 2011). Plants were grown in a cabinet using an 8/16 h (light/dark) photoperiod and a constant temperature of 23◦C. Developing seedlings were transferred to 96-well plates filled with 1/4 strength Murashige and Skoog (MS; Murashige and Skoog, 1962) culture medium. All treatments were carried out by supplementing the culture medium with the corresponding agent. More specifically, the PAs Put, Spd, and Spm were added as aqueous solutions at a final concentration of 1mM. Control plants were mock treated with dH2O.

#### **POLAROGRAPHIC MEASUREMENT OF RESPIRATORY OXYGEN CONSUMPTION**

The rate of oxygen consumption was essentially determined as previously described (Andronis and Roubelakis-Angelakis, 2010). In brief, polarography was performed at 30◦C with a Clark type electrode system (Hansatech Instruments, Kings's Lynn, Norfolk, UK), in the presence and absence of the alternative respiratory inhibitor salicylhydroxamic acid (SHAM). Oxygen consumption was measured for a period of 5 min. For inhibitor treatments, leaves were incubated in 15 mM SHAM in 3% (v/v) methanol for a period of 10 min prior to measurement. Control leaves were incubated in dH2O or 3% (v/v) methanol. In all cases, the rate of oxygen consumption was expressed as per g fresh weight.

#### **NADPH OXIDASE NATIVE PAGE AND ACTIVITY STAINING**

Separation of NADPH oxidase isoenzymes and activity staining were carried out according to Carter et al. (2007). *Arabidopsis* leaf tissue was collected and ground using liquid N2. The powder was homogenized in a buffer containing 50 mM sodium phosphate, pH 6.8, supplemented with 0.5% (v/v) Triton X-100. 100 μg of protein were separated using native PAGE at 40 mA. Gels were then incubated in 0.5 mg mL−1 nitroblue tetrazolium (NBT) in 10 mM Tris, pH 7.4, and 134 mM NADPH until bands were detected.

#### **IN SITU DETECTION OF H2O2 AND O**•− **2**

*In situ* accumulation of H2O2 was detected using the method of Thordal-Christensen et al. (1997) and of O•− 2 according to Jabs et al. (1996). *Arabidopsis* seedlings were destained using boiling pure ethanol and photographed using a Nikon Coolpix 4500 digital camera.

#### **PROTEIN GEL BLOT ANALYSES AND IN-GEL ACTIVITIES OF APX AND SOD**

Protein extractions and gel blots were performed as previously described (Moschou et al., 2013). One hundred mg of leaf material was mixed with 100 μL of urea extraction buffer [4 M urea, 100 mM DTT, and 1% (v/v) Triton X-100] and incubated in ice for 10 min. The samples were boiled with Laemmli sample buffer for 10 min and centrifuged at 13,000 rpm for 15 min. Equal amounts of the supernatants were loaded on 10% (v/v) polyacrylamide gels and blotted on a polyvinylidene difluoride (PVDF) membrane.

For the activity staining of ascorbate peroxidase (APX), 10 mM ASA were added to isoelectric focusing electrophoresis buffer (Rao et al., 1995), and 10% gels were pre-run for 30 min at 20 mA. Subsequently, the gels were incubated in the dark in a solution containing 50 mM potassium phosphate buffer, pH 7.0, and 2 mM ASA; the gels were incubated in the dark for another 30 min in 50 mM potassium phosphate buffer, pH 7.0, 4 mM ASA, and 2 mM H2O2. Bands were visualized after the incubation of gels in coloring solution (50 mM potassium phosphate buffer, pH 7.8, 14 mM tetramethylethylenediamine, TEMED; and 1.2 mM NBT). The activity staining of superoxide dismutase (SOD) has been previously described (Beauchamp and Fridovich, 1971).

#### **IMAGE AND STATISTICAL ANALYSIS**

The image analysis was performed using ImageJ v 1.41 software1. Statistical analysis was performed with SPSS v142 or JMP v 9 software3. We used Dunett's test with alpha values set at α = 0.1.

#### **RESULTS**

#### **EXOGENOUS PAs STIMULATE OXYGEN CONSUMPTION RATE**

Our previous work established the effect of abiotic stress on the respiratory activity of WT tobacco plants (Andronis and Roubelakis-Angelakis, 2010). Under abiotic stress conditions, cytc, an electron carrier located between complexes III and IV of the ETC, dissociates leading to malfunction of the mitochondrial ETC and accumulation of ROS. As a result, the AOX pathway is activated in order to dissipate the excess electrons "leaking" from the ETC. Taking into consideration the link between PAs and plant

responses to stresses, we attempted to reveal a potential correlation between PAs and respiratory activity in *Arabidopsis thaliana*.

Two-week old Col-0 *Arabidopsis* seedlings were treated with exogenous Put, Spd, and Spm and oxygen consumption rate was determined 10 min post-treatment. Respiration rate increased in the presence of all PAs used, in terms of oxygen consumption. Spd exerted the strongest effect on the respiration rate, resulting in a 2.6-fold increase compared to the mock treated plants (**Figure 1**). Put and Spm increased respiration, by 1.8- and 2.1 fold, respectively, compared to mock treated plants. These findings revealed an apparent link between PAs and ETC regulation in plants.

#### **STIMULATION OF OXYGEN CONSUMPTION RATE DEPENDS ON PA-INDUCED GENERATION OF O**•− **2**

Previously, we have shown that exogenous application of Spd to tobacco plants leads to a significant increase in H2O2 content generated by the action of PAO (Moschou et al., 2008b). A plausible hypothesis could be that PAO-dependent ROS production is a component of the pathway which is responsible for the observed increase of the oxygen consumption rate. To test this hypothesis, we firstly examined whether exogenous Spd could induce H2O2 and O•− 2 accumulation in *Arabidopsis*. To this end, we employed *in situ* detection protocols of H2O2 and O•− 2 . Indeed, a 10 min treatment with exogenous Spd (1 mM) led to a significant accumulation of H2O2, as a result of PA oxidation. The use of 1 mM Spd was based on our previous findings that this concentration is enough to enter peroxisomes and be backconverted to Put in *Arabidopsis* (Fincato et al., 2011). Surprisingly, we found a significant increase in O•− 2 content, as well (**Figures 2A,B**). From the aforementioned it is evident that the increase in plant respiration coincided with elevated H2O2 and/or O•− 2 in treated plants.

To prove the link between the produced ROS and increased respiration, we tested whether quenching of ROS would alleviate the

**FIGURE 1 | Effect of exogenous polyamines on oxygen consumption rate in** *Arabidopsis***.** *Arabidopsis* Col-0 seedlings were treated with 1 mM Put, Spd, or Spm for a period of 10 min, and oxygen consumption rate was estimated by a Clark-type electrode. Data are the means of three independent experiments ±SD. Asterisks indicate statistical significant differences (\*\*\**P* < 0.001).

1http://rsb.info.nih.gov/ij/index.html

2www.spss.com

3www.jmp.com

effect of Spd on respiration. Spd was added in combination with ASA, a scavenger of O•− 2 and H2O2, catalase (CAT), a scavenger of H2O2 and SOD, a scavenger of O•− 2 . Addition of ASA to the medium failed to produce a significant effect on Spd-induced respiration, whereas CAT led to a 37% decrease over the rate found in Spd treatments (**Figure 3A**). The addition of SOD to the Spdcontaining medium alleviated the effect of Spd to a greater extent leading to an overall reduction of 57% over the Spd treated plants, to a rate similar to that determined for the untreated plants (control). Finally, treatment with both, SOD and CAT in addition to Spd further reduced the respiration rate, rendering it lower than that determined in the untreated plants. These results suggest that PA-dependent ROS and particularly O•− 2 are required for induction of the increased oxygen consumption rate.

Previous work from our laboratory established the role of PAs and NADPH-oxidase in the developmental fate of isolated protoplasts (Papadakis and Roubelakis-Angelakis, 2005). Considering that Spd led to a more significant increase of O•− 2 , than of H2O2 we hypothesized that O•− 2 is produced *via* the activation of the NADPH- oxidase. Indeed simultaneous treatment of Col-0 *Arabidopsis* plants with Spd and the NADPH-oxidase blocker DPI led to a significant reduction in both H2O2 and O•− 2 in the treated plants (**Figures 2A,B**), providing strong evidence for the participation of the NADPH-oxidase in the Spd-induced ROS accumulation.

So far there are strong indications that there is interplay between Spd and NADPH- oxidase in the generation of ROS which induces enhancement of respiration in *Arabidopsis*. To further verify this result we studied the effect of Spd as a doseresponse on NADPH-oxidase activity in Col-0 *Arabidopsis* plants. Treatment with a range of Spd concentrations resulted to a dose-dependent effect in the increase of NADPH-oxidase activity up to a saturation point, and decreased thereafter as indicated by an in gel enzymatic assay (**Figure 3B**). Lower concentrations of Spd (0.1, 0.2, and 1 mM) increased NADPH-oxidase activity compared with the control plants, whereas higher concentration (5 mM and above) decreased NADPH-oxidase activity. These results suggest that low concentrations of Spd can induce NADPH-oxidase.

#### **AtPAO3 REGULATES THE BALANCE BETWEEN H2O2 AND O**•− **2**

Recent data have shed light on the biochemical role of plant intracellular PAOs, which interconvert Spm to Spd and Spd to Put, reversing the PA biosynthetic pathway (Fincato et al., 2011; Ahou et al., 2014). We have previously shown that plants overexpressing *AtPAO3* efficiently oxidize Spd to Put producing H2O2 (Moschou et al., 2008c; Wu et al., 2010; Fincato et al., 2011). S-*AtPAO3* plants overexpressing the peroxisomal*AtPAO3* and lossof-function *Atpao3* seem to be valuable tools in the study of PA-induced respiration in *Arabidopsis* due to the localization of AtPAO3 in the peroxisomes which are in proximity to mitochondria. Indeed, *in situ* detection of ROS in the S-*AtPAO3 Arabidopsis* plants showed that they exhibited a higher but balanced production of H2O2 and O•− 2 compared with Col-0 plants (**Figure 4A**). In contrast, *Atpao3* plants accumulated significantly lower levels of H2O2 when compared with Col-0 plants, but increased levels of O•− 2 . Therefore, S-*AtPAO3* and *Atpao3* plants are good models

**FIGURE 4 |** *In situ* **ROS inWT, S-***AtPAO3,* **and** *Atpao3 Arabidopsis* **plants. (A)** *In situ* ROS detection in WT, S-*AtPAO3* and *Atpao3* plants. Data are from a single representative experiment, repeated three times, and densitometric analysis. Data are the means of three different positions on a leave. Asterisks indicate statistical significant differences from the Col-0 (\*\*\**P* < 0.001). Scale bars, 2.8 cm. **(B)** Native electrophoresis and activity staining of SOD and densitometric analysis of isoenzymes. **(C)** Native electrophoresis and activity staining of APX and densitometric analysis.

for studying the differential effects of H2O2 and O•− 2 in oxygen consumption.

Next, we examined a possible contribution of the antioxidant machinery in the observed differences in ROS levels in S-*AtPAO3* and *Atpao3* plants. We determined the contribution of SOD and APX, a well-established H2O2 scavenger, in the observed differences, between Col-0, S-*AtPAO3* and *Atpao3* in H2O2 and O•− 2 levels. Accumulation of O•− 2 in S-*AtPAO3* and *Atpao3* plants coincided with increased SOD activity in mitochondria and chloroplasts (**Figure 4B**). In contrast, APX was elevated in the *Atpao3*, but reduced in S-*AtPAO3* plants (**Figure 4C**). APX has a high affinity for H2O2 and therefore is downregulated at higher levels of H2O2 (Asada, 1992), which perhaps can explain the decrease of APX observed in S-*AtPAO3* plants, with increased H2O2 levels. These results suggest that at least APX and SOD mirror the changes of ROS levels observed in S-*AtPAO3* and *Atpao3*.

#### **DEREGULATION OF AtPAO3 RESULTS TO CHANGES IN OXYGEN CONSUMPTION RATE**

Overall the data presented so far support the role of Spd as an inducer of mitochondrial respiration *via* the NADPH-oxidase generated O•− 2 in Col-0 plants. This response is alleviated mostly by the action of the NADPH-oxidase blocker, DPI. Furthermore, loss-of-function mutant plants for the peroxisomal *AtPAO3* gene accumulate O•− 2 , but not H2O2 in contrast to S-*AtPAO3* overexpressing plants, which accumulate both O•− 2 and H2O2. Considering the above, we determined the oxygen consumption rate in the S-*AtPAO3* transgenics and the *Atpao3* mutants to test whether the differential accumulation of ROS in the two genotypes leads to altered oxygen consumption. Indeed, the three tested genotypes exhibited notable differences in their capacity to consume oxygen (**Figure 5A**). The *Atpao3* plants exhibited the highest rate of oxygen consumption among the tested plants, showing a 2.7-fold increase over the Col-0 plants and a 4.3-fold increase over the S-*AtPAO3* plants. Interestingly, the increase in oxygen consumption of *Atpao3* plants resembled the effect of exogenous Spd in Col-0 plants.

Next, we examined whether the increase in oxygen consumption of *Atpao3* plants is O•− 2 -dependent. To test this, we treated *Atpao3* with DPI, and as a control, we treated Col-0 plants simultaneously with Spd and DPI (**Figure 5A**). Treatment of *Atpao3* plants with DPI reduced the respiratory activity to the control levels, suggesting that increase of oxygen consumption in *Atpao3* plants depends on the production of O•− 2 by NADPH-oxidase. Similarly, Spd plus DPI treated Col-0 plants showed similar oxygen consumption rate to the untreated Col-0 plants, producing an effect equivalent to the DPI-induced decrease in the O•− 2 accumulation reported earlier.

We hypothesized that the increase observed in the*Atpao3* plants could be due to an increased contribution of the AOX pathway. To test this, we used the AOX pathway blocker SHAM. Indeed, application of SHAM to the *Atpao3* plants exerted a dramatic decrease in the oxygen consumption rate, suggesting the participation of the AOX pathway in the O•− 2 -induced oxygen consumption (**Figure 5A**). In addition, the levels of the immunoreactive AOX protein in *Atpao3* were significantly higher compared to the rest

immunoreactive protein in **(B)**. Data are from a single representative

experiment, repeated three times.

of the tested plants (**Figures 5B,C**). These results suggest that the O•− 2 -dependent increase of oxygen consumption in *Atpao3* plants is exerted through the AOX pathway.

#### **DISCUSSION**

Exogenous Spm application to tobacco plants leads to mitochondrial dysfunction and to transcriptional activation of the AOX pathway, while small molecular weight antioxidants efficiently attenuate this induction, suggesting a possible involvement of ROS in this Spm-signaling pathway (Takahashi et al., 2003). A long standing notion proposed that PA oxidation results in induction of signaling cascades through H2O2, since H2O2 is a direct product of PA oxidation (Moschou et al., 2008b). However, recent evidence suggests that other ROS types may as well contribute to PA-dependent signaling cascades and that the PAs-ROS crosstalk stretches beyond H2O2 (Zepeda-Jazo et al., 2011;Velarde-Buendia et al., 2012).

In this work, application of exogenous PAs to Col-0 *Arabidopsis* plants stimulated the oxygen consumption rate; the stronger effect was exerted by Spd. Exogenous application of Spd is expected to increase H2O2 content through the PA oxidation/backconvertion pathway (Yoda et al., 2003; Moschou et al., 2008b). Therefore, we hypothesized that this product could be responsible for the increase in oxygen consumption. Surprisingly, H2O2 scavenging did not significantly attenuate the PA-dependent oxygen increase. On the other hand, exogenous SOD or DPI ameliorated the oxygen increase, caused by the exogenous Spd application. This denotes that the increase in oxygen consumption relies on O•− 2 production, and suggests that exogenous PA induces O•− 2 production by NADPH-oxidase intriguingly, DPI exerts a stronger effect than SOD. This may be due to the fact that SOD is expected to scavenge intercellular O•− 2 produced by NADPH-oxidase, giving rise to H2O2, while DPI is a plasma membrane permeable suicidal NADPH-oxidase inhibitor, that could efficiently prevent production of higher amount of O•− 2 . Surprisingly, CAT mimicked the DPI effect. Nevertheless, it should be noted that recent evidence suggests that CAT participates in the induction of cell death (Hackenberg et al., 2013), therefore perplexing the interpretation of the data obtained with the use of CAT. ASA, on the other hand, did not alleviate oxygen consumption increase. It should be noted that ASA is a reducing agent, thereby directly affecting the ETC, and scavenges both O•− 2 and H2O2 (Foyer and Noctor, 2011). These results suggest that the PA-derived oxygen consumption increase depends mostly on O•− 2 production by the PA-induced NADPH-oxidase.

Exogenous PA at relatively high concentrations stimulated production of O•− 2 in human neutrophils (Guarnieri et al., 1987). In our study, exogenous application of Spd in *Arabidopsis* increased the content of H2O2 produced through PAO, as previously suggested for other plant species (Yoda et al., 2006; Wu et al., 2010; Moschou and Roubelakis-Angelakis, 2011; Tisi et al., 2011). Nevertheless, exogenous Spd induced a significant increase in the levels of O•− 2 along with an increase in NADPH-oxidase activity. Interestingly, previous studies suggested that Spd induces autophagy (self-consumption) in non-plant models and is an important surveillance mechanism that rather restricts ROS production (Eisenberg et al., 2009). In accordance, during protoplast isolation from tobacco (Papadakis and Roubelakis-Angelakis, 2005), induction of NADPH-oxidase and the concomitant production of O•− 2 was highly suppressed by PAs. This discrepancy between our current and previous work could be due to the fact that in Papadakis and Roubelakis-Angelakis paper, PAs were added to the protoplasts and then NADPH-oxidase was purified

and assayed. On the contrary, in this study PAs were added exogenously and NADPH-oxidase assessment was performed by an in-gel assay omitting further purification steps. This allows us to hypothesize that exogenous PAs may antagonize for Ca2+in NADPH-oxidase preparations. Calcium is required for NADPHoxidase activation. Therefore, we assume that NADPH-oxidase cannot accommodate Ca2+ in the presence of PAs on its binding. In addition, in the previous study O•− 2 content was determined by a chemiluminescence assay, while in this work we used an *in situ* detection protocol, which seems to be a more accurate and reliable method (Song et al., 2006). Therefore, PAs and especially Spd seem to positively affect NADPH-oxidase *in planta*, unlike in *in vitro* systems. In addition, a species-related differential effect on NADPH-oxidase of PA cannot be ruled out (tobacco versus *Arabidopsis*).

From the aforementioned, it is conceivable that production of O•− 2 may depend on a PAO system. There are two main PA oxidation regimes: an apoplastic and an intracellular one. Several plant species, especially monocots, possess the ability to oxidize higher PAs in their apoplastic compartments. The oxidative reaction is executed by DAOs and PAOs residing in this compartment. However, the former enzymes show low affinity for higher PAs such as Spd. Notably, apoplastic PAOs in *Arabidopsis* are missing. There are five genes encoding for PAOs in *Arabidopsis.* The inducible *AtPAO1* (Tavladoraki et al., 2006) and constitutively expressed *AtPAO5* (Ahou et al., 2014) encode for cytoplasmic enzymes, oxidizing Spm, while *AtPAO2, 3, 4* encode peroxisomal proteins (Kamada-Nobusada et al., 2008; Moschou et al., 2008c), oxidizing Spd (AtPAO2, 3) and Spm (AtPAO2,3,4). On the other hand, in *Arabidopsis* there are ten genes encoding DAOs, four of which have been characterized. One of them is an apoplastic enzyme; however, six remain to be characterized (Moschou et al., 2013; Planas-Portell et al., 2013). These suggest that most likely the contribution of the apoplast to the Spd-dependent production of O•− 2 is rather minimal in *Arabidopsis*.

To further examine whether intracellular PAOs are responsible for inducing production of O•− 2 , we employed genetic means. The best characterized so far enzymatic activity that oxidizes mostly Spd and to a smaller extent Spm in *Arabidopsis* is that of the peroxisomal AtPAO3 (Moschou et al., 2008c; Fincato et al., 2012). In mammals and plants, PA oxidation has been implicated in the execution of PCD (Yoda et al., 2003; Tavladoraki et al., 2006; Moschou and Roubelakis-Angelakis, 2013). It has been exemplified that there is a direct relationship between PCD and the levels of cytotoxic PA catabolic products, i.e., H2O2 and aminoaldehydes. For example, during *Helicobacter pylori* infection that contributes to gastric cancer, PA-derived H2O2 coincides with PCD induction (Chaturvedi et al., 2004). However, PA-derived H2O2 seems to be a double-edged sword since oxidation by SMO could perhaps contribute to the eradication of tumor cells (Babbar et al., 2007). In tobacco, overexpression of apoplastic PAO is accompanied by premature cell death of xylem tissue (Tisi et al., 2011). Interestingly, exogenous supply of Spd to maize root tips highly expressing *PAO* alters cell cycle distribution, toward quiescence and induces PCD (Tisi et al., 2011). In addition, premature cell death of xylem hinders the proper differentiation of the secondary cell wall, which is normally deposited before PCD induction in xylem.

Importantly, a H2O2 scavenger partially ameliorates Spd-induced effects. In addition, 4-aminobutanal which is an additional oxidation product of Spd, failed to mimic Spd effects, indicating that PAO-derived H2O2 is sufficient to induce PCD independently of aminoaldehydes.

In contrast to the previous, the PA backconversion pathway seems to have completely distinct functions, which remain largely elusive. It was shown that AtPAO3 is an important component of pollen tube elongation (Wu et al., 2010). More specifically, AtPAO3 generates H2O2 which positively affects the permeability of a plasma membrane-residing Ca2+influx channel. As a result, the intracellular concentration of Ca2+ increases, thereby promoting pollen tube elongation. In loss-of-function *Atpao3* reduction of pollen tube elongation, and in a physiological context reduced fertility was evident. In addition, a role for the PA backconversion pathway was hypothesized with respect to dehydration response of *Arabidopsis* (Alcazar et al., 2011a). The putative paralog of *AtPAO3* gene, *AtPAO2* is upregulated by drought stress in a similar fashion as *RD29A* and *RD22*.

A number of PAOs have been implicated in the PA backconversion pathway in *Arabidopsis* and, unlike in mammals, plant PAOs did not require acetylated derivatives (Moschou et al., 2008c). We observed that plants overexpressing *AtPAO3* showed increased content of H2O2 consistent with its role in PAs oxidation. Surprisingly, this H2O2 production led to a significant O•− 2 increase, while *Atpao3* mutants showed reduced levels of H2O2 but increased O•− 2 . This implies that loss of AtPAO3 caused an increment of O•− 2 *versus* H2O2. In animal cells, ROS have also been shown to play an important role in maintaining the balance between cell proliferation and differentiation. A redox-dependent signaling pathway controls the induction of cell division through the regulation of *cyclinD1* expression (Burch and Heintz, 2005). Distribution of specific ROS appears to act as an important signal at the transcriptional and posttranscriptional levels during cellcycle progression (Menon and Goswami, 2007). In *Drosophila*, changing ROS balance can switch the status of hematopoietic cells from proliferation to differentiation (Owusu-Ansah and Banerjee, 2009). In *Arabidopsis*, it was shown that O•− 2 accumulates primarily in the root meristematic zone, whereas H2O2 accumulates mainly in the elongation zone (Tsukagoshi et al., 2010). Moreover, it has been shown that Mn-SOD activity regulates cell-cycle progression through modulation of ROS levels, which control expression of both the *cyclinB1* and *cyclinD1* genes in mouse cells (Sarsour et al., 2008). The authors proposed that O•− 2 regulates the proliferative cycle, whereas H2O2 induces quiescence and differentiation. Therefore, in the root elongation zone, the ratio between O•− 2 and H2O2 is decreased (Tsukagoshi et al., 2010).

In our study, AtPAO3 was shown to be an important factor for balancing O•− 2 and H2O2. Increased levels of O•− 2 *versus* H2O2 were detected in the absence of AtPAO3, perhaps due to the increased activity of APX, which scavenges H2O2. S-*AtPAO3* plants show reduced expression of APX but increased expression of mitochondrial and chloroplastic SOD isoenzymes, while*Atpao3* show significantly increased expression of APX, mitochondrial and chloroplastic SOD. Noteworthy, the increased isoenzymes are in proximity to peroxisomes. These changes are in accordance with the ROS levels detected in these plants. However, further studies are required to elucidate whether the increased/decreased expression of these antioxidants controls ROS levels or alternatively, whether ROS levels control the induction/reduction of these genes/enzymes. Although this may sound like a "chicken or the egg" question it merits careful examination to further understand the regulation of ROS homeostasis. We can speculate that similar regulation of the O•− 2 /H2O2 ratio takes place during other developmental transitions, apart the ones reported in the root (Tsukagoshi et al., 2010) like for example during pollen tube growth, which could contribute to the failure of *Atpao3* pollen tube elongation (Wu et al., 2010).

Overexpression of *SSAT* in mice, an acetylase required to direct PAs in non-plant models toward the PAO pathway, leads to increased H2O2 and carbonyl content, and reduced SOD, CAT, and cyt CYP450 2E1 expression, responsible for xenobiotic metabolism. This suggests that transgenic mice are hypersensitive to stress, leading to cell death, and they also are sluggish and less hostile (Kaasinen et al., 2004). Interestingly, although S-*AtPAO3* plants accumulate significantly higher amounts of ROS, they do not show symptoms of chronic stress. Tobacco plants overexpressing apoplastic PAO exhibit increased SOD and CAT expression, which do not exert a protective effect, but rather this increased expression represents an attempt to scavenge surplus H2O2 produced by continuous PA oxidation. The previous suggests that as in animals, constitutive apoplastic PA oxidation in plants can lead to chronic oxidative stress (Moschou et al., 2008a).

On the contrary, the AtPAO3 backconversion pathway seems to have a completely different function. We show that Spd oxidation by AtPAO3 is required for a balanced respiration through the cyt-c and AOX pathways. Notably, overproduction of PAderived H2O2 in the S-*AtPAO3* plants results in a small decrease of oxygen rate consumption, but not in induction of the AOX pathway. To the contrary, *Atpao3* plants show increased oxygen consumption through the AOX pathway. Interestingly, this increase is attenuated by application of DPI, which specifically blocks O•− 2 generation by NADPH-oxidase. It was reported that a microtubule associated kinesin and a mitochondrial channel are able to regulate the balance between cyt-c and AOX pathways (Yang et al., 2011). In addition, it has been hypothesized that the ratio of (singlet + O•− 2 )/H2O2 determines PCD initiation during stress (Sabater and Martin, 2013). The previous allow us to propose that an increased ratio of O•− 2 /H2O2 leads to increased oxygen consumption through the AOX pathway. Likewise, it has been reported that O•− 2 is sufficient to induce *AOX1a/b* genes in rice (Li et al., 2013). These results demonstrate that depletion of AtPAO3 leads to higher production of O•− 2 , which in turn activates the AOX pathway.

In conclusion, our results allow us to propose that AtPAO3 is required for balancing O•− 2 /H2O2 production. An imbalance of the O•− 2 *versus* H2O2 production leads to activation of AOX pathway and increases oxygen consumption. The next critical step to advance our understanding on the role of PA backconversion, and its interplay and crosstalk with ROS will be the genetic dissection of PA backconverting pathways, and their molecular effectors.

#### **ACKNOWLEDGMENTS**

This work is implemented in the frame of ABISTOLE project KA3571 and the EU COSTFA1106 action.

#### **REFERENCES**

regulates cell polarity and cytokinesis in *Arabidopsis*. *Plant Cell* 25, 2171–2186. doi: 10.1105/tpc.113.113043

**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.

*Received: 20 January 2014; paper pending published: 16 February 2014; accepted: 20 March 2014; published online: 03 April 2014.*

*Citation: Andronis EA, Moschou PN, Toumi I and Roubelakis-Angelakis KA (2014) Peroxisomal polyamine oxidase and NADPH-oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana. Front. Plant Sci. 5:132. doi: 10.3389/fpls.2014.00132*

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

*Copyright © 2014 Andronis, Moschou, Toumi and Roubelakis-Angelakis. 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) or licensor 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.*

## Changes in free polyamine levels, expression of polyamine biosynthesis genes, and performance of rice cultivars under salt stress: a comparison with responses to drought

#### *Phuc T. Do1†, Oliver Drechsel 1†, Arnd G. Heyer 2, Dirk K. Hincha1 and Ellen Zuther 1 \**

*1 Infrastructure Group Transcript Profiling, Max-Planck-Institute of Molecular Plant Physiology, Potsdam, Germany 2 Department of Plant Biotechnology, Institute of Biology, University of Stuttgart, Stuttgart, Germany*

#### *Edited by:*

*Ruben Alcazar, Universitat de Barcelona, Spain*

#### *Reviewed by:*

*Joseph M. Jez, Washington University in St. Louis, USA Igor Pottosin, Universidad de Colima, Mexico*

#### *\*Correspondence:*

*Ellen Zuther, Infrastructure Group Transcript Profiling, Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam, Germany e-mail<EMAIL_ADDRESS>
#### *†Present address:*

*Phuc T. Do,Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi, Vietnam; Oliver Drechsel, Center for Genomic Regulation (CRG), Barcelona, Spain*

Soil salinity affects a large proportion of rural area and limits agricultural productivity. To investigate differential adaptation to soil salinity, we studied salt tolerance of 18 varieties of *Oryza sativa* using a hydroponic culture system. Based on visual inspection and photosynthetic parameters, cultivars were classified according to their tolerance level. Additionally, biomass parameters were correlated with salt tolerance. Polyamines have frequently been demonstrated to be involved in plant stress responses and therefore soluble leaf polyamines were measured. Under salinity, putrescine (Put) content was unchanged or increased in tolerant, while dropped in sensitive cultivars. Spermidine (Spd) content was unchanged at lower NaCl concentrations in all, while reduced at 100 mM NaCl in sensitive cultivars. Spermine (Spm) content was increased in all cultivars. A comparison with data from 21 cultivars under long-term, moderate drought stress revealed an increase of Spm under both stress conditions. While Spm became the most prominent polyamine under drought, levels of all three polyamines were relatively similar under salt stress. Put levels were reduced under both, drought and salt stress, while changes in Spd were different under drought (decrease) or salt (unchanged) conditions. Regulation of polyamine metabolism at the transcript level during exposure to salinity was studied for genes encoding enzymes involved in the biosynthesis of polyamines and compared to expression under drought stress. Based on expression profiles, investigated genes were divided into generally stress-induced genes (*ADC2, SPD/SPM2, SPD/SPM3*), one generally stress-repressed gene (*ADC1*), constitutively expressed genes (*CPA1*, *CPA2, CPA4, SAMDC1, SPD/SPM1*), specifically drought-induced genes (*SAMDC2*, *AIH*), one specifically drought-repressed gene (*CPA3*) and one specifically salt-stress repressed gene (*SAMDC4*), revealing both overlapping and specific stress responses under these conditions.

**Keywords: polyamines, salt stress, drought stress, gene expression, rice, natural variety**

## **INTRODUCTION**

Cultivation of rice (*Oryza sativa* L.) is limited by environmental stresses, of which salinity and drought represent some of the most devastating ones. Erosion, soil degradation and salinization affect approximately 3.6 billion of the world's 5.2 billion ha of dryland used for agriculture (Riadh et al., 2010). 10% of the land surface (950 Mha) and 50% of all irrigated land (230 Mha) are salt-affected (Ruan et al., 2010). Global annual losses from soil salinity are estimated at US\$12 billion (Qadir et al., 2008). Rice (*Oryza sativa* L.) is considered to be moderately sensitive to salinity (Akita and Cabuslay, 1990) with a clear distinction between initial effects of salinity and long-term effects that result from the accumulation of salt in expanded leaves (Yeo et al., 1991). Salt stress causes reduction in leaf expansion, relative growth rate (Akita and Cabuslay, 1990) and photosynthesis (Nakamura et al., 2002; Cha-Um et al., 2006), as well as enhanced senescence (Lutts et al., 1996). Three major processes have been considered to participate in protection against high cytosolic Na+: (1) the minimization of Na+ entry into cells; (2) the compartmentation of Na+ into the vacuole; and (3) the increased efflux of Na+ out of the cell driven by specific ion transporters (Chinnusamy et al., 2005).

Polyamines are involved in a wide range of biological processes, including growth, development and programmed cell death (Galston and Sawhney, 1990; Bouchereau et al., 1999; Kaur-Sawhney et al., 2003; Moschou and Roubelakis-Angelakis, 2013), as well as abiotic stress responses (for recent reviews see Alcázar et al., 2010; Gill and Tuteja, 2010; Hussain et al., 2011; Gupta et al., 2013). Nevertheless, reported responses of polyamines to salt stress are often contradictory, even within one species. In rice, either a decrease of putrescine (Put) and/or spermidine (Spd)

**Abbreviations:** ABA, abscisic acid; ADC, arginine decarboxylase; AIH, agmatine iminohydrolase; CPA, N-carbamoylputrescine amidohydrolase; ODC, ornithine decarboxylase; Put, putrescine; qRT-PCR, quantitative RT-PCR; ROS, reactive oxygen species; SAMDC, S-adenosyl-methionine decarboxylase; SPD, spermidine synthase; Spd, spermidine; SPM, spermine synthase; Spm, spermine.

(Lin and Kao, 1995; Maiale et al., 2004), or of all three major polyamines (Prakash et al., 1988), but also a salt-induced increase of Put (Basu et al., 1988; Basu and Ghosh, 1991), Spd, and spermine (Spm) (Katiyar and Dubey, 1990; Maiale et al., 2004) has been reported. Also a differential modification of polyamines upon salt stress depending on the tolerance level was described (Krishnamurthy and Bhagwat, 1989). Considering water stress in rice in general, including osmotic stress, dehydration, and withholding water, accumulation of polyamines was reported for most scenarios (Capell et al., 2004) but also a decrease of Put with a parallel increase of Spm (Do et al., 2013). Modifications of polyamines in response to osmotic stress were considered to be affected by dose and time of treatment (Lefèvre et al., 2001). A transcriptome analysis of polyamine over-accumulators revealed that endogenous polyamines participate in stress signaling through a crosstalk with abscisic acid (ABA), Ca2+ signaling and other hormonal pathways in plant defense and development (Marco et al., 2011).

Positively charged polyamines are able to interact with negatively charged molecules, such as nucleic acids, acidic phospholipids, proteins, and cell wall components (Martin-Tanguy, 2001; Kakkar and Sawhney, 2002). The physiological function of the various polyamines in stress response is not resolved yet, but their involvement in protein phosphorylation, conformational transitions of DNA (Martin-Tanguy, 2001), maintenance of ion balance, radical scavenging and prevention of senescence, stabilization of membranes (Bouchereau et al., 1999), and gene regulation by enhancing DNA-binding activity of transcription factors (Panagiotidis et al., 1995) was shown and a role as compatible solutes is discussed. Additionally, polyamines affect ion channel conductivity due to their positive charge and are able to block vacuolar channels, e.g., calcium channels (Hussain et al., 2011).

Polyamine catabolism in the apoplast is a common mechanism within reactive oxygen species (ROS) signaling (Pottosin et al., 2014). Furthermore Spd and Spm were described as nitric oxide (NO) inducers in plants which are part of a complex network also containing ABA and H2O2 (Hussain et al., 2011; Pottosin et al., 2014). In addition, polyamines are major players in the turnover of nitrogenous compounds in plants under optimal as well as stress conditions (Moschou et al., 2012).

Put biosynthesis either from ornithine or indirectly from arginine via agmatine is catalyzed by ornithine (ODC; EC <IP_ADDRESS>) and arginine decarboxylase (ADC; EC <IP_ADDRESS>), respectively. Agmatine is then sequentially converted to Put by agmatine iminohydrolase (AIH; EC<IP_ADDRESS>) and Ncarbamoylputrescine amidohydrolase (CPA; EC <IP_ADDRESS>). Spd and Spm are synthesized from Put by the addition of aminopropyl groups, transferred from decarboxylated Sadenosylmethionine (SAM), which is produced from SAM by S-adenosylmethionine decarboxylase (SAMDC; EC <IP_ADDRESS>). Spd synthase (SPD; EC <IP_ADDRESS>) and Spm synthase (SPM; EC <IP_ADDRESS>) catalyze the final steps of the Spd and Spm synthesis. Polyamine metabolism and transport have been recently reviewed in Gupta et al. (2013), and the integration with other metabolic networks was shown in *Arabidopsis* (Bitrián et al., 2012).

An induction of the transcript level and/or activity of ADC could be shown for rice (Chattopadhyay et al., 1997) as well as for other species (Mo and Pua, 2002; Urano et al., 2004; Hao et al., 2005a; Legocka and Kluk, 2005; Liu et al., 2006) under salinity. Transcript levels of other polyamine biosynthesis-related genes are also increased under salt stress, e.g., *SAMDC* in rice (Li and Chen, 2000b), soybean (Tian et al., 2004), wheat (Li and Chen, 2000a), *Arabidopsis* (Urano et al., 2003) and apple (Hao et al., 2005b), *SPD* and *SPM* in *Arabidopsis* (Urano et al., 2003), and maize (Rodríguez-Kessler et al., 2006). Tolerance to drought was improved by constitutive over-expression of oat *ADC* in rice (Capell et al., 1998), with a simultaneous effect on plant development. When polyamine accumulation was induced by over-expression of oat *ADC* or *Tritodermum SAMDC* under the control of an ABA-inducible promoter, rice plants were more resistant to high salinity (Roy and Wu, 2001, 2002). Furthermore, over-expression of the *Datura stramonium ADC* gene under the control of the stress activated maize ubiqitin-1 promoter conferred tolerance to osmotic stress in rice (Capell et al., 2004). For a broader overview of transgenic approaches; see Gill and Tuteja (2010) and Marco et al. (2012).

Here we investigated changes in polyamine content and expression levels of all genes encoding enzymes involved in polyamine biosynthesis in a wide range of rice cultivars under long-term moderate salt stress using two different NaCl concentrations and we explored the possible correlations between physiological parameters, polyamine content, and gene expression levels and salt sensitivity of those 18 rice cultivars. In addition, by comparing polyamine levels and changes in gene expression with results obtained under mild drought stress conditions, we were able to classify the different genes into either salt- or drought- or generally stress-responsive.

#### **MATERIALS AND METHODS**

#### **PLANT MATERIAL, CULTIVATION, AND SALT STRESS TREATMENT**

Eighteen rice (*Oryza sativa* L.) cultivars originating either from the IBT (Institute of Biotechnology, Hanoi, Vietnam) or from the IRRI (International Rice Research Institute, Manila, Philippines) {Nipponbare (IRGC accession 12731) (NB), Taipei 309 (IRGC accession 42576) (TP), IR57311-95-2-3 [IRGC accession 17509 (INGER)] (IR) and Zhonghua} were grown under control and salt stress conditions in three independent experiments in a climate chamber. For a complete list of cultivars see **Table 1**. The design was a randomized complete block design with five blocks, each containing one hydroponic culture box with 0, 50, and 100 mM NaCl, respectively. Boxes were randomized within the blocks.

Seeds were germinated at 28◦C for 10 days. Plantlets were transferred to a climate chamber with 12 h light phase at a photon flux density of 600µE m−2 s −1 (Lamps: Iwasaki Eye MT 400 DL/BH E40, DHL Licht, Wülfrath, Germany); temperature was 26◦C (day) and 22◦C (night), with a relative humidity of 70%. Plants were grown hydroponically in 10 l polypropylene boxes filled with medium according to Yang et al. (1994) and covered with a lid. Plantlets were fixed to holes in the lid with a piece of foam material and covered with a transparent lid for 2 days. After 14 days the growth medium was exchanged for medium with the

*For genotyping of the Vietnamese cultivars marked with an asterisk, see Degenkolbe et al. (2013). IBT, Institute of Biotechnology, Hanoi, Vietnam; IRRI, International Rice Research Institute, Manila, Philippines.*

appropriate salt concentration (0, 50, 100 mM NaCl), which was renewed after an additional week.

After 14 days of salt stress treatment, plants were sampled 4–6 h after the beginning of the light period. The middle sections of leaves were selected for physiological measurements to avoid taking material from the elongation zone at the base of the leaf blade or senescent tissue at the top of the leaves, especially in stressed plants. Fully expanded green leaf blades were harvested and immediately frozen in liquid nitrogen for transcript and polyamine analysis. The remaining plant was harvested to determine shoot fresh (FW) and dry weight (DW, 48 h, 80◦C).

For a detailed description of the drought stress experiments please refer to Do et al. (2013).

21 rice cultivars were grown under control and drought conditions in controlled climate chambers in 12 h days (600µE m−2 s −1) at 26◦C and 75% relative humidity and 12 h nights at 22◦C and 70% relative humidity. Plants were grown in 540 g sand mixed with 8 g of Lewatit HD 50 (Lanxess, Langenfeld, Germany) and 0.4 g Fetrilon Combi (Compo, Münster, Germany). Pots were positioned. Rice plants were grown in boxes filled with water to the level of the substrate surface. 26 days after sowing drought stress was applied by water removal from half of the boxes. When the permanent wilting point for 50% of the plants was reached soil water content was kept constant for 14 days by weighing each pot at the end of the light period and adding the amount of water lost during the last 24 h.

#### **PHYSIOLOGICAL CHARACTERIZATION OF THE PLANTS AND SAMPLING**

The leaf phenotype of stressed and control plants was visually assessed for individual plants before and during salt stress treatment 24 DAS (before stress treatment), 30 DAS (6 days after stress treatment), and 37 DAS (13 days after stress treatment) based on the stress damage score of the IRRI (Mitchell et al., 1998). An average stress damage score considering chlorosis and necrosis and using a scale from 1 to 9 was given to every plant with "1" representing plants with undamaged leaves, "9" almost or completely dead plants. The number of tillers was counted, and plant height (Wopereis et al., 1996) measured at the same time.

Chlorophyll-*a* fluorescence and leaf temperature were measured with a pulse-amplitude-modulated Dual- PAM-100 fluorometer (WALZ, Effeltrich, Germany) on the middle section of the second fully expanded leaf during mid-day without dark adaptation and under climate chamber conditions. The effective quantum yield of PS II (-F/Fm ) was determined from the maximum light-adapted fluorescence yield (Fm ) and the current fluorescence yield (Ft) as [-F/Fm = (Fm - Ft)/Fm ].

Salt score values were ranked and the average rank calculated within each experiment. Results of three experiments were combined and mean and standard error of the average ranks was calculated for every cultivar. The relative DW of the shoot was calculated by dividing the average shoot DW under salt condition by the respective control value for each cultivar and experiment.

A description of the physiological characterization and the ranking of cultivars under drought stress conditions are given in Do et al. (2013).

#### **POLYAMINE ANALYSIS**

Free polyamines (Put, Spd, and Spm) were quantified by High Performance Liquid Chromatography (HPLC) as described in Do et al. (2013). Data were analyzed using the Chromeleon software (Dionex, Germering, Germany) and calibration curves obtained from the pure substances. Detailed data on polyamines levels under drought conditions are given in Do et al. (2013).

#### **QUANTITATIVE RT-PCR (qRT-PCR)**

qRT-PCR was performed with the ABI Prism 7900HT (Applied Biosystems, Foster City, CA) as described in Do et al. (2013). Primers were designed using PrimerExpress (Version 2.0, Applied Biosystems) and all primer sequences are given in Do et al. (2013). Data were analyzed using the SDS 2.0 software (Applied Biosystems) and normalized based on the expression of the housekeeping genes actin 1 and cyclophilin. Normalized gene expression was calculated by dividing the average relative expression (primer efficiency P to the power of cycle number Ct) of the two housekeeping genes (H1 and H2) by the relative expression of the gene of interest (GOI) (Degenkolbe et al., 2009). Primer efficiency was calculated using LinRegPCR (Ramakers et al., 2003). Fold change was calculated as log2 of the ratio of relative expression of genes under stress conditions to relative expression under control conditions. Detailed gene expression data of droughttolerant and drought-sensitive cultivars are given in Do et al. (2013).

#### **STATISTICS**

Spearman correlations between the mean rank of the salt stress damage score and the respective means of all performance parameters across three experiments were determined using the rcorr function provided by the R-package "Hmisc" after a correction for outliers. The significance of differences between treatments was analyzed using unpaired, two-sided *t*-tests in Microsoft Excel. Significance levels in the figures are indicated as: 0.05 > *p* > 0.01 (∗), 0.01 > *p* > 0.001 (∗∗), *p* < 0.001 (∗∗∗).

#### **RESULTS**

#### **PHENOTYPING OF RICE CULTIVARS UNDER SALT CONDITIONS**

18 rice cultivars representing either *japonica* or *indica* subspecies, including cultivars from a Vietnamese breeding program and four well characterized IRRI cultivars (**Table 1**), were subjected to two different salt stress conditions, 50 and 100 mM NaCl, in the vegetative stage using hydroponic culture in controlled growth chambers. Before salt treatment and after 6 and 13 days of salt stress plants were characterized and compared to control plants by scoring their leaf phenotype and by measuring plant height, tiller number, FW, and DW. Differences between cultivars were highest after 14 days at 100 mM NaCl. At this time point cultivars could be clearly classified based on their salt sensitivity using the average rank of visual scoring (**Figure 1**). Mean ranks from 13.8 to 73.7 indicated a large natural variation for salt tolerance between the selected cultivars. The *indica* cultivars Cham bien (27), Cham (26), and Nuoc man (21) were the most tolerant with scoring values from 13.8 to 14.9, whereas the *japonica* cultivars Nipponbare (50), Taipei 309 (51), and Zhonghua (53) were the most sensitive, with scoring values ranging from 58.3 to 73.7. All cultivars of the *japonica* subspecies could be classified as sensitive. The most sensitive *indica* cultivars were C70 (4), DR2 (2), and Lua Man (22).

Integrity of the photosynthetic machinery was investigated by measuring the effective quantum yield of photosynthesis after 13 days of stress for both NaCl concentrations (**Figure 2**). While in most cultivars quantum yield was not impaired by salt treatment, a significant reduction occurred at 100 mM NaCl in the six most sensitive cultivars, including both *indica* and *japonica* subspecies. At mild salt stress conditions of 50 mM NaCl no significant reduction of effective quantum yield was observed.

Plant height was cultivar dependent under control conditions with largest plants in cultivar 25 and smallest in cultivar 4. A reduction due to 100 mM NaCl treatment occurred in four cultivars (14, 8, 22, 50) and showed no tolerance dependent pattern (not shown). Tiller number under control conditions was also highly cultivar dependent with the highest number in cultivar 22 and the lowest in 51, and it was reduced by 14 to 41% across cultivars under salt stress (data not shown). FW was significantly affected by salt treatments of 50 and 100 mM NaCl in most cultivars (**Figure 3A**). In the most sensitive cultivar Nipponbare (50) it was reduced to 22% of the control value. Strikingly, relative DW as %FW was increased at 100 mM NaCl with descending tolerance between no change in the most tolerant and an increase of 39% in the most sensitive cultivar (**Figure 3B**). The most tolerant cultivars showed no significant change of relative DW under salt stress. Spearman correlation analysis for the sensitivity rank at 100 mM NaCl and the ratio of all available growth parameters in comparison to control conditions revealed significant negative correlations between sensitivity rank and photosynthetic quantum yield at 50 and 100 mM NaCl and FW at 100 mM NaCl, and a significant positive correlation with the relative DW at 100 mM NaCl (**Table 2**).

#### **POLYAMINE CONTENT OF RICE CHANGES DURING SALT STRESS**

Pool sizes of predominant free polyamines (Put, Spd, Spm) were measured in leaves of all cultivars after 14 days of salt stress in comparison to the control. **Figure 4** shows the respective values in all investigated cultivars sorted by their tolerance. Put showed the highest genotypic variation, especially under control conditions, with values ranging from 120 to 4230 nmol g−1 DW. Under control conditions, Put content was significantly higher in the more sensitive cultivars including *indica* and *japonica* ssp. compared to more tolerant ones. For the eight most sensitive cultivars, Put values under control conditions ranged from 1450 to 4230 nmol g−1 DW, whereas values in the eight most tolerant cultivars reached only values from 120 to 1166 nmol g−1 DW. After 14 days of salt treatment a strong decrease of Put at both NaCl levels was restricted to the eight most sensitive cultivars

with 11 and 1.5% of the control values for cultivars 2 and 50 at 100 mM NaCl, respectively. All other cultivars showed either an increase (26, 27) or no significant change. The ratio of Put at 50 and 100 mM NaCl in comparison to control conditions was negatively correlated with the salt sensitivity score, confirming a stronger relative decrease of Put content in sensitive as compared to tolerant cultivars (**Table 2**). Strikingly, the most sensitive cultivars contained the highest absolute amounts of Put under control **Table 2 | Spearman correlation analysis between the mean sensitivity rank at 100 mM NaCl and the ratio of all available growth parameters under stress compared to control conditions, absolute polyamine content in nmol g−1 DW and ratio of polyamine content under stress in comparison to control conditions.**

*P-values below 0.05 are highlighted in italic, below 0.01 highlighted in boldface and italic, and below 0.001 highlighted in boldface numbers.*

conditions, indicated by a positive correlation of Put content with the salt sensitivity score (*r* = 0.82, *p* = 0.00003).

In contrast to Put, Spd content was not changed under salinity, except for a significant reduction at 100 mM NaCl in the most sensitive cultivars 51 and 50 (to 40 and 28%, respectively) and an increase at 100 mM NaCl in the most tolerant cultivar 27 (to 181%) that was, however, not significant (**Figure 4**). Surprisingly, absolute Spd levels at 100 mM NaCl showed a significant negative correlation (*r* = −0.56, *p* = 0.0155) with salt sensitivity and also the ratio of Spd at 100 mM NaCl in comparison to control was negatively correlated with salt sensitivity (**Table 2**).

General increases of Spm levels were observed in all cultivars under both salt stress conditions with up to 3-fold increases at 100 mM NaCl. However, the predominant compound in most cultivars under salinity conditions (100 mM) was still Put followed by Spd and Spm with the exception of the three most sensitive cultivars 53, 51, and 50 with a higher Spm content, followed by Spd and Put.

#### **EXPRESSION ANALYSIS OF POLYAMINE BIOSYNTHESIS GENES**

Expression of 16 genes encoding enzymes involved in polyamine biosynthesis was analyzed in leaves of eight cultivars after 14 days of salt treatment (50 and 100 mM NaCl) using qRT-PCR. The eight cultivars were selected based on their widely differing salt tolerance and included both subspecies with cultivar 52, 14, and 1 belonging to the more tolerant, 5, 4, 22, 2, and 50 to the more and most sensitive ones. The log2 fold change between gene expression under salt compared to control conditions is shown in **Figure 5**. In general, increased gene expression is more obvious at 50 mM than at 100 mM NaCl with the exception of *ODC1* in some cultivars and *SPD/SPM2* and *SPD/SPM3* in all cultivars. At 50 mM NaCl *ADC2*, a gene involved in the synthesis of Put, was induced in almost all cultivars except cultivar 5 and the most sensitive cultivar 50. Also *SPD/SPM2* and *SPD/SPM3* were induced in almost all cultivars under this condition. There are two alternative pathways to synthesize Put, either indirectly from arginine, catalyzed by the enzymes ADC, AIH, and CPA, or directly from ornithine, catalyzed by ODC. In two of the tolerant cultivars, genes encoding enzymes for both pathways (*ADC2, ODC1*) were induced by salt stress, whereas in sensitive cultivars the expression of only one of these genes, either *ADC2* or *ODC1* was increased.

The fold change in the expression of genes encoding enzymes involved in the following steps of the polyamine biosynthesis pathway, leading to the synthesis of Spd and Spm, varied for cultivars and genes with *SAMDC2* being induced in most of the cultivars at both salt levels. Additionally, *SPD/SPM2* and *SPD/SPM3* were induced in all cultivars under both conditions. No correlations could be found between the log2 fold change of gene expression and the salt sensitivity of the cultivars.

#### **COMPARISON OF THE SALT AND DROUGHT RESPONSES OF POLYAMINE METABOLISM AND GENE EXPRESSION**

For comparison of the responses of rice to salt and drought stress at the level of polyamine metabolism, data from three salttolerant (52, 14, 1) and three salt-sensitive cultivars (22, 2, 50) were summarized and averaged. These cultivars were chosen to allow comparison of their responses to drought stress investigated in a previous study (Do et al., 2013). Do and co-workers analyzed drought tolerance and the response of polyamine metabolism in 21 rice cultivars with an overlap of nine cultivars between the two studies. For the comparison, three drought-sensitive cultivars (22, 2, 50) and three drought-tolerant cultivars (1, 4, 52) were selected. Cultivar 14 in our selection of salt-tolerant cultivars, had to be replaced by 4 in the selection of drought-tolerant cultivars, because cultivar 14 was not drought-tolerant.

Average polyamine levels (Put, Spd, Spm) of the three tolerant and sensitive cultivars were compared under drought and salt (50, 100 mM NaCl) conditions (**Figure 6**). Under control conditions Put levels were similar for the different growth conditions (growth in sand for drought and hydroponic culture for salt stress) with values between 100 and 800 nmol g−1 DW except for sensitive cultivars in hydroponic culture with over 4150 nmol Put g−1 DW. Put levels were clearly reduced under both drought and salt conditions with the exception of tolerant cultivars at 50 and 100 mM NaCl. Spd values were slightly lower in hydroponic culture compared to plants grown in sand under control conditions. While Spd was significantly reduced after 18 days of drought stress in all cultivars, no significant changes were observed under salt stress. Spm levels were also similar for both growth conditions in the absence of stress and were significantly elevated in sensitive

cultivars under drought and both salt treatments, while they rose only at 100 mM NaCl in the tolerant cultivars.

In summary, tolerant cultivars are able to keep their Put levels constant under salt stress and increase their Spm levels only slightly at the high salt concentration, whereas in sensitive cultivars Put levels are strongly decreased and Spm levels increased. Under drought conditions, polyamine levels show the same pattern in sensitive cultivars with reduced Put and increased Spm levels. In contrast, the pattern in drought tolerant cultivars under drought is different from the salt response with strongly decreased Put and Spd levels and no changes of Spm.

To compare gene expression levels under different culture conditions, we calculated the average gene expression of the three tolerant and sensitive cultivars, respectively. A positive value of relative expression (log2) represents a higher expression of a GOI in comparison to the housekeeping genes and a negative value a lower expression. Differences in gene expression between growth conditions could be observed for *ADC1*, with a lower expression in sand-grown plants compared to plants grown hydroponically,

conditions. Each value represents the mean (±s.e.m.) of three cultivars with five replicate plants each. Significance levels are indicated as: *p* < 0.001 (∗∗∗), 0.001 < *p* < 0.01 (∗∗), 0.01 < *p* < 0.05 (∗) in comparison to the respective control.

and *ODC1*, showing a higher expression in salt-sensitive cultivars grown in hydroponic culture (**Figure 7**).

Additionally, log2 fold change values of average gene expression of the selected tolerant and sensitive cultivars under both stresses were calculated (**Figure 8**). At drought and salt conditions compared to control *ADC1* expression was reduced in both groups with the highest reduction in sensitive cultivars at 100 mM NaCl, while *ADC2* was highly induced under stress with the exception of sensitive cultivars at 100 mM NaCl. Log2fold change of gene expression of *ADC3* showed a fluctuating pattern with the strongest reduction again in sensitive cultivars at 100 mM NaCl. This seemed to be partly compensated by a high induction of *ODC1*, which catalyzes an alternative pathway of Put synthesis. On the other hand, *ODC1* is also induced under all other conditions except for drought in tolerant and at 50 mM NaCl in sensitive cultivars. Whereas *SAMDC2* was always induced in sensitive cultivars under all conditions, this induction was only observed under drought in tolerant cultivars. Log2 fold change of gene expression of *SAMDC4* showed a clear stress specific pattern, whereas *SPD/SPM2*, *SPD/SPM3,* and *SPD/SPM4* (with one exception) were generally stress induced in both tolerance groups.

Based on expression profiles of cultivars differing in tolerance, the response in gene expression after drought and salt stress could be differentiated for 13 genes. The investigated genes were divided into generally stress-induced genes *(ADC2*, *ODC1*, *SPD/SPM2*, *SPD/SPM3*) with two genes showing tolerance related differences under salt (*ADC2*) or drought conditions (*ODC1)*, one generally stress-repressed gene *(ADC1*), constitutively expressed genes *(ADC3, CPA1, CPA2, CPA4*, *SAMDC1, SPD/SPM1*), one specifically drought-induced gene *(AIH*), one specifically drought-repressed gene *(CPA3*), and one specifically

housekeeping genes is shown for drought- and salt-tolerant (52, 4, 1) and

salt-stress repressed gene *(SAMDC4*) revealing both overlapping and specific stress responses.

## **DISCUSSION**

Despite several published studies on the response of polyamine metabolism to salt stress in rice, comparative analyses of a large number of cultivars are rare. For the present investigation of the polyamine response in 18 cultivars, a detailed physiological characterization at two different salt concentrations in an early vegetative stage was performed and revealed a large variation of salt tolerance among the cultivars based on a rank of scoring data that indicated a higher salt sensitivity of *japonica* as compared to *indica* cultivars. It was previously reported that salt tolerance of *indica* cultivars was higher than that of *japonica* cultivars, denoted by a lower reduction of growth and a better Na+ exclusion (Lee et al., 2003). Among the cultivars investigated in the present study, Nipponbare was known to be salt-sensitive (Karan et al., 2012), while for all other cultivars no salt tolerance classification was available. Our phenotypic ranking was supported by a decrease of photosynthetic quantum yield and a reduction of FW under salt stress, especially in sensitive cultivars. Decreased photosynthetic yield is well known from previous studies (Lutts et al., 1996; Yamamoto et al., 2004) and the lack of an effect in a salt tolerant cultivar (Pokkali) was also shown (Dionisio-Sese and Tobita, 2000). Reduced FW under salinity was also described by Su and Wu (2004), who used this parameter for the classification of salt tolerance. The three most sensitive cultivars, 50, 51, and 53, all *japonica* ssp., as well as the two most tolerant cultivars, 26 and 27 (*indica*), were also clustered according to their metabolomic profiles considering changes of four metabolite pools under salinity suggesting a similar metabolic state (Zuther et al., 2007).

tolerant cultivars.

**FIGURE 8 | Log2 fold change of expression of genes, encoding enzymes involved in polyamine biosynthesis, under drought and salt conditions as compared to control in three tolerant vs. sensitive cultivars of the respective stress condition.** Log2 fold change of gene expression is shown for drought- and salt-tolerant (T) (52, 4, 1) and drought- (22, 2, 50) and salt-sensitive cultivars (S) (22, 2, 50). Data represent the means of three cultivars from either one or two biological replicates with two technical replicates each.

#### **EFFECTS OF SALINITY STRESS ON FREE POLYAMINE CONTENTS IN LEAVES**

A general increase of Spm was observed under salinity conditions for all cultivars in the present study, which is in agreement with previous results obtained in rice (Krishnamurthy and Bhagwat, 1989; Maiale et al., 2004) and other species (Sanchez et al., 2005). Spm may be involved in the stabilization of membranes (Tassoni et al., 1998; Rajasekaran and Blake, 1999) and nucleic acids (Hultgren and Rau, 2004), scavenging of free radicals (Lester, 2000), osmotic adjustment (Aziz et al., 1999), regulation of ion nutrition (Chattopadhyay et al., 2002), and regulation of senescence (Lahiri et al., 2004). It can improve the viability of protoplasts (Tiburcio et al., 1986) and participates in the prevention of electrolyte leakage and chlorophyll loss (Chattopadhyay et al., 2002) as well as enhanced stem elongation growth (Rajasekaran and Blake, 1999). Polyamines are also able to block ion channels thereby reducing NaCl-induced K+ efflux proportional to their charge (Zhao et al., 2007). Furthermore Spm can provoke a net Ca2+-efflux which might influence ROS and PA signaling (Pottosin et al., 2012).

These multiple functions suggest a beneficial role for Spm under stress conditons, independent of the tolerance level. Spm was also identified as the polyamine responsible for salt acclimation in *Arabidopsis* using transgenic lines overexpressing oat ADC (Alet et al., 2011b). Furthermore it was shown that *Arabidopsis* mutants, defective in the synthesis of Spm (*spms-1*), accumulated more Na+ and were impaired in survival experiments compared to control (Alet et al., 2012).

Despite only slight changes of Spd content in most of the cultivars under salinity, a significant negative correlation of Spd levels and of the changes under salt stress with salt sensitivity was found. This is in agreement with findings of Krishnamurthy and Bhagwat (1989) who reported that salt-tolerant rice accumulates high levels of Spd and Spm. Over-expression of *SPD* from *Cucurbita ficifolia* in *Arabidopsis* results in significantly increased Spd content in leaves and in enhanced tolerance to various abiotic stresses (Kasukabe et al., 2004). Spd treatment also induced recovery from salinity-induced damage of the plasma membrane and PM-bound H+-ATPase in salt-tolerant as well as salt-sensitive cultivars (Roy et al., 2005).

Strikingly, polyamine levels changed differently in tolerant and sensitive cultivars, with higher levels of Put under control conditions and a stronger decrease under salt stress in sensitive cultivars. This resulted in a shift of the predominant polyamine at 100 mM NaCl from Put in tolerant to Spm in the three most sensitive cultivars. These results may help to explain contradictory findings for changes of Put levels in the literature. It was earlier suggested that endogenous levels of Put might be limiting for salt resistance (Gupta et al., 2013). Our results are in agreement with Maiale et al. (2004), who also found a larger decrease of Put in sensitive as compared to tolerant cultivars. However, it has also been reported that Put strongly accumulates in response to osmotic (Flores and Galston, 1982; Aziz and Larher, 1995; Liu et al., 2004) and salinity stress (Basu et al., 1988; Krishnamurthy and Bhagwat, 1989; Katiyar and Dubey, 1990; Lefèvre et al., 2001). This accumulation was considered to be protective, conferring a selective advantage to the stressed plants. Put was reported to stabilize membranes (Prakash and Prathapsenan, 1988) and to counteract the Na+ and Cl− accumulation and induction of the K+ efflux (Prakash and Prathapsenan, 1988; Ndayiragije and Lutts, 2006). Polyamine specificity is more and more discussed with dominant polyamines generating a specific signature for the response to a specific stress (Pottosin et al., 2014). Tolerance dependent differences of Put levels under control conditions were independent of subspecies and might be used as potential markers for future breeding efforts.

## **EXPRESSION LEVELS OF GENES ENCODING ENZYMES INVOLVED IN POLYAMINE BIOSYNTHESIS UNDER SALINITY CONDITIONS**

To elucidate the molecular basis for changes in polyamine biosynthesis in response to salinity conditions, expression levels of genes encoding enzymes involved in polyamine biosynthesis were analyzed. The expression of many of these genes under different stress conditions and at different growth stages has been analyzed before (Li and Chen, 2000a,b; Kwak and Lee, 2001; Piotrowski et al., 2003; Tian et al., 2004; Hao et al., 2005a; Rodríguez-Kessler et al., 2006). However, the expression profiles of all genes involved in polyamine biosynthesis were previously only studied in rice under drought (Do et al., 2013), but not under salt stress. In *Arabidopsis*, the expression of all genes was investigated under dehydration (Alcázar et al., 2006b), while studies with *Arabidopsis* under salt, dehydration, cold and ABA treatments (Urano et al., 2003) and with maize under salt stress (Rodríguez-Kessler et al., 2006) did not include expressions of *AIH* and *CPA*.

We found expression levels of *ADC2*, *SAMDC2*, *SPD/SPM2,* and *SPD/SPM3* induced at 50 mM NaCl in most of the cultivars. Among the three *ADC* genes, mainly *ADC2* was up-regulated. *ADC2* was also found to be stress-induced in *A. thaliana* (Soyka and Heyer, 1999), and mustard (Mo and Pua, 2002). Whereas we found no correlation between the log2 fold change of gene expression and the salt sensitivity of the cultivars, another study reported a correlation of the accumulation of an *ADC* transcript with salt stress tolerance in rice (Chattopadhyay et al., 1997). In addition, tolerant cultivars activated two pathways to synthesize Put via arginine and ornithine by higher expression of *ADC2* and *ODC1*, whereas sensitive cultivars only induced one of these genes. Increased synthesis of Put catalyzed by two alternative pathways might be advantageous for the further accumulation of Spm catalyzed by, e.g., *SAMDC2*, *SPD/SPM2,* and *SPD/SPM3*. Enzyme activity measurements will be necessary to confidently link gene expression data to polyamine pool sizes.

#### **COMPARATIVE ANALYSIS OF POLYAMINES UNDER DROUGHT AND SALT STRESS**

Our results clearly show that Spm content was significantly increased under both, drought, and salinity conditions, except for tolerant cultivars under drought, which kept their already high initial levels. Under drought stress Spm became the most prominent polyamine, whereas this was only true for the most sensitive cultivars at 100 mM NaCl. The increase in Spm content is consistent with a report by Maiale et al. (2004) for rice under salinity conditions, but contradictory to reports by Krishnamurthy and Bhagwat (1989) for salt and by Liu et al. (2004) for osmotic stress. In these studies, tolerant rice cultivars accumulated higher levels of Spd and Spm, while sensitive rice cultivars showed low levels of these substances and an increase in Put levels. Nevertheless, Spm accumulation seems to be a general feature of plant responses to drought and salinity stress, although its physiological role under stress is still partly unknown. Elevated polyamine levels under salt stress seem to have self-protecting effects due to the modulation of ion channels thereby mediating ion flux homeostasis (Zhao et al., 2007). In roots the immediate effect of polyamines on NaCl-induced K+ efflux was dependent on the plant and the polyamine and ranged from beneficial to detrimental (Pottosin et al., 2014). In barley alterations of K+-homeostasis, caused by interaction between polyamines and ROS, contributed substantially to genetic variability in salt-sensitivity (Velarde-Buendía et al., 2012).

Together with Spm, Spd may also be involved in the response of plants to stress, e.g., through the induction of stomatal closure (Liu et al., 2000), prevention of chlorophyll loss (Chattopadhyay et al., 2002), stabilization of membranes (Rajasekaran and Blake, 1999) and scavenging of free radicals (Velikova et al., 1998). Several studies reported an accumulation of Spd under salt (Krishnamurthy and Bhagwat, 1989; Basu and Ghosh, 1991) and osmotic stress (Tiburcio et al., 1986; Li and Chen, 2000a). In contrast, decreased Spd levels in response to stress were also reported under salt (Maiale et al., 2004; Sanchez et al., 2005), osmotic (Aziz et al., 1997), and drought stress (Turner and Stewart, 1986). In this study, a reduction of Spd was only observed under drought conditions, whereas under salinity condition the Spd content was unchanged. The ability of Spd to prevent the uptake of Na+ and the loss of K+ (Chattopadhyay et al., 2002) may suggest that high Spd levels could be more important under salt than under drought stress.

Put levels decreased in our experiments in leaves under drought stress independent of tolerance of the cultivar, while under salinity conditions they were not changed in tolerant and sharply decreased in sensitive cultivars. For salt-sensitive cultivars threefold higher Put levels were observed under control conditions in hydroponic culture compared to cultivation in sand, with Put levels at 50 mM NaCl reaching values comparable to values under control conditions in sand grown plants. Nevertheless this will not affect the comparison between different stress conditions due to the restriction to relative changes in Put levels in comparison to control levels. The decrease of Put levels in all cultivars under drought and in sensitive cultivars under salinity conditions could be caused by the higher substrate need for the Spm synthesis. A strong metabolic canalization of Put into Spm synthesis induced by drought was also described for *Arabidopsis* and *Craterostigma plantagineum* but did not lead to Spm accumulation in *Arabidopsis* due to a Spm-to-Put back-conversion (Alcázar et al., 2011).

A Spm-to-Put back-conversion by polyamine oxidase (PAO) might have also occurred, indicated by the fact that Spm accumulation after salt stress was lower in salt sensitive than in salt tolerant cultivars. On the other hand, Put levels in poplar and tomato did not affect Spm levels, while Spd and Spm levels are inter-dependent (Mattoo et al., 2010). Reduced Put levels could be also reached by the action of DAO yielding pyrroline, H2O2 and ammonia (Moschou et al., 2012) but avoiding high Put levels, which might be toxic for plants (Slocum et al., 1984; Panicot et al., 2002). For an estimation of degradation processes an analysis of gene expression and enzyme activities of DAO and PAO would be necessary. Due to the involvement of polyamines in stress response as well as programmed cell death the balance between intracellular polyamine concentrations and polyamine catabolism resulting in ROS generation in the apoplast will be crucial for the survival of plants (Pottosin et al., 2014).

Another interesting finding of our study was a descending gradient of Put levels from sensitive to tolerant cultivars already under control conditions. This was also shown previously for the Put levels and drought tolerance of a set of 21 rice cultivars (Do et al., 2013). The higher Put content in sensitive cultivars under control conditions might be a useful tool for breeders to select against sensitivity in breeding programs. In conclusion, we have shown that polyamines are strongly involved in the response of rice to drought and salinity stress. From our results and previous reports we hypothesize that Spm contributes to the drought and salinity tolerance of rice, while the involvement of Spd and Put particularly in salt tolerance still needs to be clarified.

## **COMPARISON OF EXPRESSION OF GENES ENCODING ENZYMES INVOLVED IN POLYAMINE BIOSYNTHESIS BETWEEN SALT AND DROUGHT STRESS**

The comparison of tolerant and sensitive cultivars under the different stress conditions indicated a general up-regulation of *ADC2*, except for sensitive cultivars at 100 mM NaCl. This higher expression could provide an advantage for stress adaptation, e.g., through the *de novo* synthesis of Put as a substrate for longer chain polyamines. Arabidopsis plants over-expressing the *ADC* gene from oat under the control of a stress-inducible promoter were more resistant to dehydration stress associated with an increase of putrescine levels (Alet et al., 2011a). *ADC2* was additionally identified as salt- and drought-induced in *Arabidopsis* in several studies, as reviewed in Alcázar et al. (2006a). Under salt stress at 100 mM NaCl cultivars of the sensitive group seem to compensate the lower *ADC2* induction by the induction of *ODC1*, while tolerant cultivars activated both pathways under salinity which is in contradiction to reports showing that the ADC pathway for polyamine biosynthesis is predominant in higher plants (Birecka et al., 1985; Rajam, 1993; Rodríguez-Kessler et al., 2006). The existence of two alternative routes for the synthesis of Put could be explained by the differential compartmentation of the two enzymes resulting in the specific regulation of different plant processes. With the localization of ADC in chloroplasts, polyamines synthesized via the ADC pathway seem to play a role in maintaining photosynthetic activity (Borrell et al., 1995). Additionally, ADC is thought to be the enzyme primarily responsible for abiotic stress-induced Put accumulation (Galston and Sawhney, 1990; Tiburcio et al., 1997). ODC is found in the nucleus (Slocum, 1991) and mitochondria (Acosta et al., 2005). It has been suggested that ODC is involved in the regulation of cell division/proliferation in growing plant tissues, while ADC is involved in cell expansion (Cohen, 1998).

The transcript level of *AIH* was reported to be up-regulated under drought stress in tolerant as well as sensitive cultivars (Do et al., 2013), but was unchanged in response to salt stress. One report is available for *A. thaliana* under dehydration stress (Alcázar et al., 2006b), where the expression level of *AIH* was slightly increased, in agreement with the results from rice under drought stress.

The *CPA* genes of rice show different expression levels under control conditions both in plants grown in sand and in hydroponic culture. The expression levels of the four genes are related as follows: *CPA*1 > *CPA*2 ≥ *CPA*4 > *CPA*3. Except for a reduction in the expression level of *CPA3* under drought stress (Do et al., 2013), no change was detectable for any *CPA* gene under drought or salt stress, showing that these genes are constitutively expressed. This result is in accordance with the observation that the transcript level of the *CPA* gene in *A. thaliana*, which is similar to *CPA1* from rice, was not altered by osmotic stress (Piotrowski et al., 2003). A drought-dependent repression of *CPA3* in rice has not been reported before.

**FIGURE 9 | Changes of polyamine metabolism in response to salt (A) and drought stress (B) in rice leaves of tolerant and sensitive cultivars.** Changes of polyamines are shown for tolerant (solid line) or sensitive cultivars (dotted line). A decrease is shown in red, an increase in blue. Changes in gene expression under stress conditions are color coded as

follows: constitutively expressed—gray, induced by drought and salt stress—blue, induced by drought—purple, repressed by salt and drought stress—orange, repressed by drought stress—yellow, repressed by salt stress—pink. Arg, arginine; Orn, ornithine; SAM, S-adenosylmethionine; dcSAM, decarboxylated S-adenosylmethionine.

Several enzymes are involved in the pathway from Put to Spd and Spm, including *SAMDC* and *SPD/SPM*. In response to drought and salt stress, the expression levels of *SAMDC2*, *SPD/SPM2*, and *SPD/SPM3* were up-regulated with the exception of *SAMDC2* in tolerant cultivars under salt stress. Increased expression of *SAMDC* was also reported in rice (Li and Chen, 2000b; Kawasaki et al., 2001; Rabbani et al., 2003; Shiozaki et al., 2005), wheat (Li and Chen, 2000a), soybean (Tian et al., 2004), and *Arabidopsis* (Alcázar et al., 2006b) under drought and salt, and in maize (Rodríguez-Kessler et al., 2006) for *SPD/SPM* under salt conditions. In addition, a tolerant rice cultivar responded more quickly to salt and reached the highest expression level of the *SAMDC* gene under short-term stress, while the sensitive cultivar reached higher levels after a prolonged time of stress (Li and Chen, 2000b). In our study, a differential regulation of *SAMDC* genes among the different tolerance groups was not observed. However, *SAMDC4* was specifically down-regulated at 100 mM NaCl. Under all conditions and over all tolerance groups an induction of *SPD/*SPM*2*, *SPD/SPM3*, and *SPD/SPM4* genes was observed. A spermine synthase was also shown to be saltand drought-induced in *Arabidopsis*, as reviewed in Alcázar et al. (2006a), and a spermine synthase mutant of *Arabidopsis* was shown to be more sensitive to drought and salt stress (Yamaguchi et al., 2007).

Changes in gene expression and in polyamine levels in differentially tolerant cultivars under salt or drought stress conditions are summarized in **Figure 9**. By comparing the gene expression analyses under salt and drought stress in tolerant and sensitive cultivars, three genes that had previously been classified as drought-induced (Do et al., 2013) are now identified as generally stress-induced (*ADC2*, *SPD/SPM2*, *SPD/SPM3*), whereas *AIH* was confirmed as specifically drought-induced. *SAMDC2*, on the other hand, was induced by drought and was only induced by salt stress in sensitive cultivars. *ADC1*, which was previously classified as drought-repressed could now be identified as generally stress-repressed, whereas *CPA3* was confirmed as specifically drought-repressed. Only *SAMDC4* could be identified as saltstress repressed gene. Five genes that were previously classified in drought stress experiments (Do et al., 2013) as constitutively expressed (*CPA1, CPA2, CPA4, SAMDC1, SPD/SPM1*) were confirmed under salt stress.

#### **ACKNOWLEDGMENTS**

We thank Prof. Le Tran Binh from the Institute of Biotechnology, Hanoi, Vietnam, and Ruaraidh Sackville Hamilton and Gary Atlin from the IRRI, Manila, Philippines, for providing rice germplasm. We thank Ulrike Seider for excellent technical assistance. This project has received financial support by the German Federal Ministry of Education and Research through the project 0312854 (Improving the quality of rice with respect to abiotic stress resistance and nutritional parameters). Phuc T. Do was financially supported by a PhD fellowship from the Vietnamese Ministry of Education and Training and the Max-Planck-Society.

#### **REFERENCES**

Acosta, C., Perez-Amador, M. A., Carbonell, J., and Granell, A. (2005). The two ways to produce putrescine in tomato are cell-specific during normal development. *Plant Sci.* 168, 1053–1057. doi: 10.1016/j.plantsci.2004.12.006

L.) cultivars as affected by salinity stress. *Plant Mol. Biol.* 34, 477–493. doi: 10.1023/A:1005802320672

Zuther, E., Koehl, K., and Kopka, J. (2007). "Comparative metabolome analysis of the salt response in breeding cultivars of rice," in *Advances in Molecular Breeding Toward Drought and Salt Tolerant Crops*, eds M. A. Jenks et al. (Dordrecht: Springer Publishing, Inc.), 285–315.

**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.

*Received: 18 February 2014; accepted: 17 April 2014; published online: 08 May 2014. Citation: Do PT, Drechsel O, Heyer AG, Hincha DK and Zuther E (2014) Changes in free polyamine levels, expression of polyamine biosynthesis genes, and performance of rice cultivars under salt stress: a comparison with responses to drought. Front. Plant Sci. 5:182. doi: 10.3389/fpls.2014.00182*

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

*Copyright © 2014 Do, Drechsel, Heyer, Hincha and Zuther. 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) or licensor 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.*

## Physiological and molecular implications of plant polyamine metabolism during biotic interactions

## *Juan F. Jiménez-Bremont1, María Marina2 , María de la Luz Guerrero-González1, Franco R. Rossi 2 , Diana Sánchez-Rangel1, Margarita Rodríguez-Kessler 3 , Oscar A. Ruiz 4 and Andrés Gárriz 2 \**

*1 División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, México*

*2 UB3, Instituto de Investigaciones Biotecnológicas, Instituto Tecnológico de Chascomús, Universidad Nacional de San Martín, Consejo Nacional de Investigaciones Científicas y Técnicas, Chascomús, Argentina*

*3 Facultad de Ciencias, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México*

*4 UB1, Instituto de Investigaciones Biotecnológicas, Instituto Tecnológico de Chascomús, Universidad Nacional de San Martín, Consejo Nacional de Investigaciones Científicas y Técnicas, Chascomús, Argentina*

#### *Edited by:*

*Ruben Alcazar, Universitat de Barcelona, Spain*

#### *Reviewed by:*

*Francisco Marco, Universitat de València, Spain Panagiotis N. Moschou, Swedish University of Agricultural Sciences, Sweden*

#### *\*Correspondence:*

*Andrés Gárriz, UB3, Instituto de Investigaciones Biotecnológicas, Instituto Tecnológico de Chascomús, Universidad Nacional de San Martín, Consejo Nacional de Investigaciones Científicas y Técnicas, Intendente Marino Km 8.2, Chascomús, Buenos Aires 7130, Argentina e-mail<EMAIL_ADDRESS>
During ontogeny, plants interact with a wide variety of microorganisms. The association with mutualistic microbes results in benefits for the plant. By contrast, pathogens may cause a remarkable impairment of plant growth and development. Both types of plant– microbe interactions provoke notable changes in the polyamine (PA) metabolism of the host and/or the microbe, being each interaction a complex and dynamic process. It has been well documented that the levels of free and conjugated PAs undergo profound changes in plant tissues during the interaction with microorganisms. In general, this is correlated with a precise and coordinated regulation of PA biosynthetic and catabolic enzymes. Interestingly, some evidence suggests that the relative importance of these metabolic pathways may depend on the nature of the microorganism, a concept that stems from the fact that these amines mediate the activation of plant defense mechanisms.This effect is mediated mostly through PA oxidation, even though part of the response is activated by non-oxidized PAs. In the last years, a great deal of effort has been devoted to profile plant gene expression following microorganism recognition. In addition, the phenotypes of transgenic and mutant plants in PA metabolism genes have been assessed. In this review, we integrate the current knowledge on this field and analyze the possible roles of these amines during the interaction of plants with microbes.

**Keywords: polyamines, spermidine, spermine, putrescine, thermospermine, plant pathogen, mutualism**

## **INTRODUCTION**

The aliphatic polycationic compounds collectively known as polyamines (PAs) have proven to be essential for growth and development in all living organisms (Cohen, 1998). PAs participate in many fundamental processes such as chromatin condensation, DNA replication and transcription, RNA processing, protein synthesis and post-translational modification (Igarashi and Kashiwagi, 2000; Childs et al., 2003; Wallace et al., 2003). The most common PAs in nature are the diamine putrescine (Put), and those traditionally called higher PAs as the triamine spermidine (Spd) and the tetraamines spermine (Spm) and thermospermine (TSpm). In addition, other compounds are also regarded as PAs, such as the diamines 1,3-diaminopropane (DAP) and cadaverine (Cad). Uncommon PAs show a limited distribution in nature and consists of Spd and Spm-related molecules as nor-Spd, nor-Spm, homo-Spd, homo-Spm, and longer pentaand hexaamines (Bagni and Tassoni, 2001; Fuell et al., 2010; Sagor et al., 2013).

In plant tissues, the relative abundance of PAs depends on the species and the developmental stage. They are detected in actively growing plant tissues and in plants exposed to biotic or abiotic stress (Jimenez-Bremont et al., 2007; Rodriguez-Kessler et al., 2008; Alcázar et al., 2010; Gill and Tuteja, 2010; Hussain et al., 2011). It has been demonstrated that PAs are important for the normal course of diverse plant ontogenic processes such as cell division and elongation, organogenesis and somatic and zygotic embryogenesis (Hummel et al., 2004; Paschalidis and Roubelakis-Angelakis, 2005; Mattoo et al., 2010). In general terms, the levels of Put in plants are higher than those of the other PAs (Cohen, 1998). Cad can also be found in considerable high concentrations even though it seems to be limited to legumes and a few species belonging to other plant families (Tomar et al., 2013). Additionally, PAs may exist in soluble as well as insoluble fractions. The soluble fraction consists of free PAs and PAs conjugated to organic acids, mainly hydroxycinnamic acid. In turn, the insoluble fraction is formed by PAs bound to macromolecules such as proteins, cell wall polysaccharides and plasma membrane (Martin-Tanguy, 2001).

Although the exact mechanism of action of PAs remains still elusive, many authors have proposed that the spatial separation of their positive charges under physiological pH plays a crucial role. This feature gives them the capacity to bind negatively charged molecules such as nucleic acids, phospholipids, and proteins; and it is thought that by these means PAs affect the structure and function of these macromolecules. Moreover, the formation of these compounds protects macromolecules

from degradation and modification (Ruiz-Herrera et al., 1995; D'Agostino et al., 2005).

There is a considerable amount of information about the role played by PAs during the interactions of plants with either pathogenic or beneficial microbes. These data indicate that changes in PA metabolism constitute a key adaptive response of the plant, and that their occurrence determines the development of the interaction. In turn, evidence suggests that some microorganisms are able to perturb plant PA metabolism in order to adjust it to their own requirements.

In this review, we integrate the current knowledge on the modulation of plant PA metabolism upon pathogen recognition and its importance for plant defense, giving particular emphasis in the advances achieved by the scientific community in the last few years. In addition, we discuss the importance of PAs in beneficial plant interactions, as those established by nitrogen-fixing bacteria and mycorrhizalfungi. The impact of biotechnological approaches and genetic engineered plants with altered PA levels in plant–microbe interactions is also reviewed.

#### **PA METABOLISM IN PLANTS**

The concentration of PAs is finely controlled in eukaryotic cells in order to fulfill the requirements at any time. Precise regulation of PA levels is important since not only its depletion, but also extensive PA accumulation may be deleterious (Ditomaso et al., 1989; He et al., 1993; Poulin et al., 1993). Therefore, several processes such as PA synthesis, catabolism, transport, compartmentalization, and conjugation act in concert to maintain the concentration of these compounds in suitable levels (reviewed by Kusano et al., 2007; Carbonell and Blázquez, 2009). We briefly summarize below the principal aspects of plant PA metabolism.

#### **BIOSYNTHESIS OF PAs**

In plants, the synthesis of Put may be conducted by two pathways (Martin-Tanguy, 2001). One of these metabolic routes starts with the decarboxylation of the amino acid arginine by the enzyme arginine decarboxylase (ADC, EC <IP_ADDRESS>), located mainly in the chloroplast in photosynthetic cells but directed to the nucleus in non-photosynthetic cells (Borrell et al., 1995; Bortolotti et al., 2004). This reaction renders agmatine, which is then metabolized to N-carbamoylputrescine by agmatine iminohydrolase (AIH, EC <IP_ADDRESS>). Consequently, N-carbamoylputrescine is converted in Put by the enzyme N-carbamoylputrescine amidohydrolase (NCPAH, EC <IP_ADDRESS>). The other metabolic pathway begins with the amino acid ornithine converted into Put in a single-step reaction catalyzed by the enzyme ornithine decarboxylase (ODC, EC <IP_ADDRESS>), which is mainly found in the plant cytosol. The synthesis of higher PAs requires decarboxylated S-adenosylmethionine (dcSAM) as aminopropyl donor, generated by the action of the enzyme S-adenosylmethionine decarboxylase (SAMDC, EC <IP_ADDRESS>). dcSAM is then used by the enzyme spermidine synthase (SPDS, EC <IP_ADDRESS>) to add an aminopropyl moiety to Put, hence forming Spd. In a consecutive aminopropylation reaction, the enzyme spermine synthase (SPMS, EC <IP_ADDRESS>) adds an aminopropyl group to Spd converting it to Spm. Alternatively, thermospermine synthase (tSPMS, EC <IP_ADDRESS>) forms TSpm by adding the

aminopropyl group to the opposite end of the molecule of Spd.

The relative importance of ADC and ODC activitiesfor Put synthesis varies according to the plant species and the tissue involved (Cohen,1998; Hanfrey et al.,2001; Flemetakis et al.,2004). In some plant species the *ODC* gene is absent, as occur in many members of the *Brassicaseae* family including *Arabidopsis thaliana*, and in nonvascular plants such as *Physcomitrella patens* (Jiménez-Bremont et al., 2004; Fuell et al., 2010). In addition, whereas in most plants there is only one gene coding for ADC, two paralogous genes with different patterns of expression occurs in *Brassicaseae* members (Galloway et al., 1998; Hummel et al., 2001). The disruption of each *ADC* gene in *Arabidopsis* does not cause a major phenotypic change, but knocking down both has a lethal effect (Urano et al., 2005).

The *Arabidopsis* genome contains four genes coding for SAMDCs, two SPDSs, one SPMS and one tSPMS (known as *ACAULIS5*; Fuell et al., 2010). SPDS and SPMS seem to be localized in the cytoplasm and the nucleus (Belda-Palazón et al., 2012), while SAMDC appears to be mostly cytoplasmic (Torrigiani et al., 1986; Belda-Palazón et al., 2012). Plant SAMDCs are usually regulated at the translational level by PAs (Hanfrey et al., 2002, 2003). It has been shown that a small upstream open reading frame (uORF) in the SAMDC mRNA 5 leader sequence is responsible for RNA translation repression mediated by PAs. This mechanism regulates the availability of dcSAM for Spd and Spm synthesis, which seems to constitute the main point of regulation for the synthesis of higher PAs in these organisms (Fuell et al., 2010). The double mutants *atsamdc2*(*bud2*)-*atsamdc1* (Ge et al., 2006) and *atspds1-atspds2* (Imai et al., 2004b) are embryo lethal, indicating that higher PAs are essential for plant embryogenesis. This effect could be due to a deficiency in Spd synthesis and not Spm, since no phenotypic effect is evident in *spms* plants (Imai et al., 2004a). In turn, disruption of the *ACAULIS5* gene in *Arabidopsis* plants lead to a notable dwarf phenotype, demonstrating the importance of TSpm synthesis for plant growth (Imai et al., 2004a).

## **CATABOLISM OF PAs**

Polyamine catabolism is mediated by diamine oxidases (DAOs, EC <IP_ADDRESS>) and PA oxidases (PAOs, EC <IP_ADDRESS>). DAOs are homodimers with copper as the prosthetic group. These enzymes show a preference for diamines such as Put and Cad, and have low affinity for Spd and Spm. The oxidation of Put renders H2O2, NH4 + and γ-aminobutanal, the latter being spontaneously converted to -1-pyrroline. DAOs from legumes and *Arabidopsis* have been described to be directed to the extracellular space, remaining in this compartment loosely associated to cell walls (Federico and Angelini, 1991; Moschou et al., 2008b). Recently, it was demonstrated that *Arabidopsis* has at least ten putative *DAO* genes, four of which have been characterized: *AtCuAO1* and *AtAO1* encode for apoplastic proteins, whereas the products of *AtCuAO2* and *AtCuAO3* are found at the peroxisome (Planas-Portell et al., 2013). Interestingly, *DAO* genes show different expression profiles in response to external stimuli (Planas-Portell et al., 2013).

Polyamine oxidases use FAD as cofactor and oxidize mainly Spd and Spm. These enzymes may be involved in two different catabolic pathways: (1) terminal catabolism and (2) backconversion of PAs. PAOs acting in the terminal catabolism pathway oxidize Spd or Spm using molecular O2 as electron donor. In this case, DAP and H2O2 are released as final products, besides either 4-aminobutanal or (3-aminopropyl)-4-aminobutanal depending on whether Spd or Spm act as substrate, respectively. Alternatively, enzymes back-converting PAs transform Spm into Spd, and Spd into Put with the concomitant production of 3-aminopropanal and H2O2. Both types of reactions produce H2O2 as final product, which plays an essential role in plant–microbe interactions. Five genes coding for PAOs have been identified in *Arabidopsis*, showing cytosolic (AtPAO1 and AtPAO5) and peroxisomal (AtPAO2, AtPAO3, and AtPAO4) localization (Moschou et al., 2008c; Takahashi et al., 2010; Fincato et al., 2011).  The peroxisomal back-converting pathway has also been described in rice (Ono et al., 2012), even though all other enzymes characterized in monocotyledonous plants so far are involved in the terminal catabolic pathway (Tavladoraki et al., 1998; Šebela et al., 2001). It was recently found that *AtPAO2* in *Arabidopsis* is negatively modulated in a PA-dependent manner by a uORF located in the 5- -UTR sequence (Guerrero-González et al., 2014). A comparative analysis of several PAO transcripts from different plant species indicated that uORF sequences are very conserved, suggesting a common regulatory mechanism for these enzymes.

#### **TRANSPORT AND CONJUGATION OF PAs**

The transport of PAs into different cell compartments is a crucial step to regulate several cell processes (Igarashi and Kashiwagi, 2010; Mulangi et al., 2012b). Only PA importers have been characterized so far at the functional level in plant cells. The first evidence of PA import was found in carrot protoplasts and isolated vacuoles, and it was suggested that PA entrance is driven by the transmembrane electrical gradient and a possible antiport mechanism (Pistocchi et al., 1988). It was found that external pH and the concentration of calcium modulate PA uptake (Antognoni et al., 1994; Pistocchi et al., 1988). Recently, the identification and characterization of specific importers has been achieved in rice and *Arabidopsis* (Fujita et al., 2012; Mulangi et al., 2012a). The first of these proteins reported was OsPUT1 (polyamine uptake transporter) from rice. Radiological uptake and competitive inhibition experiments in a heterologous system revealed that OsPUT1 functions mainly as a Spd importer and that the gene is expressed in all tissues except for mature roots and seeds (Mulangi et al., 2012b). Five additional importers from *Arabidopsis* and rice were described to form part of the same clade with *OsPUT1* and show high affinity for Spd (Mulangi et al., 2012a). A LAT (L-type amino acid transporter) protein in *Arabidopsis* named RMV1 is involved in the proton-dependent incorporation of PAs with a high affinity for Spm (Fujita et al., 2012). Lastly, PA transport into phloem and xylem tissues has been reported (Friedman et al., 1986), but the cell components involved in this processes have not yet been characterized.

Conjugation of PAs is an additional mechanism used by plant cells to regulate the intracellular concentration of the free forms, and these conjugates may comprehend up to 90% of the total PA cell content in some plant species (Bagni and Tassoni, 2001). It has been shown in *Nicotiana attenuata* that different hydroxycinnamoyl transferases (HCTs, EC <IP_ADDRESS>) catalyze the conjugation of caffeoyl, cinammoyl, and feruloyl-CoA to Put or Spd (Onkokesung et al., 2012). It is unknown whether the deconjugation of these compounds is catalyzed by the same transferases. Interestingly, the genes coding for HCTs in *N. attenuata* are induced by the transcription factor R2R3-NaMYB8, which is known to mediate the defense responses against herbivore attack (Kaur et al., 2010).

## **PAs AND PLANT–MICROBE INTERACTIONS**

Plant PA metabolism undergoes remarkably changes during plant– microbe interactions (Walters, 2003; Hussain et al., 2011). The roles played by PAs during these processes are considerably intricate. This is due to the fact that PAs are not only essential to maintain cell viability, but function as signaling molecules regulating many of the responses that help the plant to cope with biotic stress. As will be discussed later, this effect may be exerted either directly by PAs or being mediated through their catabolism. The modulation of PA metabolism is so important for the outcome of the pathogenic interactions that not only the plant modifies PA concentration in response to pathogens, but some microorganisms have developed mechanisms to induce modifications of PA levels in host tissues. Therefore, it seems that when plants are attacked by pathogens, the organism that takes control of the PA machinery has a great opportunity to take the lead. The interaction of plants with beneficial microorganisms also induces changes in PA metabolism, and the establishment of mutualism with symbionts such as Rhizobia and Mycorrhizae appear to depend on PA levels. In the following section we aboard the evidences generated so far regarding the role played by PAs in plant associations with both pathogenic and beneficial microorganisms.

## **CHANGES IN PLANT PA METABOLISM DURING PATHOGENIC INTERACTIONS**

Research indicates that PAs participate actively during the interactions between plants and pathogens. It has been shown that PAs levels and the activity of PA metabolic enzymes augment in infected tissues during microbial colonization, which seems to be independent of the nature of the pathogen, i.e., biotroph or necrotroph (Walters, 2003; Hussain et al., 2011).

Biotrophic microorganisms feed on nutrients produced by the host living cells (Glazebrook, 2005) and the establishment of this type of interaction leads to the accumulation of PAs in plants. For instance, Greenland and Lewis (1984) reported an increase of Spd in barley (*Hordeum vulgare* L.) leaves infected with the rust fungus *Puccinia hordei*, while Spd and Put were increased in this plant species during the infection caused by the powdery mildew fungus *Blumeria graminis* f. sp. *hordei* (Walters et al., 1985). Similar effects were demonstrated to occur in the interaction between wheat and *Puccinia graminis* f. sp. *tritici* (Machatschke et al., 1990; Foster and Walters, 1992) and in bean plants treated with fungal

elicitors (Broetto et al., 2005). Importantly, the main increments in PA concentration during the interaction with biotrophs are found in leaf areas close to the pathogen entry point. These areas have been termed "green islands" and are correlated with the induction of photosynthesis and delay in senescence. The accumulation of PAs was explained on the basis of higher activities in plant PA biosynthetic enzymes, in particular that of ADC (Walters and Wylie, 1986; Coghlan and Walters, 1990). Nevertheless, this is not necessarily true for all the interactions involving microorganisms of this kind. For instance, the ODC seems to be responsible for the increment of PAs in wheat leaves infected by *Puccinia graminis* f. sp. *tritici* (Foster and Walters, 1992). In this particular case, the authors reported a reduction of ADC activity but a significant induction of ODC activity in the pustules, the sites were fungal spores brake through the plant epidermis. On these grounds, it was hypothesized that the ODC from the pathogen would be responsible for PA production at the infection site.

Variations in PA levels between resistant and susceptible cultivars in response to pathogens has been described. In this trend, the analysis of two genotypes of barley during the interaction with *B. graminis* f. sp. *hordei* revealed that the resistant cultivar accumulates higher Put and Spd levels than the susceptible one following pathogen infection (Cowley and Walters, 2002a,b; Asthir et al., 2004). Similarly, PAs are accumulated in resistant (but not susceptible) tobacco plants in response to tobacco mosaic virus (TMV; Marini et al., 2001). Accordingly, highly susceptible sugarcane buds infected with the smut fungus *Ustilago scitaminea* reduce free PA contents while increase their conjugation to phenolic acids (Legaz et al., 1998). The authors attributed the susceptibility of the plant to a reduction in the availability of free phenolic compounds with importance for defense rather than a diminution in free PA concentration. In contrast, *Arabidopsis* ecotypes Col-0 and Bur-0, which are susceptible and partially resistant to *Plasmodiophora brassicae* infection, respectively, did not show significant differences in PA contents when challenged with the pathogen. However, susceptible plants displayed a transient accumulation of the PA precursor agmatine and strong arginase activity (the enzyme catalyzing the transformation of arginine in ornithine), whereas partially resistant plants showed continuous agmatine production and weak arginase activity. Based on these observations, it was proposed that symptom development is associated to differences in arginine metabolism and agmatine production (Jubault et al., 2008).

Many studies have shown that any situation leading to free PA accumulation in plants is usually accompanied of a rise in the concentration of conjugated PAs and the induction of PA oxidase activity. This is important because both phenomena could account for the improvement in plant tolerance. Thus, conjugated PAs are accumulated in plant tissues in response to viral and fungal infections (Walters, 2003; Rodriguez-Kessler et al., 2008; Muroi et al., 2009), and it was shown that these compounds affect microbial growth and prevent the development of disease in plants (Martin-Tanguy et al., 1976; Mackintosh et al., 1997; Walters et al., 2001). Muroi et al. (2009) reported the accumulation of conjugates of agmatine and Put in leaves of *A. thaliana* infected with *Alternaria* *brassicicola*. The importance of these compounds in the defense against this pathogen was clearly demonstrated by the interruption of the *AtACT* gene, encoding an enzyme that conjugates PAs to hydroxycinnamic acid, which enhanced the plant susceptibility. In addition, it has been suggested that PA conjugates contribute to the formation of a phenolic barrier that makes cell walls more resistant to microbial enzymatic hydrolysis (Walters et al., 2001). In turn, PA oxidation could also have a positive effect on plant defense. DAO and PAO activities were induced accompanying the accumulation of free PAs in tobacco and barley plants resistant to TMV and *B. graminis* f. sp. *hordei*, respectively (Marini et al., 2001; Cowley and Walters, 2002a,b). Interestingly, plant resistance to pathogens in these cases depends on the activation of the hypersensitive response (HR),which is characterized by host cell death at the infection site. As one of the key steps triggering the HR is the production of H2O2, it is conceivable that the activity of PA catabolic enzymes contributes to elicit this response. In addition, H2O2 may promote plant resistance by HR-independent mechanisms, involving cell wall reinforcement, MAPK activation, and modulation of gene expression (Cona et al., 2006a; Nanda et al., 2010). In this regard, PA oxidation was also induced in barley against an incompatible strain of *B. graminis*, where resistance is not associated to HR induction but depends on the ability to avoid penetration of leaf epidermal cells (Cowley and Walters, 2002a). In the same line of evidences, DAO activity is induced in chickpea in response to the necrotrophic pathogen *Ascochyta rabiei*, and its inhibition by 2-bromoethylamine reduces plant resistance (Rea et al., 2002). Necrotrophic pathogens kill the host cells to feed on the remaining, and it has been demonstrated that the activation of HR ultimately favors host colonization (Govrin and Levine, 2000). Therefore, it might be expected that the resistance to *Ascochyta rabiei* mediated by PA oxidation would be due to a mechanism other than HR.

## **DIRECT MODULATION OF HOST PA METABOLISM BY PHYTOPATHOGENIC MICROBES**

Plant tumor-like structures provoked by pathogens show a remarkably induction of PA synthesis and increase in PA concentration. For instance, PAs accumulate in turnip roots infected by *Plasmodiophora brassicae Woron*., a microorganism that produce hypertrophy and hyperplasia of infected tissues in members of the plant family *Brassicaceae* (Walters and Shuttleton, 1985). Similar increments in free and conjugated PAs were described in maize tumors produced by the fungus *Ustilago maydis*(Rodriguez-Kessler et al., 2008), where ADC and SAMDC activities played a major role. These observations raise an interesting question that has remained elusive so far: are pathogens able to modulate plant PA metabolism in their own favor? Recent studies demonstrate that it might be the case for some interactions. Cytokinins produced by *Rhodococcus fascians* induce Put accumulation in *Arabidopsis* by activating *ADC* expression, which contributes to symptom development (Stes et al., 2011). Moreover, PA synthesis is induced in cabbage protoplasts infected by TYMV and it was demonstrated that these newly synthesized PAs are incorporated by viral particles (Balint and Cohen, 1985). It has not been proven yet if this is triggered by signals generated by this pathogen to assure survival in the host cells. In turn, perhaps one of the most direct evidences demonstrating the modulation of plant PA metabolism by a pathogen is the interaction between the protein 10A06 secreted by the nematode *Heterodera schachtii* and the product of the *SPDS2* gene of *Arabidopsis*. Interestingly, expression of 10A06 in *Arabidopsis* enhanced plant susceptibility, which was associated to the activation of *SPDS2* gene expression, the increment in Spd concentration and the activation of PAO activity (Hewezi et al., 2010). Surprisingly, over-expression of *SPDS2* was sufficient to affect plant defense. A further examination of these phenotypes demonstrated that *10A06* or *SPDS2* expression increases the abundance of several transcripts coding for host antioxidant enzymes. Based on this, the authors hypothesized that the induction of PAO activity imposes oxidative stress to plant cells and that this is counteracted by the activation of the antioxidant machinery, which might create a less hostile environment for the pathogen. In this same line, it was demonstrated that the protein C2 of the beet severe curly top virus (BSCTV) interacts with SAMDC1 in *Arabidopsis*, which contributes to increment plant susceptibility (Zhang et al., 2011b). In this case, it was found that C2 interacts with a region of SAMDC1 containing a PEST sequence (enriched in the amino acids Pro, Glu, Ser, and Thr), thereby attenuating its degradation at the proteasome. In this context, the stabilization of SAMDC activity leads to a low SAM/dcSAM ratio, thus reducing host and viral DNA methylation. It is though that this hypomethylated environment facilitates viral replication. Whether direct interaction with host PA metabolism enzymes is a general virulence mechanism for pathogens has not been further evaluated. Interestingly, a recent work by Kim et al. (2013a) demonstrated that the interaction between PA metabolic enzymes and pathogen effectors could also be used by plants to trigger defense. Thus, the effector protein AvrBsT of *Xanthomonas campestris* pv. *vesicatoria* is recognized by the ADC1 protein at the cytoplasm of pepper cells, and this interaction enhances AvrBsT-mediated cell death and plant resistance.

## **TRANSGENIC MODULATION OF PLANT PA LEVELS TO IMPROVE THE TOLERANCE TO PATHOGENS**

Several genetically engineered plant lines with altered PA levels show increased tolerance to microorganism attack. This fact constitutes another line of evidence demonstrating the importance of PAs during plant–pathogen interactions. In relation to this, tobacco plants over-expressing the human *SAMDC* gene accumulate free and conjugated PAs and show higher tolerance to pathogens (Waie and Rajam, 2003). Similarly, transgenic eggplants over-expressing the oat *ADC* gene acquired resistance to wilt disease caused by the fungus *Fusarium oxysporum* (Prabhavathi and Rajam, 2007)*.* These plants showed increased ADC activity and accumulation of PAs, particularly the conjugated forms of Put and Spm. As the DAO activity was also enhanced, it was suggested that the acquisition of resistance might be due to an integrated mechanism involving PA biosynthesis and degradation rather than a single event. In turn, over-expression of human *SAMDC* gene in tomato conferred resistance to the fungal pathogens *F. oxysporum* and *Alternaria solani* (Hazarika and Rajam, 2011), and the over-expression of an apple *SPDS* gene in sweet orange enhances plant tolerance to *Xanthomonas axonopodis* pv. *citri* (Fu et al., 2011; Fu and Liu, 2013). At last, the over-expression of the *SPMS* gene in *A. thaliana* resulted in higher Spm levels and enhanced resistance against *Pseudomonas viridiflava* (Gonzalez et al., 2011). Promoting PA oxidation might also favor plant defense. Regarding this, the over-expression of an extracellular PAO from maize in tobacco plants leads to an increment in the expression of genes related to systemic acquired resistance and cell wall-based defense (Moschou et al., 2009). These changes confer disease resistance against *Pseudomonas syringae pv. tabaci* and *Phytophthora parasitica*, but not against infections caused by the *cucumber mosaic virus*.

#### **PAs AND DEFENSE HORMONE METABOLISM**

Polyamine metabolism is modulated by exogenous application of phytohormones known to be associated to plant defense. However, only a few works addressed the relationship between PAs and defense hormones in a plant-pathogen system. For instance, treatment of barley primary leaves with methyl-jasmonate (MeJA) induces the increment in free and conjugated Put and Spd, as well as in ODC, ADC, SAMDC, and DAO activities (Walters et al., 2002). Conjugated PA accumulation was extended to non-treated leaves and correlated with a reduction in powdery mildew infection in these tissues. It was also shown that application of MeJA entails PA accumulation in loquat fruits and reduction of the symptoms associated to anthracnose rot caused by *Colletotrichum acutatum* (Cao et al., 2014). Similarly, treatment of wheat with MeJA increases the concentrations of both free and conjugated PAs and induces ODC and PAO activities. The change in PA metabolism was correlated with the induction of pathogenesis related (PR) proteins such as peroxidases and chitinases and a reduction in the incidence of infections by *Puccinia recondita* (Haggag and Abd-El-Kareem, 2009). The over-accumulation of conjugated PAs and induction of PA oxidation mediated by jasmonates (Jas) was also reported to occur in *Hycoscyamus muticus* L. root cultures, tobacco leaf disks and chickpea plants (Biondi et al., 2000, 2003; Biondi and Scaramagli, 2001; Rea et al., 2002). The effect of MeJA on conjugated PA levels is exacerbated in tobacco transgenic lines expressing a *SAMDC* gene in antisense, which showed low SAMDC activity and a relative accumulation of Put (Torrigiani et al., 2005). Interestingly, treatment of Arabidopsis plants with MeJA increases the expression of *ADC2* while *ADC1* remains unaltered, suggesting the existence of different regulatory pathway for both genes (Perez-Amador et al., 2002). Importantly, *ADC2* is regulated by a stress-induced kinase cascade involving MPK3 and MPK6 and is important for defense against *P. syringae* pv. tomato DC3000 (Kim et al., 2013b). Two *ODC* genes are also induced in tobacco in response to MeJA, but its importance for plant defense has not been explored (Xu et al., 2004). The effect of MeJA on PA metabolism may be quite different in other plant species. Thus, it has been shown that MeJA caused a transient inhibition at the transcriptional levels of the expression of *ADC*, *SAMDC,* and *SPDS* in rice (Peremarti et al., 2010). These data could be explained by the existence of contrasting regulatory mechanisms operating over the metabolic enzymes in different plant species. In addition, it should also be considered that the expression of PA metabolism genes not only

depends on the action of one hormone, but it is influenced by several signaling pathways. Thus, it has been demonstrated that the effect of MeJA on ODC and SAMDC activities function synergistically with that of auxins in tobacco leaf disks, but it is counteracted by cytokinins (Biondi et al., 2003). In *Arabidopsis*, MeJA induces the expression of *CuAO1*, *CuAO2,* and *CuAO3*, and it was shown that *CuAO1* and *CuAO3* also respond to abscisic acid (ABA; Wimalasekera et al., 2011b; Planas-Portell et al., 2013).

Salicylic acid (SA) plays a fundamental role in plant defense (Glazebrook, 2005). Unfortunately, the current information concerning the relationship between SA and PAs is scarce. It has been shown that SA is able to induce the accumulation of PAs by activating the expression of *ADC* and *ODC* in maize, tobacco, and tomato (Németh et al., 2002; Jang et al., 2009; Zhang et al., 2011a). Nevertheless, it has not been evaluated yet whether JA and SA act synergistic on the activities of these enzymes. In chickpea plants, however, the application of SA repressed the induction of PA oxidation mediated by JA (Rea et al., 2002). In turn, ABA has been recently demonstrated to induce DAO expression in *Arabidopsis* (Wimalasekera et al., 2011b). ABA is also able to induce Put oxidation at the apoplast of *Vicia faba* (An et al., 2008). This was demonstrated to be important for stomatal closure, a known mechanism contributing to prevent plant colonization by bacterial pathogens. Future research is necessary to understand the connections between SA and ABA metabolism with PAs during plant biotic stress.

The infection of tomato and purple passion (*Gynura aurantiaca*) plants with the citrus exocortis viroid (CEVd) induces ethylene production and represses PR expression. This is accompanied by a reduction in ODC activity, which leads to a decline in Put contents (Belles et al., 1991, 1993). Importantly, the reduction in Put concentration is absent with the use of ethylene biosynthesis inhibitors or compounds that prevent the action of the hormone, suggesting that ethylene exerts an inhibitory effect on ODC activity. Conversely, PAs may also interfere with ethylene metabolism. This was demonstrated in a tomato transgenic line expressing the yeast *SPDS* gene and showing relatively high levels of Spd (Nambeesan et al., 2012). This line exhibits high susceptibility to the attack of the fungus *Botrytis cinerea*, which seems to be due to an attenuated expression of ethylene biosynthetic and signaling genes.

#### **PAs AS MEDIATORS OF PLANT DEFENSE ACTIVATION**

Increasing evidences demonstrate that PAs and their catabolic products (i.e., H2O2) can act as defense signaling molecules. This function has been mostly assigned to Spm, even though it has been shown that in some systems either Put, Spd, or TSpm participate in defense activation.