IntroductionTop

The plant cell is enclosed by the apoplast that consists of the cell wall fibrillar structure and the intercellular liquid and gas spaces. Generally, apoplastic enzymes and reactive oxygen species (ROS) play a role in cell wall synthesis and loosening, processes involved in plant growth regulation (Passardi et al., 2004). Also, apoplast, especially the root apoplast, as the exposed compartment of the plant cell, is important for all of the plant’s interactions with its environment (Zhou et al., 2011). It can detect environmental changes and stress signals and transfer them into the cell interior to trigger a whole cell response (Zhou et al., 2011). Cell-wall-located enzymes are considered as active sites for apoplastic ROS production. Depending on the concentration, ROS (superoxide anion radical, hydrogen peroxide, hydroxyl radical and singlet oxygen), have a dual role. At high concentration ROS cause damage to biomolecules, whereas at low/moderate concentration they act as secondary messengers in intracellular signaling cascades that elicit various cellular responses. Cell wall enzymes, such as class III peroxidases (PODs) in their oxidative cycle, malate dehydrogenase (MDH), oxalate- and amine oxidases are related to H₂O₂-production necessary for polymerization processes such as lignification and oxidative coupling of cell wall proteins or for direct defense against abiotic and biotic stresses (Gross et al., 1977; Vuletić & Hadži-Tašković Šukalović, 2000; Laurenzi et al., 2001; Mika et al., 2004; Ros-Barcelo et al., 2004). By enabling rapid induction of chemical defenses against stress, such enzymes represent a part of induced plant defense. Being exposed to various abiotic and biotic stresses, apoplastic production of ROS in many adverse environmental conditions has to be tightly controlled in order to avoid oxidative damage of plant cell. Therefore, root cell walls have evolved protective systems to eliminate or reduce the excess of ROS, including enzymes such as POD, superoxide dismutase, ascorbate oxidase (AO) and non-enzymatic low molecular weight antioxidants ascorbate and phenolics (Takahama & Oniki, 1992; Mika et al., 2004; Pignocchi et al., 2006; Kukavica et al., 2009). PODs and superoxide dismutase are directly involved in ROS scavenging, removing hydrogen peroxide, and dismutating superoxide anion radical to hydrogen peroxide, respectively. AO oxidizes ascorbate to water and ascorbate-free radicals, contributing to the regulation of the ascorbate redox state and coupled redox pairs and radical species. Apoplastic ascorbate is involved in the regulation of cell expansion and elongation due to its ability to impair the catalytic reaction of POD by re-reducing the intermediate phenoxyl radical produced during the peroxidative reaction (Takahama & Oniki, 1992).

Plant phenolics are secondary metabolites involved in the defense mechanism of plants and growth regulation. Their accumulation is a striking example of metabolic plasticity against abiotic and biotic stress factors, enabling plants to adapt to changing environments (Boudet, 2007). Phenolics esterified with cell wall polysaccharides, lignin and wall proteins, constitute cell walls, and in such a way are involved in mechanical system of defense, efficient in response to stress conditions (Santiago et al., 2013). As cell wall constituents, phenolics contribute to overall antioxidant capacity in many ways: as electron donors to H₂O₂ in reactions catalysed by class III POD, free radical scavengers (Rice-Evans et al., 1997) and regulators of H₂O₂ and ascorbate content through the ASC/phenolic/H₂O₂ system catalysed by POD (Takahama & Oniki, 1997; Hadži-Tašković Šukalović et al., 2008).

Nitrogen availability and the use of NO₃^- or NH₄⁺ affects plant growth (Marschner, 1995; Stitt, 1999), intracellular pH (Raven & Smith, 1976), cation and anion content of plant tissues (Kirkby & Mengel, 1967), protein, organic acids, and carbohydrates content (Marschner, 1995; Raab & Terry, 1995; Stitt, 1999), as well as the activity of enzymes involved in the N assimilation and organic acid synthesis (Hadži-Tašković Šukalović & Vuletić, 1998). Increased N supply, favoring plant growth, reduces accumulation of total phenolic compounds (Leser & Treutter, 2005) while N deficiency increases the content of phenolic compounds (Kováčik & Bačkor, 2007). However, there are only a few reports, in which the state of the antioxidant enzymatic systems was studied as the function of N nutrition. Polesskaya et al. (2004) demonstrated that NH₄⁺ induced increased antioxidant enzyme activities. Wang et al. (2008) in a study with Vallisneria natans leaves have shown that NH₄⁺ induce generation of ROS and up-regulate antioxidant enzymes (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase and glutathione reductase). Podgórska et al. (2013) showed that ammonium nutrition changes mitochondrial electron transport chain activity, increasing mitochondrial reactive oxygen species production. Fernández-Crespo et al. (2012) also reported that the ammonium-fed citrus plants showed increased levels of H₂O₂ accumulation.

Previously, we demonstrated a variable response of maize root soluble antioxidant enzymes and phenolic compounds to the presence of increased nitrate or nitrate plus ammonium content in the growth medium (Vuletić et al., 2010). The goal of this work was to study whether N nutrition could modify antioxidant systems of cell walls. The activity of enzymes involved in regulation of ROS level and cross linking of cell walls, protein content, total phenolic content, reducing capacity and free radical quenching activity of isolated walls were analysed. Since N metabolism is tightly coupled to root growth, we performed the experiments on basal non-growing root regions and intensely growing lateral roots, the aim being the determination of an eventual metabolic shift in the above mentioned enzyme and antioxidant systems in the early stages of response before any visible morphological changes in the appearance of the plant occur.

Material and methodsTop

ResultsTop

The effect of nitrogen sources on plant growth

To examine the possible modification of antioxidant systems by different N sources, we used nutrient solutions containing only NO₃^- or the mixture of NO₃^-+NH₄⁺ in a 2:1 ratio, in order to alleviate the effect of ammonia toxicity as a sole N source (Hadži-Tašković Šukalović & Vuletić, 1998). When NO₃^- was the only N source, maize plants produced longer roots and shoots, increased shoot FW and slightly decreased root FW (Table 1). However, the differences noted were not statistically significant.

Table 1. Root length (RL), shoot length (SL) and fresh weight (FW) of maize plants grown on nutrient solution containing a NO₃^-+NH₄⁺ or just NO₃^-.

Protein content of fraction ionically and covalently bound to the cell walls

The content of proteins ionically and covalently bound to the cell walls is shown in Table 2. Covalent fraction contained 4-5 fold more proteins than the corresponding ionic fraction. Plants grown on mixed N had higher protein content (up to 25%) in comparison with nitrate grown plants in all fractions, except the ionic fraction isolated from primary roots.

Table 2. Protein content (mg/g FW) in cell walls isolated from maize roots grown on different nitrogen containing solutions.

Activity of enzymes ionically and covalently bound to the cell walls

Since POD enzymes are closely coupled to the oxidative metabolism by utilizing and/or producing reactive oxygen species during their peroxidative and oxidative cycles, respectively, both activities were evaluated. Ferulic acid, as natural substrate for cell wall PODs, was used to measure the peroxidative activity. Oxidative activity was assayed with NADH (widely used substrate for the in vitro experiments) or IAA (generally accepted as a naturally occurring reductant in the apoplastic place). Activity measurements show that POD enzymes are predominately associated with cell walls by ionic interaction. The ratio between ionically and covalently bound peroxidative activities (calculated on the root FW basis) was almost constant (≈ 1.35), except in lateral roots of nitrate-grown plants (≈ 2.1). Both activities, peroxidative (Table 3) and oxidative (Table 4), were generally higher in plants grown in the presence of mixed N in comparison with nitrate-grown plants. Thus, in most isolates, the peroxidative activity was 30% greater, with exception of covalent fraction isolated from lateral roots where it was doubled. Oxidative activity increased 7-22%. Activity of MDH was predominately detected in the covalent fraction. In lateral roots isolated from covalent fraction, the activity was about 2.8 times higher than in ionic fraction, irrespective of the N treatment. Also, both cell walls fractions isolated from lateral roots of plants grown on mixed N had about 1.4 times higher MDH activity than the corresponding isolated from plants grown on nitrate only (Table 5). In both cell wall fractions, mixed N induced the increase of MDH activities (>40%) in lateral roots only, while in primary roots the activity was decreased (Table 5).

Table 3. Peroxidative activity of peroxidase (µmol ferulic acid oxidized/(g FW·min)) ionically and covalently bound to the cell walls of maize roots grown on different nitrogen forms.

Table 4. Oxidative activity of peroxidase (µmol NADH oxidized/(g FW·min)) ionically and covalently bound to the cell walls of maize roots grown on different nitrogen sources.

Table 5. Activity of cell wall ionically and covalently bound malate dehydrogenase (µmol NADH oxidized/(g FW·min)) in maize roots grown on different nitrogen forms.

Ascorbate oxidase activity was found to be predominantly covalently bound (~ 95% of total activity) to the cell walls (Vuletić et al., 2014), and thus the effects of N forms were analyzed in NaCl-washed cell wall suspension. The activity was higher in nitrate-grown plants (Table 6), in both lateral (27% increase) and primary roots (24% increase).

Table 6. Activity of root cell wall covalently bound ascorbate oxidase (nmol O₂/(g FW·min)) in maize plants grown on different nitrogen forms.

Since one could assume that cell wall enzyme activities are regulated in situ by variation in the concentration of apoplastic metabolite, we analysed the effect of malate and oxaloacetate on POD activity, and phenolic POD substrates on MDH activity, as well. Peroxidative reaction was inhibited with oxaloacetate in a concentration-dependent manner (Fig. 1). Much higher inhibition was detected in ionic fraction than in the covalently bound fraction. Such a strong inhibitory effect of oxaloacetate on oxidative POD activity was not detected (the activity was measured as IAA oxidation). Also, 10 mM malate did not change POD activities. Activity of MDH was not changed in the presence of 0.1 mM ferulic or p-coumaric acid. Effects of oxaloacetate and malate on POD activities as well as the effect of phenolics on MDH activity are presented in Fig. 2.

Figure 1. Inhibitory effect of oxaloacetate on peroxidative peroxidase activity measured with ferulic acid in both cell wall fractions (PODi-ionic and PODc-covalent). Residual POD activities are expressed as percent of maximal POD activity, obtained in the absence of oxaloacetate.

Figure 2. The effect of 10 mM malate on peroxidative peroxidase activity (PODper), 10 mM malate and 1 mM oxaloacetate on oxidative peroxidase activity (PODox) and the effect of 0.1 mM ferulic and 0.1 mM p-coumaric acid on MDH activity. Ionic fraction of cell walls isolated from lateral roots grown on NO3-+NH4+ nutrient solution was used for experiments. Residual enzyme activities are expressed as percent of maximal activities, measured without effectors added to the assay mixture. Data are means ± SE of three independent experiments.

Generally, the activities of all studied enzymes and protein content in both N treatments were higher in cell walls of lateral than in the investigated segment of primary roots, as presented in Fig. 3. The ratio of POD activities ionically bound to lateral and primary roots was similar irrespective of N forms. Slightly higher POD activity ratio was detected in the covalent fraction isolated from mixed N-grown plants. The difference between lateral and primary roots was most pronounced for MDH in both N treatments, especially in plants grown on mixed N.

Figure 3. The ratio of enzyme activities and protein content between lateral and primary root cell walls of maize plants grown in the presence of NO3- or mixture NO3-+NH4+. Experiments were performed in ionic (a) and covalent fractions (b) of root cell walls. PODper, peroxidative peroxidase activity; PODox, oxidative peroxidase activity; MDH, malate dehydrogenase activity; AO, ascorbate oxidase activity. Data are means ± SE of three independent experiments. Different letters above the histogram bars indicate significant differences between groups (p≤0.05).

Phenolic content, DPPH radical scavenging activity and reducing power of cell walls

The phenolic content, DPPH radical scavenging activity and reducing power of salt-washed cell walls isolated from lateral roots of plants grown solely on nitrate were higher than of roots grown on mixed N (42, 97 and 22%, respectively), as opposed to enzyme activities and protein content. Phenolic content and radical scavenging activity were slightly higher (≈10%) in primary roots grown on mixed N or were unaffected in the case of reducing power, compared to nitrate-grown plants (Table 7). Cell walls of lateral roots grown on nitrate had much higher phenolic content and exhibited stronger free radical scavenging properties against DPPH radical than walls of primary roots (≈70%). Such a difference was not observed in mixed N-grown plants (Fig. 4).

Table 7. Total phenolic content, DPPH radical scavenging activity and reducing power of NaCl-washed cell wall suspension. Cell walls were isolated from maize roots grown on different nitrogen forms.

Figure 4. The ratio of DPPH radical scavenging activity, reducing power (Red. Power) and total phenolic content (Phenol) between lateral and primary root cell walls of maize plants grown in the presence of NO3- or NO3-+NH4+. Data are means ± SE of three independent experiments. Different letters above the histogram bars indicate significant differences between groups (p≤0.05).

DiscussionTop

Nitrogen, a major constituent of plant macromolecules, is available in the soil as nitrate (NO₃^-) or ammonium (NH₄⁺) ions or as organic N derived from the degradation of plant and animal residuals. The degree of efficiency for two N forms on plant growth and nutrient uptake, when both sources of N are present in the solution, is dependent on plant species and NO₃^-/ NH₄⁺ ratio (Errebhi & Wilcox, 1990). Although ammonium was considered to be toxic when applied as the sole N source (Britto & Kronzucker, 2002), it has been shown that in combination with nitrate it provides maximal growth rates of maize plants (Schrader et al., 1972). Previous results of our laboratory indicated that the mixture of NO₃^-+NH₄⁺ in a 2:1 ratio positively regulates root mitochondrial respiration and tricarboxylic acid metabolism (Hadži-Tašković Šukalović & Vuletić, 1998), root soluble antioxidant enzyme activities and proline content, but negatively total phenolics (Vuletić et al., 2010), when compared with plants grown on NO₃^-as the sole N source. Positive effect of mixed N on plant growth (longer roots with more developed lateral roots), manifested during prolonged cultivation was reported previously (Vuletić et al., 2010). However, in this work, in order to analyse the zone of intense growth rate, eleven-day old plants, 2 days after the initiation of lateral roots, were used for experiments. Since the experiments were performed in an early developmental stage of maize plants, during the shift from seedling heterotrophy to autotrophy (Enns et al., 2006), different N treatments exhibited no significant effect on growth parameters. Nevertheless, the effect of applied treatments on the enzymatic and non-enzymatic antioxidant systems of cell walls was evident, and point that at this stage of growth, the chemical analysis is a more reliable indicator of initial metabolic changes prior to first visible morphological parameters. An enhancement of protein content, POD and MDH activities, accompanied by the AO activity decrease, was observed in the cell walls isolated from mixed N-grown plants, as compared to that from nitrate-grown plants. On the other hand, cell wall fragments isolated from lateral roots grown on nitrate exhibited increased antioxidant capacity, detected as radical scavenging activity and reducing power.

Generally, higher POD and MDH activities and lower AO activity in plants grown on mixed N could point to the response against more intense ROS production. Although Wang et al. (2008) and Podgórska et al. (2013) indicated that ammonium ions induce an increase in ROS production, Domínguez-Valdivia et al. (2008) demonstrated that ammonium stress is not an oxidative stress, moreover, it diminishes oxidative damage to proteins. The increase in amino acid synthesis and protein turnover (resulting in C deficiency) in plants fed with ammonium (Domínguez-Valdivia et al., 2008), could be the explanation of increased cell wall protein content and consequently enzyme activities in our experimental system. The increase of cell wall enzyme activities, as well as root soluble antioxidant enzyme activities (Vuletić et al., 2010) under similar growing conditions, point to the increased levels of preformed cell wall enzymes in symplast and their possible export to the apoplastic compartment.

Higher activity of POD in cell walls of plants grown on mixed N, suggests that this enzyme is not a limiting factor for phenolics cross-linking of cell wall polymers under mixed N conditions. It could be supposed that phenolic synthesis and/or delivering to the cell wall compartment could be one of the reasons for its lower content detected in the cell wall structure. Reduced accumulation of total phenolic compounds and higher protein content in cell walls of plant grown on mixed N could be explained by more intensive amino acid synthesis (Schrader et al., 1972), leading to the decrease of the C/N ratio and limited production of secondary metabolites, according to carbon/nutrient balance hypothesis (Bryant et al., 1983; Karowe & Grubb, 2011).

An additional reason for lower phenolics content in cell walls of mixed N-grown roots could be the in situ regulation of enzyme activities, implicated directly (POD) or indirectly (MDH and AO) in formation of phenolic cross-links between cell wall polymers. Although the regulation of POD activities in phenolic cross-linking to the cell wall structures could be closely and positively regulated with MDH activity through the providing NADH for H₂O₂ generation in oxidative POD reaction for lignification process (Gross, 1977; Gross & Janse, 1977), our results point to the possibility of inhibition of ferulic and p-coumaric acid oxidation with H₂O₂ in peroxidase reactions by oxaloacetate produced in MDH reaction. On the other hand, reductants such as ascorbate (regulated by the AO in the apoplast) or NADH (as a product of malate oxidation), as secondary electron donors, could generate ascorbate/phenolic/H₂O₂ or NADH/phenolic/H₂O₂ peroxidase system functioning solely as H₂O₂ scavengers (Hadži-Tašković Šukalović et al., 2008). In either case, the result would be the prevention of phenolic binding to cell wall structures. In the light of such considerations, lower phenolic content in lateral roots grown on mixed N compared to nitrate grown plants could be explained by suppression of POD activity by oxaloacetate, produced in MDH reaction, highly stimulated by mixed N, as well as higher ascorbate content in apoplastic compartment as a consequence of lower AO activity. As opposed to that, higher phenolic content in lateral roots of nitrate grown plants could be due to the increased AO activity in combination with decreased MDH activity that allows POD enzyme to function efficiently in the cross-linking of phenolics. Similar IEF patterns in both N treatments and almost constant ratio of ionically to covalently bound POD activities, indicating that POD enzyme binding to the cell wall was unaffected by N forms, argues in favor of POD regulation with metabolites.

The increased content of total phenolics together with increased radical scavenging activity and reducing power in nitrate-grown roots argue that phenolic esters bound to the wall polymers contribute significantly to the antioxidant capacity (Ohta et al., 1994). The most abundant phenolic constituents of maize root cell walls are ferulic and p-coumaric acids (Vuletić et al., 2014) esterified with arabinoxylan (Hatfield et al., 1999) and lignin (Hatfield & Chaptman, 2009), respectively. Much greater increase in DPPH scavenging activity than in reducing power in the same cell wall isolates could be ascribed to the presence of ferulic acid esters, being extremely potent as DPPH radical scavengers and possessing weaker reducing power than free ferulic acid (Szwajgier et al., 2005). The effect of p-coumaric acid could be minimal, due to its lower antioxidant activity in any form (Szwajgier et al., 2005). The difference in antioxidant capacity observed in roots grown on different N media was evident only in lateral roots, clearly demonstrating the differences in the structure of cell walls.

Nitrogen form slightly influenced the enzyme activity ratio between lateral and primary roots, with the exception of MDH, which increased in roots grown on mixed N. Increased MDH activity in intensively growing lateral roots, as demonstrated previously (Hadži-Tašković Šukalović et al., 2011), supports the proposal of Gross (1977) that MDH is implicated in growth regulation.

Our results confirm that in growing tissue mixed N stimulates metabolic processes leading to increased protein (and enzymes) availability, while nitrate highly improves antioxidative capacity by increasing constitutive phenolics content. It could be argued that plants with increased level of cell wall bound phenolics possess better mechanical defense, therefore exhibiting greater tolerance to biotic stress. On the other hand, increase in redox enzyme activities allows rapid induction of chemical defenses against biotic and abiotic stress.

The lack of such a difference within segments of primary roots may be explained by their formation during seedling heterotrophy.

It could be concluded that the use of nutrients, such as NO₃^- or NO₃^-+NH₄⁺ enables an increase of constitutive phenolic content or redox enzyme activities, respectively, in root cell walls. Thus, different N treatments could be applied as a tool towards improved constitutive resistance or induced chemical defenses of plants.