Treatments including the NO donor, SNP, the HO-1 inhibitor, ZnPPIX, and PEG attenuated the decrease in HO-1 activity in aleurone layers treated with PEG alone (**Figure 1B**). Treatment using PEG + SNP and PEG + SNP + ZnPPIX for 24 h did not result in any distinct differences in the HO-1 activities of the aleurone layers, whereas these, respectively, increased by 1.95 and 2.58-fold compared to that using PEG alone (**Figure 1B**), which indicates that the HO-1 inhibitor, ZnPPIX, did not prevent the NO effect. Meanwhile, the observed changes in HO-1 activity were in agreement with the detected *HO-1* expression profile.

After treatment with PEG + Ht + cPTIO for 24 h, the relative expression level of *HO-1* and the activity of HO-1 in rice aleurone layers, respectively, decreased by 33.30 and 55.94% relative to that using PEG + Ht (**Figures 1A,B**). Meanwhile, no statistically significant differences between the treatments PEG and PEG + Ht + ZnPPIX were observed. These results indicated that the NO-specific scavenger cPTIO blocked the Ht-induced expression of *HO-1* by eliminating endogenous NO under drought stress. These results thus suggest that HO-1 acts downstream of NO in the rice aleurone layers under drought stress. Xuan et al. (2012) previously showed that HO/CO possibly acts as the downstream molecule of NO during the process of adventitious rooting in cucumber.

### HO-1 Increases the Level of Endogenous NO in Rice Aleurone Cells Under Drought Stress

To further elucidate the relationship of upstream and downstream between HO-1 and NO during the PCD of rice aleurone layers subjected to drought stress, DAF-2DA, the specific NO fluorescence probe, was used to label the cells of the cultured rice aleurone layers subjected to various treatments for 24 h. The NO signal of cells was captured by LSCM (**Figures 2A–J**). The NO fluorescent signal was strong in rice aleurone cells under normal cultivation conditions, which clearly indicated that the level of intracellular NO was high (**Figure 2A**). In contrast, barely detectable NO fluorescent signals were observed under drought stress (**Figure 2B**), indicating that the formation of endogenous NO was significantly restrained under drought stress. However, the combined treatments of PEG + Ht (**Figure 2C**) or PEG + SNP (**Figure 2D**) showed stronger fluorescence signals compared to that using PEG alone, indicating that the HO inducer, Ht, induced NO synthesis in the rice aleurone cells under drought stress.

The combination of the NO donor, SNP, with the HO-1 inhibitor, ZnPPIX, effectively alleviated the decrease in the level of endogenous NO under drought stress (**Figure 2E**); therefore, the intracellular fluorescence intensity was distinct within 24 h of treatment, and was similar to that observed with PEG + SNP. These results indicated that the HO-1 inhibitor, ZnPPIX, did not reduce the synthesis of NO, which was induced by the NO donor, SNP, under drought stress. PEG + SNP + Ht (**Figure 2F**) treatments including the NO scavenger such as PEG + Ht + cPTIO and PEG + SNP + cPTIO effectively prevented NO formation, which was elicited by the HO-1 inducer, Ht, and the NO donor, SNP, in the rice aleurone layers after 24 h of treatment (**Figures 2G,H**). These findings indicated that the NO-specific scavenger, cPTIO, blocked the forming of intracellular NO, which was induced by HO-1 and SNP in the aleurone layers.

### GA-Induces the Down-Regulation of *HO-1* Gene Expression and Activity Level in Rice Aleurone Layers Subjected to Drought Stress

By fluorescent QRT-PCR relative analysis, we compared the results with those using distilled water (C, control), the PEG alone treatment significantly inhibited the expression level of *HO-1* by 44%, and exogenous GA treatment also inhibited the expression level of *HO-1* by 27%, but the treatment combined GA with PEG decreased by 33.80% compared to that observed in the GA alone treatment (**Figure 3A**). The treatments PEG + GA + Ht and PEG + GA + SNP partly eased the reduction in the *HO-1* expression level, compared to the treatment with PEG + GA, and respectively, increased by 29.79 and 42.55% (**Figure 3A**). The expression levels of the *HO-1* gene showed no significant differences among treatments PEG, PEG + GA, and PEG + GA in the presence of exogenous HO-1 inhibitor or NO scavenger, and changes in HO activity changes induced by these reagents were identical (**Figure 3B**). The HO-1 activities of rice aleurone layers treated with exogenous GA alone or PEG + GA were significantly inhibited (*P <* 0.05 or *P <* 0.01; **Figure 3B**).

Both treatments PEG + GA + Ht and PEG + GA + SNP partly, respectively, raised the HO-1 activities inhibited by the treatment PEG + GA by 2.34- and 2.04-fold (**Figure 3B**). The treatment of HO-1 inhibitor, ZnPPIX, being added into PEG + GA + Ht, compared to the treatment PEG + GA + Ht, significantly reduced the HO-1 activity that was induced by Ht, thus indicating that the response was reserved by ZnPPIX (**Figure 3B**). Because of the participation of NO scavenger cPTIO, the treatment PEG + GA + SNP + cPTIO significantly decreased the effects of NO on raising the HO-1 activity of aleurone cells treated with PEG + GA (**Figure 3B**).

### GA, HO-1, and NO Promoted or Delayed PCD in Rice Aleurone Layers Under Drought Stress

The aims of the present study were to examine, whether HO-1 performs a crucial function in the induction of PCD in germinating rice aleurone layers and to confirm whether this effect is caused by its interaction with NO and GA signaling.

FDA and FM4-64 are fluorescence dyes commonly used to identify cell viability, wherein live cells emit green fluorescence and dead cells red fluorescence. This facilitates in distinguishing between live and dead cells in the aleurone layers.

FDA and FM4-64 dual fluorescent staining combined with LSCM showed that the number of dead cells gradually prolonged the incubation time of rice aleurone layers subjected to various treatments (**Figures 4A–X**). Dead cells did not appear in rice aleurone layers within 24 h of distilled water culture (C, control), and most of cells showing green fluorescence after 48 h of incubation were still alive (**Figures 4A–C**). After treating aleurone layers with exogenous GA for 24 h, some cells died, and after 48 h, more than half of the cells were dead (**Figures 4D–F**). On the other hand, PEG and co-treatment with PEG and GA accelerated the death rate of aleurone cells; as early as 12 h after treatment, cells were dead, and within 48 h, almost all cells were dead in the aleurone layer (**Figures 4G–L**). While the treatments containing the HO-1 inducer or NO donor elevated the survival rate of the aleurone layer cells at 12, 24, and 48 h, even after 48 h, 35.67 and 48.33% of the cells remained alive (**Figures 4M–R**). On the other hand, treatments PEG + GA + Ht + ZnPPIX and PEG + GA + SNP + cPTIO showed no distinct differences at each time point, almost all cells of the aleurone layer were dead within 48 h of treatment (**Figures 4S–X**). These findings illustrated that the respective inhibitor and scavenger reversed the effects of HO-1 and NO on delaying PCD.

A coordinate curve of cell survival rate and time of various treatments was constructed (**Figure 4Y**). The aleurone layers showed a 90% cell survival rate within 48 h of distilled water treatment, whereas that treated with exogenous GA alone decreased to 45% after 24 h of drought stress, and almost no live cells were observed after 48 h. The cell survival rates of aleurone layers remained at 38 and 33% after 48 h of incubation in PEG + GA + Ht and PEG + GA + SNP, respectively, whereas when the treatments PEG + GA + Ht + ZnPPIX and PEG + GA + SNP + cPTIO were applied to the aleurone layer for 48 h, the cells remained alive (**Figure 4Y**), indicating that HO-1 and NO delayed the GA induction the cell death in rice aleurone layers subjected to drought stress.

### GA Accelerates the Development of a Large Central Vacuole in Aleurone Cells Under Drought Stress, and HO-1 and NO Slows the Effect of GA

The large central vacuole of aleurone cells is the prelude to PCD during cereal seed germination (Bethke et al., 1999). Therefore, our next goal was to explore, whether the vacuolation was associated with the PCD of aleurone layers in germinating rice seeds subjected to drought stress.

By exploring morphological changes of aleurone cells during PCD under normal cultured conditions, we determined that the morphological changes of PSVs were significant in the aleurone cells. Therefore, based on the morphological changes in PSVs, the cell death process of rice aleurone layers could be determined. From 0 to 1 day of culture, aleurone cells were hexagonal, with nearly uniform wall thickness, and with spherical-shaped aleurone grains (**Figure 5A**, arrow). When aleurone layers were cultured from 2 to 6 days, the PSVs of aleurone cells began to merge into larger vesicles (**Figures 5B–E**, arrows) accompanied by vacuolation, a large central vacuole appeared after 7 days (**Figure 5F**, arrow), and then the large central vacuole deformed and elongated, and integrity of the vacuole membrane disappeared after 8 days (**Figure 5G**, arrow). Finally, vacuole rupture (**Figure 5H**, arrow) resulted in protoplast dissolution, followed by shrinking into a ball after 10 days (**Figure 5I**, arrow).

In the GA alone treatment, several small PSVs were observed in the aleurone cells within 1/2 day (**Figure 6A**, arrow). However,

the PSVs began to fuse and formed into several bigger vacuoles in GA-treated aleurone cells after incubation for 1 day (**Figure 6B**, arrow), and then these larger PSVs fused to form a large central vacuole after 2 days (**Figure 6C**, arrow). The large central vacuole elongated and deformed, and the deformed large central vacuole showed an unclear boundary after 3 days (**Figure 6D**,

arrow), and finally, protoplasts appeared shrunken within cells after 4 days (**Figure 6E**, arrow). The death process of aleurone cells was earlier at 6 days compared to cells cultured under normal conditions. These results confirmed that exogenous GA significantly accelerates the vacuolation processes in aleurone cells.

Compared to the GA alone treatment, the PEG and PEG + GA treatments hastened the occurrence of the large central vacuole. The PSVs of aleurone cells treated with PEG and PEG + GA were larger than those treated with GA after 1/2 day (**Figures 6F,I**, arrows), exhibiting a large central vacuole with distinct deformation after 1 day (**Figures 6G,J**, arrows) and

only after 2 days, presented the phenomenon of cell protoplasts mixing together and shrinking (**Figures 6H,K**, arrows). The results certified that drought stress speeds up the formation and rupture of large central vacuoles in GA-inducing aleurone cells.

When the HO-1 inducer and NO donor were, respectively, added to PEG + GA, a large central vacuole of aleurone cell was not observed at 1 day (**Figures 7A,E**) and 2 days (**Figures 7B,F**). A large central vacuole did not emerge until

7, 8, 9, and 10 days. The scale is 20 μm.

3 days of the PEG + GA + Ht and PEG + GA + SNP treatments (**Figures 7C,G**, arrows), cell protoplast shrinking was only observed after 4 days (**Figures 7D,H**, arrows), which was then followed by cell death. The occurrence of large central vacuoles was observed two days later in both PEG + GA + Ht and PEG + GA + SNP treatments compared to that in the PEG + GA treatment (**Figures 6I–K**). These results suggest that HO-1 and NO delayed the occurrence of large central vacuoles in GA-induced aleurone cells subjected to drought stress. Therefore, HO-1 and NO contribute to the delay in GA-induced aleurone layers PCD during drought stress.

### DISCUSSION

In our previous study, HO-1 inducer Ht differently upregulated the activities of SOD, CAT, and peroxidase (POD), resulting in the apparent decrease of membrane permeability and malondialdehyde (MDA) content. These results suggested that HO-1 alleviates drought-induced oxidative damage in rice seeds germination (data not shown). Increasing evidence demonstrated that endogenous ROS, particularly H2O2*,* play a critical role in regulating the PCD of cereal aleurone layers (Bethke and Jones, 2001; Wu et al., 2011). Antioxidants and ROS scavengers such as butylated hydroxytoluene (BHT), dithiothreitol (DTT), and ascorbic acid (AsA) not only delayed PCD, but also mimicked the effect of HO-1 inducer haematin on up-regulating HO-1. In addition, haematin blocked the decrease of GA-induced ascorbate peroxidase (APX) and CAT activities, and then resulted in the decrease of H2O2 level, and these effects were reversed by HO-1 inhibitor ZnPPIX (Wu et al., 2011). The up-regulation of HO-1 would contribute to the capability of HO, act as a potent antioxidant enzyme, eliminating ROS and protecting cells from oxidative stress in wheat aleurone layers (Wu et al., 2011, 2014), which has previously been confirmed in plants (Noriega et al., 2004; Han et al., 2008). The present study showed that the upregulation of HO contributed to the delay in PCD in rice aleurone layers induced by GA under drought stress, the similar results were obtained by Wu et al. (2011, 2014). Above mentioned results revealed that HO-1 function in the PCD of cereal aleurone layers is dependent on enhancing the activities of antioxidant enzymes.

Mounting evidence has confirmed that HO-1 and NO play important physiological functions in plants, such as improving resistance and promoting seed germination. After treatment with Ht, the gene transcription and activity level of HO-1 were upregulated, as well as alleviated Cd-induced oxidative damage in alfalfa root tissue (Han et al., 2008). NO, as an antioxidant, reduced the accumulation of ROS, and alleviated the damage caused by oxidative stress, thereby enhancing the adaptability of plants to abiotic stress (He et al., 2014). In addition, exogenous NO donor SNP induced the up-regulation of HO-1 (Noriega et al., 2007; Xuan et al., 2008; Santa-Cruz et al., 2010), and NO prolonged the life of barley aleurone cells treated with GA (Beligni et al., 2002). The aleurone layers of cereal grains undergo PCD, which is regulated by GA following germination (Domínguez et al., 2004). By exploring the relationship between HO-1 and NO during PCD in rice aleurone layers, we discovered that the HO-1 inducer Ht and NO donor SNP up-regulated the gene expression and activity of HO-1 in the rice aleurone layers under drought stress, but their effects were, respectively, reversed by specific inhibitor ZnPPIX and scavenger cPTIO. Simultaneously, the effect of HO-1 was blocked by NO scavenger cPTIO, whereas HO-1 activity inhibitor ZnPPIX did not block the effect of NO on the up-regulation of *HO-1* gene expression and activity of aleurone layers (**Figures 1A,B**). In addition, treatments using HO-1 inducer or NO donor resulted in an increase in endogenous NO content in aleurone cells under

drought stress (**Figures 2A,B**). Similar results were observed using SNP and Ht, which accelerated NO emission in etiolated wheat seedling leaves (Liu et al., 2013). Further experiments showed that the NO scavenger repressed the action of the HO-1 inducer Ht and NO donor SNP. However, the HO-1 inhibitor ZnPPIX was unable to block the effect of the NO donor of increasing the level of endogenous NO (**Figures 2A,B**). Furthermore, compared to the Ht alone-treated sample, ZnPPIX and cPTIO markedly reduced the Ht-induced up-regulated NO level, suggesting that HO-1-mediated NO production is a major source of endogenous NO in aleurone layers. Based on these results, we deduced that there is a link between HO-1 and NO in GA-induced PCD in rice aleurone layers subjected to drought stress. Similarly, SNP- and Ht-triggered up-regulation of HO-1 was observed in soybean seedlings (Noriega et al., 2007; Santa-Cruz et al., 2010) and wheat aleurone layers (Wu et al., 2011). Li et al. (2015) inferred that HO-1 acting downstream of NO signaling was involved in β-CDH-induced lateral root formation in tomato. A different conclusion was also drawn in that NO acted downstream of HO-1, wherein it is involved in hemin-induced cucumber adventitious rooting (Xuan et al., 2012). Another study also showed that HO stimulated rooting of mung bean hypocotyl cuttings upstream of NOS/NO (Xu et al., 2006).

The aims of the present study were to examine whether HO-1 performs a crucial function in responding to the GA-induced PCD of germinating rice aleurone layers and to confirm whether this effect is caused by its interaction with NO. Previous studies have confirmed that GA initiated PCD in the aleurone layers of wheat (Kuo et al., 1996) and barley (Wang et al., 1996; Bethke et al., 1999). In the present study, GA and PEG, and PEG cotreated with GA significantly reduced the gene expression and activity of HO-1 (**Figures 3A,B**), thereby accelerating PCD in aleurone layers (**Figures 4D–L**), whereas the HO-1 inducer Ht and NO donor SNP alleviated the GA-induced decline in *HO-1* gene expression and activity in rice aleurone layers subjected to drought stress (**Figures 3A,B**), improved cell survival rate, and delayed the occurrence of GA-induced aleurone layer PCD in the germinating rice seeds under drought stress (**Figures 4M–R**). The effects of the HO-1 inducer and NO donor SNP were, respectively, reversed by the HO-1 inhibitor ZnPPIX and NOspecific scavenger cPTIO, whereas PCD in rice aleurone layers was accelerated (**Figures 4S–X**). A previous study suggested that the HO inducer haematin increases HO transcription and activity, and inhibits PCD of wheat aleurone layers, whereas the HO inhibitor ZnPPIX reverses the effect of haematin and contributes to cell death (Wu et al., 2011). These results ensured that intracellular HO plays a major role in postponing the PCD of aleurone layers subjected to drought stress.

The vacuole plays a crucial role in the PCD of plants (Gucciardi et al., 2004; Gadjev et al., 2008; Xiao et al., 2009; Hara-Nishimura and Hatsugai, 2011), wherein only highly vacuolated cells undergo PCD in plants (Bethke and Jones, 2001). Therefore, we wanted to know, whether the degree of vacuolation is associated with PCD in rice aleurone layers. In the present study, we determined that variation in vacuole morphology of aleurone cells sequentially occurred under normal culture conditions; it took 7 days for the large central vacuole to emerge (**Figures 5A–F**). Subsequently, the large central vacuole lost its distinct boundary and in turn underwent deformation, thereby resulting in tonoplast rupture (**Figures 5G–I**). However, GA increased the rate of vacuolation of PEG-induced aleurone layers (**Figures 6I–K**). Therefore, a large number of cells in the aleurone layers were dead (**Figure 4L**). Interestingly, HO-1 inhibitor ZnPPIX and NO scavenger cPTIO mimicked the effect of GA-promoted vacuolation and PCD, and HO-1 inducer Ht and NO donor SNP delayed the appearance of large central vacuoles (**Figures 7C,G**), ultimately resulting in the delay in the onset of PCD in rice aleurone layers subjected to drought stress. Therefore, we speculated that HO-1 and NO are involved in postponing GA-induced vacuolation of aleurone cells in rice under drought stress by preventing the formation of large central vacuoles.

In the present study, we showed that up-regulating HO alleviates GA-induced PCD in rice aleurone layers subjected to drought stress. A similar finding involving the up-regulation of HO that results in a delay in PCD in wheat has been earlier described (Wu et al., 2011).

Previous studies have confirmed that vacuoles are essential in initiating PCD in plants, and vacuole membrane rupture is a critical step in plant PCD (Higaki et al., 2011). However, previous studies did not determine, whether changes in *HO-1* gene expression affected the process of vacuolation and PCD. Therefore, we investigated the relationship of up- or down-regulation of HO-1, vacuolation, and GA-induced PCD progression in rice aleurone layers subjected to drought stress. The results indicated that GA, the HO-1 inhibitor, and the NO scavenger down-regulate the *HO-1* mRNA level and HO activity, which in turn increases the rate of vacuolation and PCD. However, the HO-1 inducer and NO donor slowed down the process of vacuolation and PCD by up-regulating *HO-1* gene expression and HO activity.

In summary, we present evidence that suggest that GA regulates the expression of HO-1 in germinating rice aleurone layers subjected to drought stress. The level of *HO-1* gene expression and HO activity plays an important role in ascertaining the process of GA-induced PCD in response to drought stress. HO-1 and NO modulate each others function in aleurone layers, similar to that in the NO donor SNP, wherein Ht-driven HO-1 promotes the level of endogenous NO. Correspondingly, the enhanced NO triggers the up-regulated expression and activity of HO-1, whereas the NO inhibitor cPTIO down-regulated the expression and activity of HO-1. Therefore, the observed mutual induction effects indicate that there might be an inseparable relationship between HO-1 and NO in delaying the PCD of GA-induced rice aleurone layers subjected to drought stress. However, the PCD of cereal aleurone layers is a complex event, and it is unable to be elucidated clearly only depending on the evidence of biochemistry, cell morphology, and pharmacology. Therefore, future studies aim to investigate the HO, NO, and GA signal transduction pathways and the molecular mechanism in the PCD of rice aleurone layers subjected to drought stress by combining with molecular methods.

### AUTHOR CONTRIBUTIONS

HC designed the experiment. HC and HW wrote the paper. HW carried out fluorescence quantitative RT-PCR. YZ carried out cell morphology observation and subcellular localization. JL helped in enzyme activity determination. HC and HZ helped in drafting the manuscript.

### REFERENCES

### ACKNOWLEDGMENTS

This work is supported by the National Natural Science Foundation of China (grant numbers 30960182 and 31560426) and the Central and Western areas Construction Projects of Hainan University (ZXBJH-XH008).

oxygenase required to phytochrome chromophore synthesis. *Plant Physiol.* 130, 1958–1966. doi: 10.1104/pp.008128

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

*Copyright © 2016 Wu, Zheng, Liu, Zhang and Chen. 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.*

# Citrus Plants: A Model System for Unlocking the Secrets of NO and ROS-Inspired Priming Against Salinity and Drought

Athanassios Molassiotis1 , Dominique Job2,3, Vasileios Ziogas1 and Georgia Tanou1 \*

1 Faculty of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece, 2 AgroParisTech, Chair of Plant Physiology, Paris, France, 3 CNRS/UCBL/INSA/Bayer CropScience Joint Laboratory-UMR5240, Lyon, France

Plants treated with chemical compounds can develop an enhanced capacity to resist long after being subjected to (a)biotic stress, a phenomenon known as priming. Evidence suggests that reactive oxygen species (ROS) and reactive nitrogen species (RNS) coordinately regulate plant stress responses to adverse environmental conditions; however, the mechanisms underlying this function remain unknown. Based on the observation that pre-exposure of citrus (Citrus aurantium L.) roots to the NO donor sodium nitroprusside (SNP) or to H2O2 prior to NaCl application can induce acclimation against subsequent stress we characterized the changes occurring in primed citrus tissues using several approaches. Herein, using this experimental model system, we provide an overview of our current knowledge of the possible mechanisms associated with NO and H2O2 priming to abiotic stresses, particularly concerning salinity and drought. The data and ideas presented here introduce six aspects of priming behavior in citrus under abiotic stress that provide knowledge necessary to exploit priming syndrome in the context of sustainable agriculture.

Keywords: abiotic stress, drought, priming, proteins, salinity

## INTRODUCTION

Environmental stress factors, such as drought and salinity, strongly affect plant growth and pose a growing threat to sustainable agriculture (Golldack et al., 2014). This has become a hot issue due to concerns about the effects of climate change on plant resources, biodiversity and global food security (Ahuja et al., 2010). Consequently, understanding the mechanisms underlying plant abiotic stress acclimation helps us to develop fruitful new agricultural strategies (Beckers and Conrath, 2007). Evidence suggests that plants are capable of inducing some stress "memory," or "stress imprinting" following a first stress exposure that leads to acclimation to a later (a)biotic stress. Through priming (also known as hardening), plants are able to induce responses to a range of stresses, providing low-cost protection in relatively high stress-pressure conditions (Borges et al., 2014). Despite priming phenomena have previously been widely described under biotic stress (Prime-A-Plant Group et al., 2006; Conrath et al., 2015) and in the invigoration of seeds (Rajjou et al., 2012), the mechanisms of long-lasting priming are still unclear, notably under abiotic stress (Tanou et al., 2012b). It has been suggested that hormone-dependent pathways and availability of signal transduction proteins along with epigenetic mechanisms, such as histone modifications and DNA

#### Edited by:

Violeta Velikova, Bulgarian Academy of Sciences, Bulgaria

#### Reviewed by:

Giridara Kumar Surabhi, Regional Plant Resource Centre, India Andrés A. Borges, Consejo Superior de Investigaciones Científicas, Spain

#### \*Correspondence:

Georgia Tanou<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science

Received: 21 December 2015 Accepted: 11 February 2016 Published: 26 February 2016

#### Citation:

Molassiotis A, Job D, Ziogas V and Tanou G (2016) Citrus Plants: A Model System for Unlocking the Secrets of NO and ROS-Inspired Priming Against Salinity and Drought. Front. Plant Sci. 7:229. doi: 10.3389/fpls.2016.00229

methylation, are involved in priming against abiotic stress (Bruce et al., 2007; Conrath, 2011). Recently, Jiménez-Arias et al. (2015) showed that Arabidopsis seed-based priming against salt stress involves epigenetic changes (DNA hypomethylation) in genes controlling proline metabolism. In this regard, it has been proposed that priming stimulates salicylic acid (SA), abscisic acid (ABA), and jasmonic acid (JA) signaling that could facilitate the transcriptional induction of defense genes and epigenetic changes; this trans-generational induced resistance is elicited by the RNA-directed DNA methylation (RdDM) pathway, which triggers heritable changes in DNA methylation that can direct priming-inducing chromatin modifications at defense gene promoters (Pastor et al., 2013).

Almost all abiotic stressors generate reactive oxygen species (ROS) and reactive nitrogen species (RNS), resulting in oxidative and nitrosative stress in plants (Molassiotis and Fotopoulos, 2011). It has been increasingly evident that RNS (in the form of nitric oxide, NO) and ROS (in the form of H2O2) play important roles in priming phenomena in various annual plants (Del Río, 2015). Nevertheless, a priming approach has a great potential if studied in non-model long-lived fruit trees species (Molassiotis et al., 2010). Citrus is widely cultivated in Mediterranean-type ecosystems where climatic change is expected to amplify drought and salinity stress (Gordo and Sanz, 2010). Basal resistance by itself is too weak to protect citrus against environmental stimuli, since it is sensitive to oxidative and nitrosative stress induced by various abiotic stress conditions (Ziogas et al., 2013). Hence, the possible implications of NO and H2O2 in the acclimation of citrus plants to adverse environmental conditions, as well as the interactions between the two molecules, were studied. In this regard, we initially documented at physiological level that NO and H2O2 are able to induce priming against salt stress by pretreating the roots of sour orange (Citrus aurantium L.) seedlings either with the NO donor sodium nitroprusside (SNP) or with H2O2 prior to NaCl application (see Tanou et al., 2009b). Using this experimental system, the mechanisms by which citrus plants respond to salinity were investigated in order to gain a wide understanding of oxidative- and nitrosative-associated priming in plants. In this review, we summarize our current knowledge of the possible mechanisms associated with NO- and H2O2 induced salinity and drought acclimation in citrus plants. Overall, this approach reveals the following six aspects regarding the mechanism of NO- and H2O2-associated priming events in citrus against abiotic challenges.

### NO AND H2O2-ORIGINATED PRIMING IS ASSOCIATED WITH INDUCED ANTIOXIDANT ACTIVITY

One of the mechanisms actively employed by primed plants to survive under abiotic stress is the induction of the antioxidant defense system (Hossain et al., 2015). In citrus plants it was evidenced that pre-treatments with NO and H2O2 prior to NaCl stress induced antioxidative defense-related enzymatic activity [e.g., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR)] (Tanou et al., 2009b) and mRNA expression [(eg., Cu/Zn-SOD, Mn-SOD and Fe-SOD, xanthine oxidoreductase (XO), and alternative oxidase (AOX)] (Tanou et al., 2012a). Another antioxidant function of NO arises from the observation that NO protects citrus leaves from DNA strand cleavage caused by hydroxyl radical •OH produced following NaCl application (Tanou et al., 2009b), thereby avoiding oxidative damage induced by most harmful ROS. It has been proposed that NO protects from •OH-stimulated oxidative stress in two possible ways: directly by scavenging ROS, and indirectly by NO-mediated induction of ferritin proteins that contributes to diminishing free-Fe2+ levels and Fentontype reactions within the organelles (Martin et al., 2009). It had been demonstrated that •OH effectively targeted DNA methylation and regulatory genes (Shen et al., 2014), implying that NO may have an important role in priming process, at least in part, by DNA methylation-based epigenetic modifications.

### THE PRIMING ACTION OF NO AND ROS IS LONG–DISTANCE AND LONG-LASTING

Systemic signals are perceived in distant plant tissues and initiate systemic stress responses through priming (Gaupels and Corina Vlot, 2012). Knowledge on such long-distance signaling has been recently documented in various plant systems (Frost et al., 2008; Chaturvedi et al., 2012; Shah et al., 2014). According to the above studies, phloem is the likely path for systemic transmission or movement of signals associated with the acclimation process (Chaturvedi et al., 2012; Ruiz-Medrano et al., 2012). Similarly, root-applied NO or H2O2 remarkably increased NO and H2O2 steady-level in the leaves of citrus, indicating that these two molecules are systemic priming elicitors at the whole-plant level (Tanou et al., 2012a). Histochemical localization of H2O2 and O•− 2 production in citrus leaves using 3,30 -diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining indicated that ROS specifically accumulated in the vicinity of the primary vein (Tanou et al., 2012a). Such preferentially topological distribution of ROS reflects the greater exposure of periveinal cells to systemic signals, and that, when diffusing out of the veins, the concentration is diluted and therefore the cells near the vascular bundles are more likely to react (Alvarez et al., 1998). Further evidence for the long-distance and long-lasting nature of NO and H2O2 arose from the observation that both NO and H2O2-derived 4,5-diaminofluorescin diacetate (DAF-2DA) and 2,0 7 0 -dichlorofluorescin diacetate (DCF-DA) fluorescence were detected in the vascular tissues (xylem and particularly phloem) and in the upper and lower epidermal cells under normal and NaCl stress conditions and occurred following 8 days of NO/H2O2 application (Tanou et al., 2012a). In citrus we also observed that the local application of NO in roots induced drought acclimation in leaves for at least 35 days after exposure to PEG stress (Ziogas et al., 2015) further suggesting that NO priming signal is memorized and systemically

transduced. However, the evidence for in planta NO and H2O2 long-lasting signaling is not strong enough to conclude their priming roles due to the short half-lives of NO and H2O2 (Neill et al., 2002). One mode of long-distance/lasting NO and H2O2 action may be associated with the auto-propagation of ROS and RNS waves throughout the plant (Mittler et al., 2011; Molassiotis and Fotopoulos, 2011). The initial abiotic stressinduced burst of ROS/RNS in a local group of plant cells triggers cell to cell communication that propagates throughout different tissues of the plant and carries a systemic signal over long distances (Miller et al., 2009). Another scenario might be the systemic NO and H2O2 signaling through binding to specific stress-related enzymes, such as mitogen-activated protein kinases (MAPKs) (Mishra et al., 2006) and S-nitrosoglutathione reductase (GSNOR) (Leterrier et al., 2011), thereby modifying their activity. In this regard, GSNOR transcripts in leaves and roots of salt-primed citrus plants were down-regulated showing that GSNOR could be considered a mechanism by which NO and H2O2 orchestrate priming signaling (Tanou et al., 2012a).

### PROTEIN REPROGRAMMING IS A KEY MECHANISM OF NO AND H2O2-INDUCED PRIMING

Changes in environmental conditions are likely to cause rapid changes in the level, composition, and structure of different metabolites, proteins, and RNA molecules that precede signal transduction or stress acclimation events in plants (Baxter et al., 2014). An interesting finding that emerged from the work in citrus is the fact that the NaClresponsive leaf proteome (85 proteins) was remarkably affected by pre-exposure to NO or to H2O2. Indeed, NO or H2O2 pretreatment prior to salt stress imposition, reversed a large part of the NaCl-responsive proteins (53 and 55 proteins, respectively; Tanou et al., 2009a). The major set of these proteins (46.7%) participate in photosynthesis and particularly in the Calvin cycle (e.g., several isoforms of Rubisco activase, Rubisco large subunit, fructose 1,6-bisphosphate aldolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, transketolase, and carbonic anhydrase; Tanou et al., 2012a), this being a probable attempt at sustaining the photosynthesis rate during salt stress. Regulatory mechanisms of photosynthetic proteins were also studied in other stress-affected plant species and crops. For example, Komatsu et al. (2014) pointed the major proteomic alternation in wheat under various abiotic stresses focused on photosynthesis-responsive proteins. In addition to NO- or to H2O2-mediated regulation of protein expression during salinity acclimation, a more recent study showed that pre-treatment with NO also modulates specifically several PEG-affected proteins (e.g., glycolate oxidase, NADP-isocitrate dehydrogenase, and UPF0603 protein Atlg54780) in citrus plants experiencing priming against drought (Ziogas et al., 2015). These findings indicate that the priming function of NO and H2O2 in citrus plants is a dynamic, photosynthetic activity-demanding process that might be, at least partially, attributed to proteome reprogramming.

### NO AND H2O2 -ALTERED PROTEIN POSTTRANSLATIONAL MODIFICATION PHYSIOGNOMY

It has been well documented that ROS and RNS exposure can prime plants against abiotic stresses through chemical reactions with specific target proteins that result in covalent posttranslational protein modifications (PTMs), altering protein function and activity (Lounifi et al., 2013). Therefore, the characterization of PTMs is crucial for a deeper understanding of NO and H2O2 priming. Particularly, protein carbonylation is a type of protein oxidation driven by oxidative stress, which occurs by direct attack on Lys, Arg, Pro, or Thr protein residues (Oracz et al., 2007). It has been shown that basal levels of protein carbonylation under control conditions increase sharply when citrus plants are grown under salinity (Tanou et al., 2009a, 2012a), inhibiting enzyme activities and increasing protein susceptibility toward proteolytic attack (Dunlop et al., 2002). Proteomic 2DE-OxyBlot-based analysis in citrus indicated that H2O2 and SNP pretreatments before salt stress prevented the NaCl-induced protein carbonylation to the levels of untreated control plants and allowed identifying 40 carbonylated proteins showing a reversal in accumulation level upon H2O2 or SNP application (Tanou et al., 2009a). Furthermore, tyrosine (Tyr) nitration, i.e., the addition of a nitro group (NO2) to one of the two equivalent ortho carbons of the aromatic ring of Tyr residues in the presence of excess levels of ROS and NO or NO-derived species, is recognized as an important redox PTM (Corpas et al., 2009). Protein Tyr nitration has been established as a biomarker of systemic "nitroxidative stress," leading plant metabolism to a pro-oxidant status that disrupts NO signaling and induces protein structural and functional changes, some of which contribute to altered cell and tissue homeostasis (Corpas et al., 2013). Similar to the pattern of protein carbonylation, Tyr-nitration increased in citrus leaves exposed to salinity or to NO or H2O2 under stressfree conditions but diminished to control basal levels when these chemical treatments were applied before the imposition of NaCl stress (Tanou et al., 2012a). These results clearly show that citrus plants adopt a common oxy- and nitrobased stress-alleviating mechanism in their leaves. In both cases the results strengthen the notion that these PTMs are not just a 'fingerprint' of oxidative and nitrosative stress, but also they are essential components of citrus priming mechanism.

S-nitrosylation is another PTM that has been validated as signaling mechanism mediated by nitrosative/oxidative stress that occurs on cysteine (Cys) residues, being redox reversible with high spatial and temporal specificity modifying protein activity and accumulation (Astier and Lindermayr, 2012). In citrus leaves subjected to NaCl, S-nitrosylation decreased whereas NO or H2O2 pre-treatments before salt stress substantially increased protein S-nitrosylation. Remarkably,

Molassiotis et al. NO and ROS Stress Priming

this response was in contrast to protein carbonylation and Tyr-nitration patterns under the same experimental conditions (Tanou et al., 2009a, 2012a), denoting differences of PTMs regulation during salinity acclimation. By studying the ROS and RNS priming input against salinity we performed a comparative analysis of carbonylated, nitrated and nitrosylated proteome in citrus plants (Tanou et al., 2012a). This approach revealed that the majority of the PTM-targeted proteins in leaves were involved in Calvin–Benson cycle followed by disease/defense mechanisms and protein destination. More interestingly, among the 92 carbonylated, 88 Try-nitrated and 82 S-nitrosylated proteins, approximately one third of them, namely 34, 26 and 36 proteins respectively were specifically carbonylated or Try-nitrated or S-nitrosylated (Tanou et al., 2012a). On the contrary, 22 citrus proteins, including Rubisco large subunit, GAPDH subunits A, B, the photosystem II 44 kDa reaction center, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, carbonic anhydrase, light-harvesting chlorophyll a/b binding protein, ribulose-5-phosphate 3 epimerase, fructose-1,6-bisphosphate aldolase, glycolate oxidase and sinapyl alcohol dehydrogenase, simultaneously targeted by these three PTMs (Tanou et al., 2012a). It is also interesting that in a previous study we observed an overlap (17 proteins) among the NO– or H2O2–targeted carbonylated (n = 40) and S-nitrosylated (n = 49) proteins in citrus leaves experiencing priming effects against salinity (Tanou et al., 2009a), thereby disclosing that these proteins are common markers of NO and H2O2 signaling. Possible explanations for the co-modulated pattern of protein carbonylation and Tyr-nitration in contrast to S-nitrosylation have been proposed. The co-occurrence of more than one PTMs could be explained by the fact that the first PTM allosterically triggers the occurrence of other(s), thereby fine-tuning the degradation of damaged proteins by the proteasomes and/or offering protection against irreversible damage (Lounifi et al., 2013). It is therefore possible that S-nitrosylation might prevent the irreversible loss of function of proteins by protein oxidation due to carbonylation and/or Tyr-nitration by locking the structure of these proteins in a state under which they are no more sensitive to ROS/RNS attack (Sun et al., 2006). Altogether, the results support the existence of a link between oxidative and nitrosative regulatory events through PTMs characterizing priming phenomena in citrus plants.

### LEAVES AND ROOTS EXHIBIT BOTH COMMON AND DISTINCT PRIMING CODES IN RESPONSE TO NO AND H2O2

When examining the expression of genes involved in NO production [e.g., nitric oxide synthase (NOS)-like proteins, nitrate reductase (NR), nitrite reductase (NiR)] as well as H2O2 generation [e.g., diamine oxidases (DAO) and polyamine oxidases (PAO)] we observed a distinctive complementary pattern in leaves and roots of citrus exposed to NaCl (Tanou et al., 2012a), likely reflecting locally and systemically differences in oxy/nitro priming signaling. Consistent with these tissue-specific transcriptional patterns, we also uncovered the existence of a tissue-dependent regulation of S-nitrosylation induced by NO and H2O2 stimuli (Tanou et al., 2012a). The S-nitrosylation was induced in leaves and depressed in roots following NO and H2O2 priming treatments, which is in agreement with the differential spatial distribution of oxidative and nitrosative stress in leaves versus roots documented in Lotus japonicus (Signorelli et al., 2013). By contrast, chemical pre-treatments with NO or H2O2 suppressed protein carbonylation and nitration in both leaves and roots exposed to NaCl, suggesting that some PTM responses are commonly regulated in the different types of citrus tissues (Tanou et al., 2012a). This is further strengthened by the fact that SNP stimulated Tyr-nitration in leaves and roots during acclimation to drought stress (Ziogas et al., 2015). Such functional differences between leaves and roots may be partially attributed to the tissue differences in the types of NO and ROS generation systems. Superoxide (O•− 2 ) and H2O2 produced through SODs up-regulation and polyamine degradation in roots of citrus just following pre-treatments with NO or H2O2 can activate specific signaling pathways distinct from those perceived by leaves (Tanou et al., 2012a). Analogously, NO accumulation via NR activation in citrus leaves subjected to NaCl or PEG stress (Tanou et al., 2012a) accompanies several different NO-signaling events which could regulate downstream pathways and stress acclimation (Baudouin and Hancock, 2014).

### SIGNALING CROSS-TALK BETWEEN NO/ROS AND OTHER PATHWAYS

Recent studies have reported that many abiotic stress responses are coordinated by various signaling networks, particularly involving phytohormones, ROS, and RNS (Considine et al., 2015). In NaCl-treated citrus, NO and ROS accumulation in local and systemic tissues showed considerable overlap whereas a large part of the NaCl-sensitive proteins were commonly modulated by NO and H2O2 (Tanou et al., 2009a), suggesting that there is a dynamic interplay between the signals regulating priming. It is noted that comparative proteomic analysis in citrus leaves under physiological non-stressful conditions revealed (i) an interlinked NO- and H2O2 modulated protein network, (ii) the carbonylation status of a very large portion of the carbonylated citrus mitochondrial proteins, which remained constant or depressed by NO and H2O2 (Tanou et al., 2010), disclosing the parallels in action between NO and ROS. Here, we also provide examples demonstrating that NO could interact with other signaling pathways in modulating stress acclimation. For example, we observed temporal–spatial interactions between NO-specific PTMs and polyamines (PAs) homeostasis/metabolism in citrus challenged with salinity (Tanou et al., 2014), thus confirming that NO and PAs displayed some overlapping functions in plants (Parra-Lobato and Gomez-Jimenez, 2011). Targeted analysis of PA-affected S-nitrosylated citrus proteins led us to propose that PAs binding to specific

proteins, such as dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR), may affect protein conformational parameters and also the environment surrounding Cys residues of protein targets (Tanou et al., 2014). Such chemical modifications of Cys residues could lead either to Cys oxidation through ROS or to S-nitrosylation through NO. Drought stress can induce accumulation of hydrogen sulfide (H2S) and its biosynthetic enzyme L-cysteine desulfhydrase (LCD) in citrus. Moreover, in citrus roots H2S can induce the expression of NiR and mitochondrial NAD(P)H dehydrogenases that are involved in NO production (Gupta et al., 2011) while H2S accumulation acts downstream of NO in PEG-induced S-nitrosylation (Ziogas et al., 2015). Meanwhile, drought stress induction of ABA accumulation is a well-known fact (Tan et al., 2003). In citrus the fact that PEG stress-induced ABA accumulation and the expression of 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthesis, were depressed in plants pre-exposed to NO or to H2S (Ziogas et al., 2015) suggests that the interplay among NO, H2S, and ABA could be considered a mechanism by which citrus orchestrates drought stress acclimation.

### CONCLUSION AND FUTURE PERSPECTIVES

Research performed over the last years documented that NO and H2O2 induce priming toward salinity and drought in citrus plants and most importantly reveals key aspects of this phenomenon. Based on these results, we propose a signaling network through which NO and H2O2 provoke priming responses in leaves and roots of citrus (**Figure 1**). While several components of the priming mechanism have been proposed, we

still lack a thorough understanding of the complex mode of action of specific signaling molecules in plant stress acclimation. In this regard, various -omics techniques investigating both roots and leaves should be combined to fully understand NO and ROS-induced priming. It will be perhaps the major challenge of priming research to test these chemical agents against multiple abiotic stresses that occur in field conditions. Such an approach would allow establishing priming technology as a tool to manage crop yield.

### REFERENCES

### AUTHOR CONTRIBUTIONS

AM, DJ, VZ, and GT wrote the paper.

### ACKNOWLEDGMENT

Authors apologize if some references related to the main theme of the current review could not be cited due to space constraints.

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

Copyright © 2016 Molassiotis, Job, Ziogas and Tanou. 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.

# Reactive Nitrogen Species in Mitochondria and Their Implications in Plant Energy Status and Hypoxic Stress Tolerance

#### Kapuganti Jagadis Gupta1 \* and Abir U. Igamberdiev2

1 National Institute of Plant Genome Research, New Delhi, India, 2 Department of Biology, Memorial University of Newfoundland, St. John's, NL, Canada

Hypoxic and anoxic conditions result in the energy crisis that leads to cell damage. Since mitochondria are the primary organelles for energy production, the support of these organelles in a functional state is an important task during oxygen deprivation. Plant mitochondria adapted the strategy to survive under hypoxia by keeping electron transport operative even without oxygen via the use of nitrite as a terminal electrons acceptor. The process of nitrite reduction to nitric oxide (NO) in the mitochondrial electron transport chain recycles NADH and leads to a limited rate of ATP production. The produced ATP alongside with the ATP generated by fermentation supports the processes of transcription and translation required for hypoxic survival and recovery of plants. Non-symbiotic hemoglobins (called phytoglobins in plants) scavenge NO and thus contribute to regeneration of NAD+ and nitrate required for the operation of anaerobic energy metabolism. This overall operation represents an important strategy of biochemical adaptation that results in the improvement of energy status and thereby in protection of plants in the conditions of hypoxic stress.

### Keywords: peroxynitrite, nitric oxide, superoxide, hypoxia, mitochondria

### FUNCTIONALITY OF MITOCHONDRIA UNDER HYPOXIC STRESS

The primary function of mitochondria is to generate ATP, thus these organelles are vital for plant survival. Since oxygen is essential for ATP, any change in its concentration can affect ATP levels; therefore it can affect all energy requirements for biochemical reactions in the cell. Since mitochondria take up oxygen for respiration, these organelles efficiently sense oxygen. Plant tissues, especially roots, experience hypoxia during flooding and waterlogging (Bailey-Serres and Voesenek, 2008). Plants experience hypoxia in roots, germinating and developing seeds and in any bulky tissues due to the restricted diffusion of oxygen into these tissues through internal cell layers (Tschiersch et al., 2011). During hypoxia, cytochrome oxidase (COX) will have a limited capacity to function using oxygen while another terminal oxidase, the alternative oxidase (AOX), practically does not function under hypoxia (Igamberdiev and Hill, 2009). This is due to the different Km values of these two terminal oxidases. For instance, the Km value of COX for oxygen is in the range from 0.1 to 0.15 µM whereas the Km value for AOX is in the order of 10 µM (reviewed in Igamberdiev and Hill, 2009). The lack of the terminal acceptor alters the

### Edited by:

Jeremy Astier, Helmholtz Zentrum München, Germany

### Reviewed by:

Francisca Sevilla, Agencia Estatal Consejo Superior de Investigaciones Científicas, Spain Natalia Correa-Aragunde, Instituto de Investigaciones Biológicas – CONICET, UNMdP, Argentina

\*Correspondence:

Kapuganti Jagadis Gupta<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Biotic Interactions, a section of the journal Frontiers in Plant Science

Received: 11 December 2015 Accepted: 10 March 2016 Published: 24 March 2016

#### Citation:

Gupta KJ and Igamberdiev AU (2016) Reactive Nitrogen Species in Mitochondria and Their Implications in Plant Energy Status and Hypoxic Stress Tolerance. Front. Plant Sci. 7:369. doi: 10.3389/fpls.2016.00369

mitochondrial functionality under hypoxia and near anoxia and results in impairment of the mitochondrial infrastructure (Vartapetian et al., 2003). The direct effect of oxygen deficiency on mitochondria is related to the lack of terminal electron acceptor in the electron transport chain and to the lack of ATP production. We will show below that plant mitochondria may have a sufficient metabolic plasticity to partially overcome these shortcomings, but their operation in such stress conditions is associated with the formation of reactive oxygen and nitrogen species.

### PLANT MITOCHONDRIA APPLY VARIOUS STRATEGIES TO SURVIVE UNDER HYPOXIA

Survival under hypoxia depends on energy production. Therefore it is very important for the cells to safeguard mitochondria during stress. Plant mitochondria adapt various strategies to retain their structure for prolonged periods of time. One of such strategies is to keep the production of ATP under low oxygen conditions. A preliminary clue about the requisition for ATP for structural integrity came from study by Coueé et al. (1992), where the exogenous supply of ATP was shown to result in the support of endogenous ATP production and protection of mitochondria. Later several studies revealed that the application of nitrate has a protective role for root mitochondria from maize and pea seedling (Müller et al., 1994; Vartapetian and Polyakova, 1999). These authors suggested that nitrate (NO3 −) can act as a terminal electron acceptor that supports the operation of electron transport chain in the absence of oxygen. But so far there is no evidence that nitrate can act as a terminal electron acceptor. How nitrate can be transported to mitochondria is also not known. Plants take up nitrate by high and low-affinity transporters (encoded by the families of genes NRT1 and NRT2) (Dechorgnat et al., 2011). After being taken up by roots, NO3 − is reduced first to NO2 − by the cytosolic nitrate reductase (cNR) where NAD(P)H is used as electron donor, and further the plastidal nitrite reductase reduces nitrite to ammonium (Simontacchi et al., 2015). But there are no reports that mitochondria contain any nitrate transporter. On the other hand, nitrate could indirectly support the functionality of seedling mitochondria under hypoxia via its reduction to nitrite, which can be imported to mitochondria by a similar transporter to that found in chloroplasts (Sugiura et al., 2007) or by the mitochondrial inner membrane anion channel (PIMAC) activated under low ATP conditions (Laus et al., 2008). In potato tuber mitochondria, 29 metabolite transporters have been identified (Salvato et al., 2014), while 58 members of the mitochondrial carrier protein family were described in Arabidopsis (Palmieri et al., 2011), and 50 members in rice (Taylor et al., 2010). Some of these transporters can potentially carry nitrite, however, further studies are needed to establish what particular carrier is used to import nitrite into mitochondria.

The mitochondria of some anoxia-tolerant plant species do not change their structure under anoxia. For instance the mitochondria of anoxia-tolerant plants Echinochloa phyllopogon and E. crus-galli retained their structure and metabolic activity during the prolonged exposure to anaerobic stress (Kennedy et al., 1987). In another study it was shown that the mitochondrial biogenesis did not alter in the anoxia-tolerant rice (Howell et al., 2007). All this suggests that plants can use a specific strategy to survive under hypoxia or anoxia by keeping their mitochondria in a functional state (Gupta and Igamberdiev, 2011; Igamberdiev et al., 2014; Shingaki-Wells et al., 2014).

### MITOCHONDRIA CAN REDUCE NITRITE TO NO

Several lines of evidence suggest that mitochondria of different and maybe all species are capable of reducing nitrite to nitric oxide (NO). For instance, the mitochondria isolated from ciliate protists and Fusarium fungus, possess a capability to reduce nitrite to NO (Tielens et al., 2002). Kozlov et al. (1999) showed that rat liver mitochondria can produce NO using nitrite. The first evidence for this reaction in photosynthetic organisms came from the study of the green alga Chlorella sorokiniana where the addition of nitrite resulted in NO formation and could be blocked by the inhibitors of the mitochondrial electron transport chain (Tischner et al., 2004). Later it was shown that tobacco cell suspensions are able to reduce nitrite to NO and that the application of mitochondrial inhibitors suppressed this reaction (Planchet et al., 2005). Then Gupta et al. (2005) conducted a detailed study and found that nitrite reduction to NO takes place in root mitochondria from various species, such as pea, barley, Arabidopsis and tobacco, and determined the Km value for nitrite reduction to NO (175 µM). This allowed estimating nitrite concentration needed for NO production. Since under hypoxia nitrite reduction to ammonium is inhibited (Botrel et al., 1996), the accumulated nitrite can act as a substrate for NO formation. This reaction is highly sensitive to oxygen, which has a Ki value of approximately 0.05% or 0.6 µM (Gupta and Igamberdiev, 2011). Gupta and Kaiser (2010) demonstrated that this process occurs in the membrane but not in the matrix of mitochondria. The complexes III and IV of the mitochondrial electron transport chain were shown to be the sites for NO production.

$$\text{NO}\_2^- + 2\text{H}^+ + \text{e}^- \rightarrow \text{NO} + \text{H}\_2\text{O}$$

The complex III can produce NO via a leakage of electrons to nitrite in a similar way as superoxide is produced by oneelectron reduction of oxygen at this site. The mechanisms of nitrite reduction to NO by COX are still under investigation and several models are available for the explanation of this mechanism (reviewed in Gupta and Igamberdiev, 2011). The availability of oxygen, nitrite and NO determines the redox state of the COX center that contains heme a3 and copper B (Fea3CuB) that in turn depends on the redox state of cytochrome c. In the absence of oxygen, Fe2+ donates the electron for nitrite reduction to NO. But still the concrete details of this mechanism remain speculative.

Other sites of nitrite reduction to NO in mitochondria may include cytochrome c itself and other hemeproteins (Basu et al., 2008). The proteins other than hemeproteins may also be

involved in NO formation. The involvement of the AOX, which is a di-iron carboxylate protein, in NO production was suggested on the basis of the effect of its inhibitor, salicylhydroxamic acid (SHAM) on NO evolution from mitochondria (Planchet et al., 2005; Gupta and Kaiser, 2010). Some proteins of a similar iron structure are found to be effective in NO metabolism (Kurtz, 2007). However, NO production in alfalfa (Medicago truncatula) nodules was fully insensitive to AOX inhibitor propylgallate (Horchani et al., 2011). The effect of AOX inhibitors reported in several studies may be explained by their action on other proteins, including peroxidases and other hemeproteins (Brouwer et al., 1986).

### NITRITE REDUCTION TO NO LEADS TO ATP GENERATION VIA PHYTOGLOBIN-NO CYCLE

Under anoxia mitochondria produce significant amounts of NO (Gupta et al., 2005; Planchet et al., 2005). One interesting puzzle is the physiological role of the mitochondrial NO production. Previously it was suggested that COX may play a role in membrane translocation of protons during nitrite reduction to NO (Castello et al., 2006). Isolated mitochondria of barley and rice were able oxidize the externally applied NADH and NADPH under anoxia when nitrite was applied, and this oxidation resulted in the detectable ATP formation (Stoimenova et al., 2007). The build-up of ATP during the oxidation of NADH and NADPH by anoxic mitochondria was sensitive to myxothiazol and KCN suggesting that the operation of the complexes III and IV is essential for this process. The anoxic production of ATP constituted only 3–5% of the aerobic ATP generation. However, the ATP produced during glycolytic fermentation together with the mitochondrial anaerobic nitrite-driven ATP production can make a major contribution for hypoxic survival.

The produced NO needs to be recycled very fast in order to avoid the nitrosative stress. Plants possess hypoxically induced hexacoordinated hemoglobins (called class 1 phytoglobins). They are the active scavengers of NO; thereby they can significantly reduce the amounts of NO to the levels that are much less toxic to plants. Class 1 phytoglobins possess the affinity to oxygen of two orders of magnitude higher than cytochrome c oxidase (Kd ∼2 nM); therefore they can operate at the oxygen levels that cannot be utilized by mitochondria (Igamberdiev et al., 2011). Scavenging of NO involves the oxidation of phytoglobin ferrous ion, forming metphytoglobin and nitrate (Igamberdiev et al., 2006, 2011). Operation of the phytoglobin-NO cycle is essential for recycling nitrate and nitrite and for oxidation of excess of NADH and NADPH (Igamberdiev and Hill, 2004, 2009; Gupta and Igamberdiev, 2011; Igamberdiev et al., 2014) (**Figure 1**). This cycle can also contribute to NAD+ recycling for the continuous operation of glycolysis. Its operation is important not only under the hypoxic conditions (that occur during germination of seeds, in flooding stress or in compact meristemic tissues) but also in the course of pathogen infection, when significant amounts of NO are formed during the response mediated by salicylic and jasmonic acids (Mur et al., 2012).

### IS NITRITE-DRIVEN ANAEROBIC ATP SYNTHESIS SUFFICIENT FOR PLANTS TO SURVIVE UNDER HYPOXIA?

In order to survive under hypoxia and anoxia, plants need to sustain the machinery to carry out transcription and translation. It has been shown that the energy budgeting occurs under hypoxia to direct a specific amount of ATP for cellular functions (Edwards et al., 2012); at the same time cell recovery processes, such as cell division and elongation, decelerate in order to save energy (Takahashi et al., 2011). Several lines of evidence suggest that the active transcription of the mitochondrial genes takes place under hypoxia (Narsai and Whelan, 2013), while RNA translation appears to be downregulated. This was evidenced by 50% reduction in polysome content (Branco-Price et al., 2008) to reduce energy costs of unwanted translation during stress condition.

For instance, plants actively switch on the ethylene responsive transcription factors which are required for hypoxia sensing and survival (Bailey-Serres et al., 2012). In comparison to transcription, protein biosynthesis is much more energy demanding process. It has been shown that the translation of catabolic proteins takes place in anoxic coleoptiles of rice (Edwards et al., 2012). Plants invest energy in making catabolic proteins, such as the enzymes involved in glycolysis, under hypoxia/anoxia to get higher energy yield. Previously we have shown that mitochondria from roots produce NO within minutes of exposure to hypoxia suggesting that energy production initiation related to NO turnover starts within minutes. The anoxia-tolerant rice produced much higher levels of anoxic ATP in comparison to anoxia-intolerant barley suggesting essentiality of the nitrite-driven ATP production in the anoxic survival (Stoimenova et al., 2007). Since nitrate reductase (NR) is a part of the hemoglobin-NO cycle, its transcript is highly induced in anoxic rice coleoptiles (Lasanthi-Kudahettige et al., 2007). The increase of NR activity under oxygen deprivation (Planchet et al., 2005) suggests that the activation of transcription and translation of NR contributes to the excess of nitrite production and to NO generation, thus helping to improve the energy status of hypoxic plants to support the transcription and translation processes. The post-translational activation of NR under hypoxia is particularly important, which is achieved through dissociation of the 14-3-3 protein inhibitor and NR dephosphorylation (Allègre et al., 2004).

### ROS IN THEIR REACTION WITH NO PRODUCE REACTIVE NITROGEN SPECIES (RNS)

Electrons enter the mitochondrial electron transport chain primarily via complex I, the alternative rotenone-insensitive dehydrogenases, and complex II (succinate dehydrogenase). These electrons are transferred to the ubiquinone pool which is a central reservoir for accumulation of electrons. From the ubiquinone pool electrons pass to complex III and complex IV,

or to the AOX. The transfer through complexes III and IV leads to the generation of electrochemical proton gradient which is used by the ATP synthase to generate ATP (Rasmusson et al., 2008). When oxygen concentrations decrease below 10 µM, AOX does not function due its high Km to oxygen, and when oxygen concentrations fall further below 1 µM, the operation of COX is also ceased. This leads to the accumulation of electrons in the ubiquinone pool and at the other sites such as complex I and III. This results in the condition when the remaining oxygen molecules accept only one electron which leads to the production of superoxide anion (O2 −). The elevated NADH/NAD+ ratio in the mitochondrial matrix under hypoxic conditions, becomes a condition leading to the increase in superoxide anion production even at low oxygen concentrations (Murphy, 2009). The reduced electron carrier proteins are able to react with O2 to form O2 −, while NO produced under hypoxia can activate or repress these proteins to control O2 − production. NO is known to inhibit respiration by competitively binding to cytochrome c oxidase (Cooper, 2002). Thus, by increasing or decreasing the rate of O2 consumption by mitochondria NO may influence O2 − production in vivo by altering the local [O2] (Borisjuk et al., 2007). Recently it was shown that the overexpression of phytoglobins leads not only to the decrease in NO but also results in the increased respiration, lowering internal oxygen concentration, and subsequent production of ROS (Gupta et al., 2014). Paradoxically, the mitochondrial O2 − increases in response to low oxygen levels (Chandel et al., 1998; Guzy and Schumacker, 2006).

The produced superoxide is responsible for the generation of other ROS and of RNS. For instance, superoxide dismutase converts superoxide to hydrogen peroxide which can act as a signal. Its excessive amounts lead to cytotoxicity. NO does not directly react with H2O2 but there are reports that NO can reduce H2O2 formation (Małolepsza and Rózalska, 2005). This can be explained, in particular, by the activation through S-nitrosylation of ascorbate peroxidase, which is the key enzyme participating in H2O2 scavenging (Correa-Aragunde et al., 2013; Begara-Morales et al., 2014; Yang et al., 2015). The reaction between superoxide and NO occurs near the diffusion controlled rates and results in the formation of peroxynitrite (ONOO−), which is a toxic RNS form causing tyrosine nitration (Poyton et al., 2009). Peroxynitrite is formed in relatively low amounts under nonstress conditions, while under stress both NO and superoxide levels increase stimulating its formation. The biological reactions of NO and superoxide that limit their availability also constrain the amount of peroxynitrite formed. SOD competes effectively with NO for superoxide by reducing its level and decreasing the amount of peroxynitrite formed. The class 1 phytoglobin also

decreases peroxynitrite concentration by reducing the availability of NO to react with superoxide. Peroxynitrite can be scavenged in plants via the pathway involving thioredoxin (Wulff et al., 2009), which results in its lower toxicity in plants as compared to animal tissues where its scavenging is likely limited by a side reaction of cytochrome c oxidase (Pearce et al., 2002). The excess of NO under hypoxia can react with peroxynitrite resulting in the formation of non-toxic nitrogen dioxide (NO2) and nitrite (NO2 −). The produced NO2 can react with NO which then leads to the formation of dinitrogen trioxide (N2O3) which plays a role in nitrosative reactions (Espey et al., 2002).

In summary, plant mitochondria in the conditions of oxygen deficiency can reduce nitrite to NO, which can help in increasing their energy efficiency for supporting active transcription and translation processes in the hypoxic cells. NO participates in NAD+ recycling via the hemoglobin-NO cycle. In the reactions

### REFERENCES

with ROS, NO forms peroxynitrite and other RNS such as N2O3 which play a role as signals during the nitrosative stress.

### AUTHOR CONTRIBUTIONS

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

### ACKNOWLEDGMENTS

This work was supported by the grants from the Ramalingaswami fellowship and IYBA award by Department of Biotechnology, Govt. of India (to KG) and from the National Science and Engineering Research Council of Canada (to AI).

growth pattern in Arabidopsis. J. Exp. Bot. 64, 3339–3349. doi: 10.1093/jxb/ ert172

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

Copyright © 2016 Gupta and Igamberdiev. 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.

# Canavanine Alters ROS/RNS Level and Leads to Post-translational Modification of Proteins in Roots of Tomato Seedlings

Urszula Krasuska, Olga Andrzejczak, Paweł Staszek, Renata Bogatek and Agnieszka Gniazdowska\*

Department of Plant Physiology, Faculty of Agriculture and Biology, Warsaw University of Life Sciences-SGGW, Warsaw, Poland

#### Edited by:

Frank Gaupels, Helmholtz Zentrum München – Deutsches Forschungszentrum für Gesundheit und Umwelt, Germany

#### Reviewed by:

Ian Max Møller, Aarhus University, Denmark Dietmar Funck, University of Konstanz, Germany

\*Correspondence:

Agnieszka Gniazdowska<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science

Received: 09 December 2015 Accepted: 27 May 2016 Published: 14 June 2016

#### Citation:

Krasuska U, Andrzejczak O, Staszek P, Bogatek R and Gniazdowska A (2016) Canavanine Alters ROS/RNS Level and Leads to Post-translational Modification of Proteins in Roots of Tomato Seedlings. Front. Plant Sci. 7:840. doi: 10.3389/fpls.2016.00840 Canavanine (CAN), a structural analog of arginine (Arg), is used as a selective inhibitor of inducible NOS in mammals. CAN is incorporated into proteins' structure in the place of Arg, leading to the formation of aberrant compounds. This non-protein amino acid is found in legumes, e.g., Canavalia ensiformis (L.) DC. or Sutherlandia frutescens (L.) R.Br. and acts as a strong toxin against herbivores or plants. Tomato (Solanum lycopersicum L.) seedlings were treated for 24–72 h with CAN (10 or 50 µM) inhibiting root growth by 50 or 100%, without lethal effect. We determined ROS level/production in root extracts, fluorescence of DAF-FM and APF derivatives corresponding to RNS level in roots of tomato seedlings and linked CAN-induced restriction of root growth to the post-translational modifications (PTMs) of proteins: carbonylation and nitration. Both PTMs are stable markers of nitro-oxidative stress, regarded as the plant's secondary response to phytotoxins. CAN enhanced H2O2 content and superoxide radicals generation in extracts of tomato roots and stimulated formation of protein carbonyl groups. An elevated level of carbonylated proteins was characteristic for the plants after 72 h of the culture, mainly for the roots exposed to 10 µM CAN. The proteolytic activity was stimulated by tested non-protein amino acid. CAN treatment led to decline of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives. Short-term exposure of tomato seedlings to CAN lowered the protein nitration level. Activity of peroxidase, polyamine oxidase and NADPH oxidase, enzymes acting as modulators of H2O2 concentration and governing root architecture and growth were determined. Activities of all enzymes were stimulated by CAN, but no strict CAN concentration dependence was observed. We conclude, that although CAN treatment led to a decline in the nitric oxide level, PTMs observed in roots of plants exposed to CAN are linked rather to the formation of carbonyl groups than to nitration, and are detected particularly after 24 h. Thus, oxidative stress and oxidative modifications of proteins seems to be of significant importance in the rapid response of plants to CAN.

Keywords: NADPH oxidase, peroxidase, polyamine oxidase, proteolysis, protein carbonylation, protein nitration, RNS, ROS

### INTRODUCTION

fpls-07-00840 June 11, 2016 Time: 12:9 # 2

In addition to 20 amino acids, known to act as building blocks of proteins, living organisms also produce non-proteinogenic amino acids (NPAAs). In plants, about 250 NPAAs have been identified (Vranova et al., 2011), which possess a rich structural diversity and some of them exhibit harmful biological effects both in plants and animals (Rosenthal, 1982, 2001; Bell, 2003). The possible role of NPAAs in plants is protection against predators or pathogens. They act also as an allelopathic weapon against neighboring organisms. Among NPAAs naturally occurring in planta L-canavanine (CAN), the L-2-amino-4- (guanidinoxy) butyric acid is identified and recognized as a compound of high toxicity (Rosenthal, 2001), but of great importance in medicine, where is used as cytotoxic agent against cancer cells in humans (Swaffar et al., 1994, 1995; Vynnytska et al., 2011). In animals the mode of action of CAN depends on the fact that this NPAA is the guanidinoxy structural analog of arginine (Arg) and its presence can lead to a production of CAN-containing proteins, which may disrupt cellular metabolism (Rosenthal and Harper, 1996). CAN affects also regulatory and catalytic reactions of Arg metabolism or uptake. According to the toxicological data, CAN is considered to be "very toxic" (Rodricks, 2007), as is linked with several serious diseases in humans, e.g., systemic lupus erythematosus (SLE), which is characterized by a defect in the immune system. The inflammatory and oxidative modification reactions are the most important events associated with complications of SLE patients (Shah et al., 2014). Recently, hyper-nitration of tyrosine residues of, e.g., histone H1 has been suggested as an etiopathogenesis of SLE and rheumatoid arthritis (Khan et al., 2014).

In mammalian tissue NO synthase (NOS) converts Arg to NO and L-citrulline (Stuehr, 1999). Acting as an antimetabolite of Arg, in animal tissue CAN is commonly used as a selective inhibitor of inducible isoform of NOS (iNOS; Abd El-Gawad and Khalifa, 2001; Li et al., 2001). Application of CAN in animal or human tissue to inhibit NOS activity results in a decrease in NO emission (Luzzi and Marletta, 2005), but also induces an oxidative burst demonstrated by an elevated level of ROS (Demiryürek et al., 1997; Riganti et al., 2003).

In plants, like in animal tissue, NO acts as a signaling molecule. But the pathways of its biosynthesis are still not clarified. Among many metabolic pathways such as polyamines (PAs) or ethylene biosynthesis, in plant cells Arg is the hypothetical substrate for NO formation. There is no doubt that in higher plants NO formation depends on reductive pathways utilizing NO2 − as a substrate, but the existence of the enzyme of NOSlike activity is controversial (Corpas et al., 2009; review by Gupta et al., 2011; Hancock, 2012; Corpas and Barroso, 2015). Nevertheless, both in animals and plants NO mode of action involves PTMs of proteins including S-nitrosylation of cysteine (Cys) and nitration of tyrosine (Tyr) residues (Lozano-Juste et al., 2011). In addition, carbonylation of bovine serum albumin (BSA) has been demonstrated in vitro after NO donor application (Krasuska et al., 2014b). Nitration of Tyr is connected with a covalent binding of a nitro (-NO2) group to one of the two equivalent ortho carbons of this amino acid and leads to the formation of 3-nitrotyrosine (NO2Tyr, 3-NT; Chaki et al., 2009). Nitration is associated with an accumulation of peroxynitrite (ONOO−), the oxidizing product of reaction of superoxide anion (O2 r−) and NO in its radical form (Arasimowicz-Jelonek and Floryszak-Wieczorek, 2011; Astier and Lindermayr, 2012).

Disturbances in the metabolism of NO or other RNS are related to an alteration in ROS level leading commonly to the induction of nitro-oxidative stress (Corpas and Barroso, 2013). The formation of carbonylated proteins is due to various types of oxidative modification of amino acids, direct ROS attacks on proline (Pro), Arg, lysine (Lys), asparagine (Asn) and threonine (Thr) residues, the incorporation of reactive carbonyl derivatives into peptides by interaction with Cys, histidine (His) and Lys or the adduction of advanced glycation end products formed by ROS reactions with reducing sugars (Møller et al., 2011; Yan and Forster, 2011). It has been proposed that carbonylation, although is a negative consequence of ROS over-accumulation, serves also as an intracellular metabolic regulatory mechanism.

Treatment of tomato (Solanum lycopersicum L.) seedlings with CAN (10 µM) for 24 or 72 h led to 50% inhibition of root growth, while the application of CAN at higher concentration (50 µM) entirely inhibited root elongation, but had no lethal effect, although no recovery was observed after seedlings transfer to Arg or water (Krasuska et al., 2016). CAN treatment did not lead to DNA fragmentation, but resulted in slight enhancement in RNA content, accompanied by 20% enlargement in total protein level. Preliminary data indicated that prolonged culture of tomato seedlings in CAN solution increased accumulation of ROS (O2 r− and H2O2) in tomato root tips detected by tissue staining with NBT or DAB (Krasuska et al., 2016). CAN inhibited also NO emission and transiently enhanced ONOO− production in root tips of tomato seedlings, suggesting its direct impact on NO biosynthetic pathway (Krasuska et al., 2016).

The aim of our work was to show another than incorporation into the protein structure mode of action of CAN. We investigated the modifications in ROS concentration or generation rate, and fluorescence of DAF-FM or APF derivatives after reaction with RNS in roots of tomato seedlings after 24–72 h long exposure to CAN, as our previously published data indicated influence of CAN on RNS content in root tips (Krasuska et al., 2016). Following the treatments, formation of carbonyl groups and 3-NT were studied, to detect stable and reliable markers of nitro-oxidative stress, which could be induced by CAN. Some PTMs, e.g., carbonylation stimulate proteolytic degradation, therefore, we investigated impact of CAN on proteolytic activity. In addition, we linked CAN toxicity with the activity of polyamine oxidase (PAO) because decline of PAs content was observed as a result of CAN treatment (Davis, 1997) and degradation of PAs leads to H2O2 formation. NADPH oxidase catalyze the apoplastic production of O2 r−, plant

**Abbreviations:** Arg, arginine; CAN, canavanine; cPTIO, 2-(4-carboxyphenyl)- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; 3-NT, 3-nitrotyrosine; NO, nitric oxide; PTMs, post-translational protein modifications; RNS, reactive nitrogen species; ROS, reactive oxygen species.

peroxidase Class III (POx) catalyzes oxidoreduction between H2O2 and various reductants (Hiraga et al., 2001; Kaur et al., 2014). Both enzymes govern also root architecture and growth, together with classical phytohormons and NO (Krasuska and Gniazdowska, 2015) therefore, could play an important role in plant response to CAN.

To summarize, the aim of the study was to find the stable markers of CAN toxicity and describe the mode of action of this non-protein amino acid, to show that application of commonly used inhibitor of NO biosynthesis may result in oxidative modifications.

### MATERIALS AND METHODS

### Plant Material

Seeds of tomato (Solanum lycopersicum L. cv. Malinowy Ozarowski) were germinated in water at 20 ˙ ◦C in darkness for 3 days. After this period seedlings of equal roots' length (5 mm) were separated and transferred to Petri dishes (Ø15 cm) containing filter paper moistened with distilled water (control) or CAN (Sigma-Aldrich) dissolved in distilled water pH 7.5 (**Figure 1**). Control seedlings and seedlings treated with CAN were cultured in a growth chamber at 23/20◦C, 12/12 h day/night regime for 24 or 72 h.

The concentration of CAN (10 µM) required for reduction of root length to 50% of the control, was accepted as IC50. The concentration (50 µM), which completely inhibited elongation growth of roots, was accepted as IC100.

### Measurement of Hydrogen Peroxide (H2O2) Concentration

The concentration of H2O2 in extracts of tomato roots was determined according to Velikova et al. (2000). Isolated roots (100 mg) were immediately homogenized by pestle and mortar in cooled 0.1% (w/v) trichloroacetic acid (TCA). After centrifugation at 15,000 g for 15 min at 4◦C, the supernatant was collected for further analysis. The concentration of H2O2 was measured in the assay mixture (0.25 ml supernatant, 1 ml freshly prepared 1 M KI in 10 mM potassium phosphate buffer pH 7.0, and 0.5 ml 10 mM potassium phosphate buffer pH 7.0) at 390 nm using a Hitachi U-2900 spectrophotometer. The standard curve was prepared using 0.88 µM H2O2 (Sigma-Aldrich). Measurements of H2O2 concentration were done in three independent experiments, each in three biological replicates and expressed as nmol g−1 FW.

Using the same method, the H2O2 concentration was measured in the liquid medium surrounding the roots. An equal number of control or CAN-treated tomato seedlings were placed in Petri dishes (Ø7.5 cm), and the culture was carried out as was described in plant material section. Then, the whole solution from the plate of the culture was collected, the volume was measured, and 0.5 ml was taken for the analyses in the reaction mixture. H2O2 concentration in the culture solution was measured at 390 nm. As a blank, water or CAN

FIGURE 1 | Tomato seedlings morphology. Equal of size, germinated seeds of tomato, with roots of 5 mm long were grown in water (control) and 10 or 50 µM CAN solution for 72 h. Increase in length of root was measured twice during the culture period: 24 h after seedlings transfer to CAN and then after additional 48 h of experiment. Values are average ± SE of five independent experiments. Asterisk (∗ ) indicate significance from control at the same time of culture period at p ≤ 0.05 based on Student's test.

solutions from the plates without plant material were used. The experiment was done in four repetitions, and expressed as nmol L−1 .

### Measurement of Oxidation of Epinephrine by Superoxide Radicals in Root Extract

Measurement of oxidation of epinephrine by superoxide radicals was done according to Misra and Fridovich (1972). Roots of tomato seedlings (60 mg) were homogenized in 0.05 M Tris-HCl pH 7.5 with addition of 2% (w/v) polyvinylpolypyrrolidone (PVPP), and centrifuged at 12,000 g for 15 min at 4◦C. The supernatant was immediately used for further analyses. The oxidation of epinephrine to adrenochrome was measured in reaction mixture (0.05 ml of supernatant, 0.05 ml 0.05 M Tris-HCl pH 7.5, 0.05 ml of 60 mM epinephrine prepared in 50 mM HCl) at 480 nm (microplate reader Sunrise, Tecan) for 2 min. Autooxidation of epinephrine in the reaction mixture (without roots extract) was done in each assay and obtained values were included in the calculations. The epinephrine extinction coefficient was ε = 4.02 mM−1 cm−1 . The measurements were done in three independent experiments, each in three biological replicates and expressed as relative units. One relative unit corresponds to the rate of epinephrine oxidation in root extracts of control tomato seedlings after 24 h of culture calculated as µmol min−1 g −1 FW.

### Measurement of Fluorescence Emission Corresponding to NO and ONOO− Level in Tomato Roots

Nitric oxide and ONOO− generation was measured as efflux of derivatives of 4-amino-5-methylamino-20 ,70 -difluorofluorescein diacetate (DAF-FM DA, Invitrogen) and 30 -(p-aminophenyl) fluorescein (APF, Invitrogen), respectively following the manufactere's instructions. APF is sensitive to ONOO− but also to OH r , and hypochlorite (Halliwell and Whiteman, 2004).

For NO analyses, roots of three plants were washed twice in distilled water and incubated for 40 min in darkness at room temperature in the mixture: 20 µM DAF-FM DA [2 mM DAF-FM DA prepared as stock solution dissolved in dimethyl sulfoxide (DMSO)] in 10 mM HEPES-KOH, pH 7.4. After incubation, roots were washed twice in 10 mM HEPES-KOH pH 7.4 and transferred to a cuvette containing 2 ml of this buffer. Fluorescence was recorded for 1,500 s (excitation 495 nm, emission 515 nm) using a Hitachi F-2500 spectrofluorimeter. A final intensity of fluorescence in the buffer above the roots, after the indicated time was taken into calculations. After measurement the roots were gently dried on filter paper and weighed.

For ONOO− determination, two roots were washed twice in distilled water and then incubated for 1 h in darkness at room temperature with 0.2% (v/v) APF [5 mM APF – stock solution dissolved in dimethylformamide (DMF)] in 10 mM HEPES-KOH, pH 7.2. After incubation roots were washed twice in 10 mM HEPES-KOH, pH 7.2 and transferred to a cuvette containing 2 ml of this buffer. Fluorescence was recorded for 1,800 s (excitation 490 nm, emission 515 nm) using a Hitachi F-2500 spectrofluorimeter. A final intensity of fluorescence in the buffer above the roots, after the indicated time, was taken into calculations. After measurement the roots were gently dried on filter paper and weighed.

Fluorescence was calculated per 1 g FW and expressed in arbitrary units (U). 1 U was estimated from the result obtained for roots isolated before treatment with CAN.

Measurements were done in 3–4 independent experiments with three repetitions in each.

### Quantitative Measurement of Protein Carbonyl Groups

Protein carbonyl groups in root extracts were measured using enzyme-linked immunosorbent assay (ELISA) according to Levine et al. (1994) and Buss et al. (1997). Roots (200 mg) were homogenized in 0.1 M Tris-HCl, pH 7.0 with 1 mM ethylenediaminetetraacetic acid (EDTA), 2% (w/v) PVPP, 1 mM dithiothreitol (DTT), 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich P9599), and 10% (v/v) glycerol in an ice bath. After centrifugation at 15,000 g 15 min at 4◦C, the supernatant was collected and incubated with 1% (w/v) streptomycin (for 20 min, at room temperature, in darkness with constant slow shaking). Then, supernatant was incubated with 10 mM 2,4-dinitrophenylhydrazine (DNPH; Sigma-Aldrich) dissolved in DMSO in the dark for 35 min at 37◦C. At the same time, samples prepared without DNPH were incubated in 2 M HCl. Proteins were precipitated 10 min with 10% (w/v) TCA, and the pellets obtained after centrifugation (10,000 g; 10 min) were washed three times with 1:1 (v/v) ethanol:ethyl acetate. After each washing step, the samples were centrifuged for 5 min at 10,000 g. Washed pellets were dissolved in 6 M guanidine hydrochloride (Sigma) in 2 M HCl. Protein concentration was measured according to Bradford (1976) using BSA as a standard. Reduction of BSA fatty acid free (Sigma-Aldrich) was done using sodium dithionite (2 mM) for 30 min at 25◦C in darkness. Solution of reduced BSA was passed through Sephadex G-25 column equilibrated with 0.1 M Tris-HCl, pH 7.0. Oxidation of reduced BSA was done using 1 µl of 3% (v/v) H2O2 with the addition of sodium perchlorate for 30 min at room temperature in darkness. Carbonyl groups in oxidized BSA were labeled with DNPH and processed as described above. Blank probes of BSA were incubated in 2 M HCl without addition of DNPH. Final BSA concentration was determined using Bradford reagent. Concentration of carbonyl groups in BSA was measured at 375 nm, and calculated from the extinction coefficient ε = 22 mM−1 cm−1 .

Before measurement, the pH of the protein samples and oxidized BSA was adjusted to 9.0 with 5 M KOH. Triplicate of 200 µl of each sample or oxidized BSA were added to wells of Nunc Immuno Plate Maxisorp (Sigma), and incubated at 4◦C overnight. Next day the samples were removed and plates were washed three times with Tris-buffered saline (TBS) with 0.05% (v/v) Tween-20 (TBST). Next, the plate wells were blocked with 0.1% (w/v) BSA in TBS (250 µl per well) for 1.5 h at room temperature. After washing step (three times with TBST), monoclonal primary antibodies (Monoclonal Anti-dinitrophenyl (DNP) antibodies, A2831 Sigma-Aldrich)

conjugated with alkaline phosphatase (dilution 1:25,000) in TBST were added, at room temperature for 1.5 h in darkness. After removal of antibodies and another washing step (three times with TBST) p-nitrophenylphosphate disodium (pNPP), prepared at concentration 1 mg ml−1 in 1 M diethanolamine (DEA) solution with 0.5 mM MgCl2 was added into each well. Incubation of the plates were carried out at 37◦C for 1 h, the reaction was stopped with 5 µl of 5 M KOH, and absorbance was read at 405 nm with referential wave 605 nm in microplate reader (Sunrise, Tecan). Measurement of protein carbonyl groups were done in three biological repetitions, each in three replicates and expressed as nmol mg−1 protein.

### Immunodetection of Carbonyl Groups

Immunodetection of protein carbonyl groups was done by immunoblotting technique. Protein samples prepared as described above were suspended in the sample buffer: 63 mM Tris-HCl, pH 6.8, 1% (w/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, 0.01% (w/v) bromophenol blue, and 20 mM DTT. After incubation at 95◦C for 10 min, 20 µg proteins were loaded per lane, and separated on 10% polyacrylamide gels with SDS (SDS-PAGE) according to Laemmli (1970), and then electrotransferred to nitrocellulose membranes (Pure Nitrocellulose Membrane, Sigma-Aldrich) according to Towbin et al. (1979) using a Bio-Rad wet blotting apparatus. Subsequently, the proteins were visualized on the membranes by incubation in 0.2% Ponceau S dissolved in 2% acetic acid. The membranes were blocked overnight at 4◦C with non-fat dry milk in TBST. After blocking, membranes were washed three times in TBST, and immunolabeling of carbonyl groups was carried out by incubating the membranes with monoclonal anti-DNP antibodies, conjugated with alkaline phosphatase at a dilution of 1:100,000 at room temperature. Visualization of carbonylated proteins was done after addition of 0.1 M Tris-HCl pH 9.5, 0.1 M NaCl, 5 mM MgCl2, 0.2 mM nitroblue tetrazolium (NBT), and 0.21 mM 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Assays were performed in 2–3 independent experiments and typical results are shown. The entire gel lanes were quantified by densitometry analysis, which was done using Image J.

### Quantitative Measurement of 3-Nitrotyrosine in Proteins

3-NT modified proteins were analyzed by an ELISA method according to Khan et al. (1998). Roots of tomato seedling (200 mg) were washed twice in distilled water and homogenized by mortar and pestle in 0.1 M HEPES-KOH, pH 7.0 with 1 mM EDTA, 2% (w/v) PVPP, 2 mM DTT, 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich) and 10% (v/v) glycerol in an ice bath. Following centrifugation at 15,000 g for 15 min, 4◦C, the supernatant was collected for protein determination.

Positive control was prepared from fatty acid free BSA dissolved in TBS. BSA was incubated with NaNO2 (1 mM) acidified with 0.2 M HCl, and in the presence of 0.1 mM NaHCO3 for 30 min at 37◦C, in darkness. After incubation, BSA was precipitated with 20% (w/v) TCA for 20 min at room temperature, centrifuged (10,000 g; 10 min), and dissolved in water adjusted to pH 9.0 with 1 M KOH. The 3-NT content of nitro-BSA was determined at 438 nm, and calculated from the extinction coefficient ε = 4.3 mM−1 cm−1 .

Root protein samples before ELISA measurement were adjusted to pH 9.0 with 1 M KOH. Nunc Immuno Plate Maxisorp (Sigma) was coated with samples or nitrated-BSA (200 µl per well), and incubated at 4◦C overnight. After incubation, the plate was washed three times with PBS. Following blocking step with 0.1% (w/v) gelatin in TBS (250 µl per well; 1.5 h at 37◦C in darkness) and washing three times with TBST, monoclonal primary antibodies (Monoclonal Anti-3-Nitrotyrosine antibodies, Sigma-Aldrich) were added at a dilution of 1:1,000 (200 µl per well), and incubated for 1 h at 37◦C in darkness. After washing three times with TBST, the plate was covered with secondary antibodies (anti-mouse IgG conjugated with Alcaline Phosphatase Sigma-Aldrich) at a dilution of 1:30,000 for 1 h at 37◦C in darkness. Visualization of nitrated groups was carried out using alkaline phosphatase substrate – pNPP, prepared as described for carbonylated protein quantitative measurement. After color development, the reaction was stopped with 5 µl of 5 M KOH and absorbance was read at 405 nm with reference wavelength 605 nm in a microplate reader (Sunrise, Tecan). Measurement of 3-NT proteins were done in three biological replicates, each in three technical replicates and expressed as nmol mg−1 protein.

### Immunodetection of 3-Nitrotyrosine Proteins

Immunoblotting technique was done for detection of 3 nitrotyrosine proteins. Isolated proteins (as described above) were mixed with sample buffer (described above). After heating at 80◦C for 5 min, samples were loaded (soluble protein – amount 25 µg per lane) and SDS-PAGE separated on 10% gels according to Laemmli (1970). An electrotrasfer of proteins to nitrocellulose membrane was done as described for carbonylated proteins. After transfer, proteins were visualized by Ponceau S Red staining. Blocking step was done using 0.1% gelatin in TBST overnight at 4◦C. After the washing step nitrocellulose membranes were incubated (1 h, at room temperature in darkness) with monoclonal primary antibodies (Monoclonal Anti-3-Nitrotyrosine antibodies, Sigma-Aldrich; 1:1,000). After washing in TBST (three times), nitrocellulose membranes were covered with secondary antibodies (anti-mouse IgG conjugated with Alkaline Phosphatase Sigma-Aldrich) diluted 1:30,000 for 1 h at room temperature in darkness. Visualization of nitrated groups was carried out as described for carbonylated proteins. Assays were performed in three independent experiments and typical results are shown. The entire gel lanes were quantified by densitometry analysis, which was done using Image J.

### POx Activity Measurement

Peroxidase activity measurement was done according to Saunders et al. (1964). Roots of tomato seedling (60 mg) were washed in distilled water, and then homogenized by pestle and mortar in

0.05 M potassium phosphate, pH 7.0 with 10% (v/v) glycerol, 5 mM DTT, 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich), and 2% (w/v) PVPP in an ice bath. After centrifugation (12,000 g for 10 min at 4◦C) supernatant was collected for further analyses. Protein extract was incubated with 5 mM pyrogallol in 0.05 M potassium phosphate pH 7.0 at 25◦C in darkness. After incubation, POx activity was measured after addition of 1 mM H2O2. POx activity was determined as absorbance increase at 430 nm using microplate reader (Sunrise, Tecan), and absorption coefficient ε = 2.47 mM−1 cm−1 . Results were expressed as nmol H2O2 min−1 mg−1 protein. Experiments were done in at least three biological replicates, each in three technical replicates.

### PAO Activity Measurement

Polyamine oxidase activity was measured as described by Luhová et al. (2003) with some modifications. Roots of tomato seedlings (60 mg) after washing in distilled water were homogenized by mortar and pestle in extraction mixture containing 0.05 M potassium-phosphate, pH 6.5, 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich), 10% (v/v) glycerol, 5 mM DTT, and 2% (w/v) PVPP in an ice bath. The homogenate was centrifuged at 12,000 g for 10 min at 4◦C. The supernatant was collected for further analyses. PAO activity was measured in 1 ml of reaction mixture containing 0.05 M potassium-phosphate, pH 6.5, 0.5 mM guaiacol, and 1 U of horseradish peroxidase (Sigma-Aldrich) in the presence of 10 mM spermine (Spm). Activity was detected as absorbance increase at 436 nm and calculated using an absorption coefficient ε = 25.5 mM−1 cm−1 . The results were expressed as nmol H2O2 min−1 mg−1 protein. Experiments were done in four biological replicates, each in three technical replicates.

### NADPH Oxidase Activity Measurement

Measurement of NADPH oxidase activity in extracts of tomato roots was done according to Paclet et al. (2007). Tomato roots were isolated, washed in distilled water and homogenized by mortar and pestle in 0.05 M Tris-HCl, pH 7.5 with 0.06 mM Tween-20, 10% (v/v) glycerol, 5 mM DTT, 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich), 2% (w/v) PVPP in an ice bath. Homogenates were centrifuged (12,000 g for 15 min at 4◦C), and supernatants were collected for further analyzes. NADPH oxidase activity was determined by reduction of cytochrome c. The reaction mixture contained: supernatant of 50 µg of soluble protein, 0.05 M Tris-HCl, pH 7.5, 60 mM cytochrome c, 1 mM CaCl2, 0.1 mM MgCl2, 0.1 mM NaCl, 0.06 mM Tween-20. In parallel, cytochrome c reduction was done in reaction mixture with addition of 50 U of superoxide dismutase (SOD; Sigma-Aldrich). After incubation at 30◦C for 5 min (when O2 r− production from endogenous reductants had ceased) the reaction was started by addition of 0.1 mM reduced β-nicotinamide adenine dinucleotide phosphate (β-NADPH) and cytochrome c reduction was measured at 550 nm (Sunrise, Tecan). The presented values were calculated from differences between cytochrome c reduction rates in the presence and absence of SOD (Sigma-Aldrich) using an absorption coefficient ε = 21.1 mM−1 cm−1 . A blank (reaction mixture without supernatant) was also done to every measurement.

The results were expressed as nmol min−1 mg−1 protein. Experiments were done in four biological replicates in three technical replicates.

### Measurement of Proteolytic Activity

Determination of proteolytic activity was done according to Pande et al. (2006) with some modifications, using azocasein as a unspecific substrate. After isolation, roots of tomato seedlings (60 mg) were washed in distilled water and homogenized by mortar and pestle in 0.1 M potassium-phosphate, pH 7.0, 0.1% (v/v) Triton X-100, 5 mM DTT, 2% (w/v) PVPP in an ice bath. After two times centrifugation (10,000 g, for 5 min, at 4◦C), the supernatant was collected, and the protein concentration was measured using Bradford (1976) reagent. Protease activity was measured at pH 5.4 and 8.8. Supernatant containing 20 µg of total soluble proteins was pre-incubated 5 min at 37◦C in 0.1 M Tris-HCl, pH 5.4 or 8.8. Subsequently 0.8% (w/v) azocasein solution was added and incubated for 30 min at 37◦C in darkness. The reaction was stopped by the addition of 10% (w/v) TCA. Samples were centrifuged (12,000 g, for 15 min), supernatant was collected and mixed with 1 M NaOH in 1:1 ratio. After 5 min incubation, the absorbance was measured at 440 nm (Sunrise, Tecan). The specific activity of proteases was expressed as enzyme units per mg protein. Units of enzyme activity were calculated from the absorbance value increase obtained after unspecific protease activity measured in buffers of different pH at 37◦C for 30 min as described above. Measurements of proteolytic activity were done in four independent experiments, each in three replicates.

### Statistics

Mean values were computed for each experiment and mean differences were calculated using analysis of variance (ANOVA) and Duncan's test. Standard deviation (SE) are also provided to indicate the variations associated with the particular mean values. Calculations were performed using the package agricolae (de Mendiburu, 2010) for the statistical freeware R version 2.14.2 (R Development Core Team, 2012). For growth parameters data were analyzed using the StatGraphics 5.1; Mean ± SE were computed for each experiment and significance of differences was assessed with Student's t-test. Differences are considered significant at p < 0.05.

### RESULTS

### CAN Inhibited Elongation Growth of Tomato Roots

Treatment of tomato seedlings with CAN resulted in drastic inhibition of root growth. Inhibition of root growth by 50% was observed in CAN at concentration 10 µM (**Figure 1**), while 50 µM CAN stopped tomato root elongation completely after 72 h (**Figure 1**). Thus, 50 µM CAN was selected as IC100 while 10 µM as IC 50 and used for further investigations.

## CAN Enhanced H2O2 Concentration and Superoxide Radicals Generation in Roots Extracts of Tomato Plants and Slightly Increased H2O2 Efflux from the Roots

Detection of superoxide radicals indicated its stable generation in roots extracts of control seedlings during the culture period (**Table 1**). In extracts of roots of control plants after 24 and 72 h of culture production of superoxide radicals was 1.125 and 1.110 µmol min−1 mg−1 FW, respectively. In roots extracts of plants treated for 24 h with 50 µM CAN generation of superoxide radicals was similar to the control, while in extracts of plants exposed to 10 µM CAN superoxide radicals generation was twice as high as in the control. Prolonged (for additional 48 h) exposure of seedlings to 10 µM CAN resulted in decline of superoxide radicals generation in roots extracts, but it was still higher than in extracts of seedlings growing in water. High generation of superoxide radicals was detected also in roots extracts of seedlings treated with 50 µM CAN for 72 h, it was twice as high as in the control (**Table 1**).

In roots extracts of control plants H2O2 concentration did not differ as culture period was prolonged, and was 75–80 pmol mg−1 FW (**Table 1**), equivalent to 78–83 µM. CAN treatment increased H2O2 level in roots extracts, leading to a doubling (about 145 µM) after application at higher concentration for 24 h. Longer (72 h) exposure of seedlings to 10 µM CAN enhanced H2O2 concentration in roots extracts to around 150% of the control (122 µM). No such effect after 72 h was observed in case of 50 µM CAN. In roots extracts of seedlings treated with 50 µM CAN, H2O2 concentration was about 116% of the control (**Table 1**), equivalent to 98 µM.

The concentration of H2O2 in the culture medium solution was very low and did not exceed 3.6 nM independently of the treatment. For control plants, during the whole experiment it was stable, and below 1.5 nM (**Table 1**). Expressed per root fresh weigh H2O2 content in the medium was significantly higher for plants treated with CAN, and after 24 h reached 461 or 547 pmol g−1 FW for 10 or 50 µM CAN respectively, in comparison to control (156 pmol g−1 FW). After 72 h culture it declined to 200 or 380 pmol g−1 FW for 10 or 50 µM CAN respectively, and 76 pmol g−1 FW in control (data not shown).

### CAN Inhibited Fluorescence of DAF-FM Derivatives Corresponding to NO Generation and Transiently Enhanced Fluorescence of APF Derivatives Corresponding to ONOO− Production in Tomato Roots

In roots of control plants growing in water fluorescence of DAF-FM declined slightly (about 25%) during prolonged culture. As expected, CAN inhibited fluorescence of DAF-FM derivatives independently of concentration (**Table 2**). After 24 h fluorescence of DAF-FM derivatives had decreased to 25%, and remained at that level after 72 h.

Fluorescence excitation of APF used for determination of ONOO− production from control roots did not differ as culture was prolonged. CAN treatment led to slight (30%) and transient enhancement in APF fluorescence after 24 h of exposure (**Table 2**). After 72 h of treatment no differences in fluorescence of APF derivatives for roots of CAN treated seedlings and control were detected.

### CAN Treatment Led to Alterations in Concentration of Protein Carbonyl Groups and the Pattern of Carbonylated Soluble Proteins

The level of protein carbonyl groups in homogenates of roots of control plants increased from 30 to 79 nmol mg−1 protein, as the culture period was extended for 3 days (**Table 3**). Short term (24 h) exposure to 10 or 50 µM CAN resulted in an increase of the level of carbonyl groups in root homogenates by 53 and 70%, respectively. As CAN treatment was prolonged, the concentration of stable DNP derivatives of the carbonyl groups in proteins isolated from roots increased to 104 nmol mg−1 protein in 10 µM CAN or 88 nmol mg−1 protein in 50 µM CAN (**Table 3**). In 50 µM CAN it was at the level of the control, while for 10 µM CAN it was 30% higher than in the control.

Detection of total soluble carbonylated proteins isolated from roots of control tomato seedlings separated by SDS-PAGE (**Figure 2A**) and transferred onto a nitrocellulose membrane stained with Ponceau S (**Figure 2B**) for protein transfer

TABLE 1 | Superoxide radicals generation, H2O2 concentration in root extracts and H2O2 concentration in culture medium of control tomato seedlings and seedlings treated with CAN (10 or 50 µM) for 24 or 72 h.

In determination of H2O2 concentration in culture medium water or CAN solution of appropriate concentration was used as a blank. One relative unit describing the rate of superoxide radicals generation corresponds to superoxide radicals generation in roots extracts of control plants after 24 h, equal to 1.125 µmol min−1 g −1 FW. Mean of values are statistically different at p < 0.05, when they share no common letter(s). The comparison were made using the Duncan test. Values are average ± SE of 3–4 independent experiments.

TABLE 2 | Fluorescence of DAF-FM derivatives (corresponding to NO) and fluorescence of APF derivatives (corresponding to ONOO−) for roots of tomato seedlings growing in water (control) or roots of seedlings treated with CAN solution at 10 and 50 µM concentration for 24 or 72 h.

Mean of values are statistically different at p < 0.05, when they share no common letter(s). The comparison were made using the Duncan test. Values are average ± SE of 3–4 independent experiments.

TABLE 3 | Content of protein carbonyl groups and 3-NT in roots of control tomato seedlings and seedlings treated with CAN (10 and 50 µM) for 24 or 72 h.

Mean of values are statistically different at p < 0.05, when they share no common letter(s). The comparison were made using the Duncan test. Values are average ± SE of at least three independent experiments.

examination showed significant modification in their pattern. In the protein extracts from roots treated with 10 µM CAN for 72 h many carbonylated proteins of molecular weight above 35 kDa were detected. After 72 h of culture thick bands of carbonylated proteins were detected at the top of the membrane corresponding to the top of the gel, both for the control and treated plants (**Figure 2A**). Bands of carbonylated proteins of high density were marked for the control and particularly CAN (50 µM) stressed roots after 72 h, and they were characteristic for proteins corresponding to the band of the molecular marker of 15 kDa. Ponceau S staining of the membrane after protein transfer is shown in **Figure 2B**. In roots of seedlings after 24 h treatment proteins of molecular weight of around 30 kDa were visible, but they were below detection range after 72 h. In contrast more proteins of molecular weight 35–50 kDa were detected in roots of seedlings cultured for 72 h (**Figure 2B**).

### CAN Treatment Led to a Slight Decline in Concentration of Total 3-NT and Modification in the Pattern of Nitrated Proteins

For control plants the level of nitrated proteins increased with root age (**Table 3**). It doubled from the value around 4– 9 nmol mg−1 protein, as the culture period was extended up to 72 h. Short term (24 h) CAN treatment resulted in slight (15–30%) increase in 3-NT concentration in root proteins, as compared to plants growing in water (**Table 3**). As CAN treatment was prolonged, 3-NT concentration increased to 7 nmol mg−1 protein, but was 25% lower than that detected in control roots (**Table 3**).

**Figures 3A,B** shows the protein patterns analyzed using Ponceau S staining and the corresponding tyrosine nitration protein patterns detected with an antibody against 3-NT in roots of control tomato plant or plants exposed to CAN for 24–72 h. In general, roots showed a similar protein tyrosine nitration pattern independently of the treatment, but it differed as experiment was prolonged. Intensity of staining of protein bands below 20 kDa differed in roots of CAN treated plants after 24 h in comparison to control (**Figure 3A**). In roots of seedlings exposed to CAN after 72 h a band of nitrated proteins of 20 kDa is not visible. The nitration profile of proteins isolated from roots after 72 h of the experiment was also different, with less intense band of around 90 kDa being detected. 3-NT-immunopositive proteins of molecular weight above 100 kDa were more abundant in roots after 72 h of the culture both in control and CAN stressed plants (**Figure 3A**).

### Activities of Enzymes Involved in Regulation of ROS Level Were Stimulated by CAN

Short-term exposure of plants to CAN did not influence POx activity in roots (**Figure 4A**). In roots of control plants and seedlings treated with CAN (10 and 50 µM) POx activity was of the same range (150–168 nmol min−1 mg−1 protein). Additional 48 h of culture resulted in enhancement of POx activity both in control and CAN stressed roots. The highest POx activity was observed in roots of seedlings treated with 50 µM CAN, and it was 160% of the control, while 10 µM CAN led to only 15% stimulation of enzyme activity (**Figure 4A**).

During the culture period PAO activity in roots of control seedlings declined from 23 to 16 nmol min−1 mg−1 protein (**Figure 4B**). CAN treatment resulted in stimulation of PAO activity, after 24 h NPAA influence was concentration independent. In roots growing in CAN for 24 h PAO activity was 60% higher than in control seedlings. Prolongation of CAN stress to 72 h led to further stimulation of enzyme activity. In roots exposed to 10 µM CAN for 72 h PAO activity was doubled as compared to control, while in roots growing in 50 µM CAN its activity was three fold higher than in non-stressed plants (**Figure 4B**).

Activity of NADPH oxidase in roots of plants growing in water was constant during the experiment (**Figure 4C**). CAN treatment stimulated activity of this enzyme. The highest activity of NADPH oxidase, twice as high as the control, was observed after short-term (24 h) application of 10 µM CAN. At 72 h its activity declined, but was still 40% higher than in the control. CAN at concentration of 50 µM led to only slight (15%) stimulation of activity of NADPH

oxidase at the beginning of the experiment, but after 72 h it was about 80% higher than in plants growing in water (**Figure 4C**).

### Proteolytic Activity in Roots of Tomato Seedlings Was Enhanced by CAN Treatment

Proteolytic activity in extract of tomato roots was determined at pH 5.4 and 8.8 (**Figures 5A,B**). In roots of control plants after 24 h of growth, proteolytic activity was around 0.3 U mg−1 protein at both pH. After 72 h it doubled at acidic pH, and was stable at basic pH. Treatment of seedlings with CAN led to stimulation of protolytic activity at both pH. The highest

proteolytic activity was observed at pH 5.4 after 72 h of culture (**Figure 5A**).

### DISCUSSION

Canavanine is a natural NPAA produced by some plants. As a compound present in seeds or leaves it could be consumed by animals or people. Due to antiviral, antifungal, antibacterial, and anticancer properties CAN is used in medicine (Ekanayake et al., 2007). Toxicity of CAN was shown for herbivores (Rosenthal, 2001; Jang et al., 2002), but also for plants, including apple (Malus domestica Borkh.) embryos (Krasuska et al., 2014a). Just recently, we demonstrated that the presence of CAN in the growing medium of young (3–4 days-old) tomato seedlings

led to an inhibition of root elongation growth. This effect was dose- and time-dependent (Krasuska et al., 2016). CAN is a substrate of arginyl-tRNA synthetases responsible for the incorporation of NPAA instead of Arg into a protein structure (Jang et al., 2002). Occurrence of dysfunctional proteins leads to a disruption of metabolism and finally stress induction. Common

physiological reaction to abiotic or biotic stressors is ROS/RNS burst (imbalance in ROS/RNS production and scavenging; Groß et al., 2013). It has also been noticed as secondary mode of action of some allelochemicals or phytotoxins (Gniazdowska et al., 2015).

We have shown that CAN, starting from 24 h of treatment, stimulated superoxide radicals generation (**Table 1**), mostly generation of superoxide anion (O2 r−), as we reported previously using root staining with NBT and correlated well to O2 consumption (Krasuska et al., 2016). CAN enhanced also H2O2 concentration in root extracts of tomato seedling (**Table 1**). It was accompanied by the exudation of H2O2, however, CAN did not disturb membrane permeability and roots viability (Krasuska et al., 2016). Thus, ROS exudation into the environment could be a root response to NPAA. The rise of ROS level and radicals production in root extracts of tomato seedlings exposed to CAN was comparable to those detected in Arabidopsis plants grown in the medium supplied with p-hydroxybenzoic acid (Guan et al., 2014) and to those in onion (Allium cepa L.) or maize (Zea mays L.) treated with cyanamide, an allelochemical of hairy vetch (Vicia villosa Roth.; Soltys et al., 2011, 2014). Enhanced O2 r− level was observed in cyanobacteria Microcystis aeruginosa (Kützing) after berberine application, and associated with growth inhibition (Zhang et al., 2011). Disruption of O2 r− and H2O2 ratio in root meristem resulted in restriction of root cells growth (Tsukagoshi et al., 2010). Therefore, inhibition of elongation of the tomato root by CAN could be explained by ROS-dependent disorder of cell differentiation or proliferation, which could be the secondary mode of action of this NPAA. The incubation of mouse glial cell lines with CAN resulted in an enhancement of ROS content, accompanied by a decline in the reduced form of glutathione (Riganti et al., 2003). CAN also stimulated the pentose phosphate pathway, a strong marker of oxidative stress. Moreover, in a cellfree system, CAN decreased DTT level (Riganti et al., 2003), indicating that this NPAA could take part in the regulation of redox state of small thiol containing compounds.

Canavanine lowered the fluorescence rate of DAF-FM, corresponding to NO formation in the tomato seedling roots (**Table 2**), similarly, as was detected previously for root tips (Krasuska et al., 2016). This effect seems to be uncommon, as stress factors usually induce secondary nitro-oxidative stress. On the other hand, it suggests that CAN could inhibit or disturb formation of NO via still unidentified Arg-dependent pathway, which seems to be important for the maintenance of regular root growth. In our experiment, a slight increase in fluorescence of APF, corresponding to ONOO− production in tomato roots was observed only after 24 h of the culture in CAN solution (**Table 2**) and matches well to microscopic localization of ONOO− in root tip cells (Krasuska et al., 2016). As compared to short-term CAN treatment, prolonged (72 h) exposure to CAN resulted in a decline in the ONOO− generation. The transient enhancement of ONOO−, measured using APF, can be linked to an elevated production of ROS (particularly OH r ).

The increase in the content of protein carbonyl groups is a commonly accepted marker for ROS imbalance. There is no information about the impact of allelopathic compounds on the level of protein carbonyl groups in tissues of acceptor plants. The treatment of tomato seedlings with CAN led to an increased concentration of carbonyl groups (**Table 3**). In CAN treated roots higher abundance of carbonylated proteins was especially observed after 72 h (**Figure 2**). Observing a band on the membrane corresponding to the front of the gel we can conclude that CAN led to increased abundance of carbonylated proteins of low molecular weight. According to the concept of Møller and Sweetlove (2010) some carbonylated peptides released from proteins, after their breakdown, can serve as signaling molecules of specific cellular compartment origin. Previously, it was demonstrated that several storage proteins, in seeds are carbonylated to accelerate their mobilization during the germination process (Job et al., 2005; Krasuska et al., 2014b). In high abundance, we have also detected carbonylated proteins that were not entering the gel. These were probably aggregates of various proteins, content of which increased after 72 h of the culture, both in CAN treated and control plants (**Figure 2**). Protein aggregates may diminish their ability for degradation (Jung et al., 2007). The physiological explanation for the accumulation of aggregates is hard to clarify, however, such phenomenon occurs also in regularly growing roots. Differences between quantitative detection of carbonylated proteins (**Table 3**) and results of Western blot assay (**Figure 2**) were due to absorption of proteins and/or fragments of proteins on Maxisorp plates. Maxisorp plates bind also small proteins, which can be lost in protein transfer during Western blot analysis. It could be also suspected that due to high proteolytic activity after treatment with CAN (**Figure 5**) fragments of small proteins, which were bonded to the surface of the plates were lost in Western blot.

During the progression of the leaves vegetative development in Arabidopsis cultured under optimal conditions, an increase of the level of carbonylated proteins was detected (Johansson et al., 2004). Similarly, we observed that the alterations in the level of protein DNP derivatives in roots of the control tomato seedling, during the progression of growth, were of the same pattern as described in the roots of apple seedlings (Krasuska et al., 2014b). The content of carbonyl groups in tomato roots at the beginning of the experiment (roots were of length of 5 mm) was higher (data not shown) than in the roots of plants growing in water for additional 24 h. During the extension of root elongation growth the amount of carbonyl groups in proteins doubled (**Table 3**). Alike, in roots of apple embryos at the termination of germination sensu stricto higher level of carbonylated peptides, than in the roots of 10-days-old seedlings cultured in water, was noticed (Krasuska et al., 2014b). Thus, it implies that accumulation of some carbonylated proteins could be due to physiological processes occurring during growth and development, not simply related to the risks of oxidative stress.

Bearing in mind that ROS act as key molecules in the regulation of root growth and development, specific carbonylated proteins may be involved is signal transduction. There is also a possibility, that carbonylation could serve as a protective mechanism, that addresses/directs putative aberrant proteins enriched in CAN to faster degradation. Isolation and identification of such proteins would be of great interest to explain CAN toxicity. The more, in HEp-2 cells it was shown that endogenous canavanyl proteins are preferentially ubiquitinated

and probably processed to degradation via proteasome (Akaogi et al., 2006).

The mode of action of RNS involves a modification of protein amino acid residues such as, e.g., Tyr (Astier and Lindermayr, 2012). Following disturbances in RNS level after CAN application we measured the 3-NT level in proteins isolated from roots of tomato seedlings (**Table 3**). Just like the content of protein carbonyl groups points at ROS metabolism, amplification of Tyr nitration can serve as a marker of nitrosative stress. CAN treatment led to a slight increase followed by a decline of 3-NT in proteins in roots of tomato seedlings (**Table 3**). The increase in the 3-NT level after short term CAN treatment is in agreement with the data obtained for ONOO− production, and can also be due to the higher abundance of small proteins. The lower (in comparison to the control) level of 3-NT groups after prolonged CAN supplementation indicated that NO cellular production was limited, or NO production was unaffected, but the reaction with O2 r− was accelerated. Increase of O2 r− (free radical burst) can be also a reason of lowering cellular NO concentration, as these compounds react rapidly. On the other hand, we have demonstrated equal concentration of NO2 − in CAN stressed and non-treated tomato seedling (Krasuska et al., 2016). ONOO− is not the only one nitration agent, as this process occurs also in the presence of NO2 − (Speckmann et al., 2016). Thus, we suspect that CAN influences other mechanisms of NO generation (probably enzymatic pathways), being beneficial during root growth and development.

Summing up these results with those of NO efflux we can conclude that CAN serves rather as inhibitor of RNS burst, and in consequence, may have a stressful effect. Therefore, the restriction of root growth in tomato seedlings by CAN could be linked to the disturbances in RNS tissue level. NO is required for an undisturbed root growth (Yu et al., 2014). Corpas et al. (2009) indicated that in roots of 71-days-old pea (Pisum sativum L.) plants growing under optimal conditions proteins with 3-NT were present at a level higher than in other organs, e.g., leaves or flowers. So, protein nitration is of physiological occurrence, similarly as we previously pointed to carbonylation. In 2-weeks-old seedlings of Arabidopsis more than 60% of the identified nitrated proteins were engaged in primary metabolism (Lozano-Juste et al., 2011), suggesting that inhibition of this PTM and/or deficiency of specific nitrated proteins could disrupt cellular homeostasis. We can also assume that peptides/proteins with 3-NT could serve as signaling molecules of specify nature and origin. Our data indicates that CAN treatment destabilizes this potent 3-NT-dependent signaling pathway. Tyr nitration increased during various stress conditions (Corpas et al., 2008). Stress factors led to a substantial strengthening of protein bands immunolabeled against 3-NT of molecular weight of 29–59 kDa isolated from pea plants. The treatment of tomato roots with CAN led to a transient (in first 24 h) increase of the intensity of 3-NT-labeling of 20 and 30 kDa protein band, while in the control samples these bands were almost at the same level up to 72 h (**Figure 3**). These proteins are probably associated with the phytotoxicity of CAN, and thus needs identification.

Canavanine treatment of tomato seedlings increased proteolytic activity (**Figure 5**). Moreover, the transient (observed after 24 h) increase in protein nitration and carbonylation (compared to the control) could favor their proteolysis. Protein nitration enhances the susceptibility to degradation by proteasome pathway, on the other hand oxidative/nitrosative stress conditions lead to an inactivation of proteasome (Souza et al., 2000; Kästle et al., 2012). Furthermore, it was shown that at high cellular ROS level oxidatively modified proteins were degraded by peroxisomal proteases (Romero-Puertas et al., 2002). In addition, proteases participate in the cellular defense system (Fowler et al., 2009), so we can conclude that a higher proteolytic activity after plant treatment with CAN is part of the detoxification machinery. Aberrant products of CAN incorporation into mitochondrial proteins of human cells lines were detected only for a short time, suggesting their enhanced degradation (Konovalova et al., 2015). Similarly, the decrease in the content of carbonylated proteins during apple embryo germination, the process stimulated by NO or hydrogen cyanide (HCN), was linked to a stimulation of proteolytic activity (D˛ebska et al., 2013; Krasuska et al., 2014b).