Prolonged root hypoxia effects on enzymes involved in nitrogen assimilation pathway in tomato plants

Abstract

In order to investigate the effects of root hypoxia (1-2 % oxygen) on the nitrogen (N) metabolism of tomato plants (Solanum lycopersicum L. cv. Micro-Tom), a range of N compounds and N-assimilating enzymes were performed on roots and leaves of plants submitted to root hypoxia at the second leaf stage for three weeks. Obtained results showed that root hypoxia led to a significant decrease in dry weight (DW) production and nitrate content in roots and leaves. Conversely, shoot to root DW ratio and nitrite content were significantly increased. Contrary to that in leaves, glutamine synthetase activity was significantly enhanced in roots. The activities of nitrate and nitrite reductase were enhanced in roots as well as leaves. The higher increase in the NH4+ content and in the protease activities in roots and leaves of hypoxically treated plants coincide with a greater decrease in soluble protein contents. Taken together, these results suggest that root hypoxia leaded to higher protein degradation. The hypoxia-induced increase in the aminating glutamate dehydrogenase activity may be considered as an alternative N assimilation pathway involved in detoxifying the NH4+, accumulated under hypoxic conditions. With respect to hypoxic stress, the distinct sensitivity of the enzymes involved in N assimilation is discussed.

Introduction

Plants grow in a dynamic environment, which frequently imposes constraints on growth and development. Among the adverse environmental factors commonly encountered by land plants, flooding is one of the most significant abiotic stresses.1 Flooding of the soil can have a tremendous impact on the growth and survival of plants, and thereby on agricultural as well as natural ecosystems. In the last decades considerable progress has been made in our understanding of the mechanisms that enable certain plant species and cultivars to withstand periods with excess soil water or even complete submergence. Much of the research has been carried out with crop plant species, such as Oryza sativa, Zea mays, Trifolium subterraneum and Medicago sativa,2–5 but wild species originating from wetland habitats have been also used, mostly for comparative studies.6,7

With transient flooding or irrigation followed by slow drainage, or in natural wetlands, plant roots can become oxygen deficient because of slow transfer of dissolved oxygen in the water-filled pore space of the soil. The decrease in ambient oxygen may not be, itself or alone, the perceived signal but could trigger metabolic responses, which then initiate the signalling cascade.8 The immediate consequence of oxygen depletion is the reduction in respiration, thus diminishing ATP generation, and leading to a decrease in the ATP/ADP ratio and the adenylate energy charge.9 Changes in ATP level itself could conceivably serve as signal for subsequent adaptive responses. Low ATP levels lead to an energy crisis to which plants can respond by increasing glycolytic flux (Pasteur Effect) and switching to alcoholic fermentation,3,10 with a variety of electron acceptors such as pyruvate and acetaldehyde replacing oxygen. This metabolic adaptation helps maintain ATP production and NAD⁺ regeneration.11 In addition, to cope with the reduction in oxygen supply, plants have developed a number of morphological adaptations that enable them to survive transient periods of complete or partial subme.^12,13 Escape from hypoxia involves shoot elongation, development of aerenchyma and adventitious root formation.14,15

Most studies on biochemical alterations in plants due to hypoxic stress have been focused on sugar metabolism.16,17 However, data regarding hypoxia effects on N metabolism steps have recently become available.18,19 The physiological roles of exogenous nitrate (NO₃⁻) and nitrate reductase (NR) under the conditions of plant hypoxia and anoxia attracted the attention of researchers since NO₃⁻ is widely applied as a N fertilizer, in particular on wetland soils abundant in many countries.20,21 The role of NO₃⁻ reduction in oxygen deficiency tolerance is the subject of many controverses. Some investigations could not verify any effect of NO₃⁻ and NR on the energy metabolism in oxygen-deficient roots.22,23 However, other investigations have shown that NO₃⁻ supply to anaerobic root tissues led to an improved redox state (NADH/NAD⁺ ratio) and a higher adenylate energy charge,24,25 through the consumption of the reducing power (NADH) generated by the glycolysis and by the NO₃⁻ reduction to nitrite (NO₂⁻). In these tissues, NR activity often increases, either through increased gene expression,26 or post-translational activation.27 These observations raised the hypothesis that NR could be a key enzyme to understand the beneficial impact of NO₃⁻ on plant survival under anoxia.

Ammonium (NH₄⁺) originated from direct absorption, NR activity, photorespiration, dinitrogen fixation or deamination of nitrogenous compounds, is assimilated by the glutamine synthetase (GS) and the glutamate synthase (GOGAT). Under special conditions, glutamate dehydrogenase (GDH) is able to generate glutamate.28 Despite its abundance in plant tissues, the precise function of GDH remains obscure. In physiological conditions, the essential function of this enzyme is the deamination of glutamate to provide carbon skeletons and reducing power for plant cells.29 The aminating activity of GDH is specially stimulated in certain stress conditions: with senescence,30 NH₄⁺ excess,31,32 dark and atmospheric CO₂ excess.33 Generally, these conditions lead to a perturbation of the activities of enzymes involved in NH₄⁺ assimilation, such as inhibition of GS,34 and GOGAT.35 However, the effects of root hypoxia on the activities of these enzymes are scarce in the literature.

In a previous study, we observed that root hypoxia affected not only the transport of nitrate from roots to shoots, but also its reduction to NO₂⁻ in clover plants.32 The effects of root hypoxia on N metabolism are highly complicated, and vary depending on plant species and experimental conditions.25 The aim of this study was to explore the effects of root hypoxia on N metabolism steps in tomato. We studied the behavior of the enzymes of nitrate and NH₄⁺ assimilation in tomato leaves and roots under prolonged root hypoxia.

Results

Plant development and biomass allocation.

Root hypoxia was applied, in the present study, at the second leaf stage. At this moment, the initial root and shoot dry weights were 0.03 ± 0.01 g and 0.13 ± 0.03 g, respectively. The changes in the root and shoot dry weights (DW) and in shoot/root ratio were investigated after a three week period of hypoxic treatment. Root hypoxia decreased root and leaf DW production by 34 and 18%, respectively. Shoot to root DW ratio increased by 26% in hypoxically treated (HT) relative to control aerated (NT) plants (Table 1). This indicates that, under these growth conditions, the dry matter was more allocated to the aerial part. Root and leaf hydrations were decreased by 23 and 18%, respectively, in HT compared to NT plants. Concomitantly, leaf chlorophyll content was decreased by about 30%, whereas no obvious difference was observed in total leaf area (Table 1).

Nitrate, nitrite, ammonium and soluble protein contents in roots and leaves.

Roots and leaves were analyzed for their NO₃⁻, NO₂⁻, NH₄⁺ and soluble protein contents (Fig. 1). Our results showed that, as for Rondello19 and Rio Grande32 varieties, root hypoxia decreased root and leaf NO₃⁻ contents. These decreases were about 70 and 50%, respectively (Fig. 1A). NO₂⁻ and NH₄⁺ contents were eight times higher in roots of HT compared to those of NT plants (Fig. 1B). Concomitantly, leaf NO₂⁻ and NH₄⁺ contents were at least 4 fold higher (Fig. 1C). Soluble protein was significantly decreased by root hypoxia both in roots and in leaves (Fig. 1D).

Effects of root hypoxia on the N-metabolizing enzymes.

In the control tomato plants, we found that leaf NR activity was equal to that measured in roots, whereas under hypoxic conditions NR activity was 5 times higher in roots than in leaves. Root and leaf NR activities were 7- and 2-fold higher in HT compared to NT plants, respectively (Table 2).

In the aerated plants, NiR activity was two times higher in roots than in leaves. Under hypoxic treatment, root and leaf NiR activities were 2- and 3- fold higher in HT compared to NT plants, respectively. In the both conditions, root and leaf NiR activities were much higher than NR activities (Table 2). This ensures a rapid nitrite metabolism, protecting cells from NO₂⁻ toxicity. The NiR to NR ratio was highly decreased in the roots after a 3 week period of hypoxic treatment, whereas in the leaves it was significantly increased (Table 2).

GS activity was significantly inhibited in the leaves and slightly enhanced in the roots of HT compared to NT plants (Fig. 2A). GDH activity was significantly enhanced by root hypoxia in roots as well as in leaves (Fig. 2B).

Proteolytic activitiy was significantly enhanced by root hypoxia treatment in the leaves and roots (Fig. 2C). This is in accordance with the decrease in soluble protein content (Fig. 1D), and the increase in NH₄⁺ accumulation (Fig. 1C).

Discussion

The primary effect of soil flooding is to slow down the oxygen transfer to the roots. This limits their aerobic respiration and dramatically depresses their metabolism.49 In the present study, the N metabolism was investigated in tomato plants after a three week period of hypoxic treatment.

Our results showed that root hypoxia led to some typical effects commonly observed in tomato36,41 and in other plant species.3,4,50,51 These included a decrease in root hydration and DW production (Table 1). The decrease in DW production was suggested to be directly related to changes in net carbon assimilation that are attributed to stomatal and non-stomatal limitations of photosynthesis.50

The consequences of oxygen deficiency were not restricted to roots. The effects of hypoxia also extended to aerial organs. Although all seedlings survived three weeks of flooding, some symptoms of hypoxic stress were observed. These included a slight chlorosis of the older leaves, a slowing of leaf initiation (data not shown) and decreases in leaf hydration and chlorophyll content (Table 1).

As NO₃⁻ is a key molecule to N metabolism, supporting the synthesis of plant N compounds, we investigated the effect of root hypoxia on NO₃⁻ reduction in tomato leaves and roots. It has been suggested that in higher plants, nitrate assimilation can take place in roots as well as in leaves, with the contribution of each organ being dependent on the species, on the external NO₃⁻ concentration and on the rate of NO₃⁻ assimilation in the aerial part of the plant.52,53 Our results showed that, under hypoxic conditions, more than 80% of NO₃⁻ reduction occurred in roots (Table 2). This result is in disagreement with the higher biomass production obtained in the leaves relative to the roots (Table 1) and may be related to a sufficient availability of reducing power in this organ.54

It is now well established that NO₃⁻ is the primary signal for the induction of its own transport system, as well as for the enzymes implicated in NO₃⁻ assimilation.55 Previous works demonstrated that exogenous NO₃⁻ supplied to the medium of anaerobic excised rice roots increased the adenylic energy charge and lowered the catabolic reducing charge.24 Both factors could contribute to root survival and suggest a possible role of NO₃⁻ reduction in oxygen deficiency tolerance. Moreover, in most plant tissues other than the rice coleoptile, NO₃⁻ reduction under flooded conditions is restricted to the conversion of NO₃⁻ to NO₂⁻⁵⁶ Our results showed that root hypoxia led to an activation or induction, of NiR in addition to the activation of NR (Table 2), which is observed in hypoxic,57 as well as anoxic roots.25,58 Such an increase in NiR activity can be considered as a direct consequence of the increase in NR activity (Table 2). In fact, we can attribute the NiR activity enhancement to a higher availability of NO₂⁻ (Fig. 1B). In addition, our results showed that under normoxic as well as hypoxic conditions, NiR activity was always much higher than NR activity (Table 2). This imbalance assures that toxic levels of NO₂⁻ are not reached in the cytosol. Ogawa et al.59 confirmed the hypothesis of a co-regulation between NR and NiR, which is indispensable to prevent nitrite toxicity and to avoid the waste of electrons by NR and NiR activities.

In a previous study,32 we showed that the increase in NiR activity under root hypoxia was restricted to roots and that no effects were observed on leaf NH₄⁺ content and NiR activity in the Rio Grande variety. By contrast, leaf NH₄⁺ content (Fig. 1C) and NiR activity (Table 2) were significantly increased by root hypoxia, in the present study. The differences between results obtained for Micro-Tom and Rio Grande varieties may be related only to the plant material used (growth type: determinate versus indeterminate variety), since the experimental conditions were almost the same in the two studies.

Our results on NH₄⁺-assimilating enzymes showed that, under hypoxic conditions, GS activity was inhibited in the leaves but enhanced in the roots (Fig. 2A). The decrease in the leaf GS activity could derive from a low accessibility of glutamate for the GS enzyme. This is consistent with the fact that, in tomato, abiotic stresses lead to a high deviation of glutamate to the organic osmoticum synthesis.60 Proline, putrescine and betaine derive from glutamate and they could restrict glutamate availability for GS activity. In the roots, GS activity increased as an attempt to overcome the GS activity decrease in the leaves. Similar results were found in tomato under cadmium stress. The GS activity decrease in leaves was due to a lower content of the chloroplastic GS isoform (GS2) protein and transcripts, whereas the increase in GS activity in roots was related to the induction of the cytosolic GS isoform (GS1) protein and transcripts.61

The significant increase in NH₄⁺ content in roots and leaves of HT relative to NT plants (Fig. 1C) was concomitant to a significant increase in the GDH activities (Fig. 2B). Excessive NH₄⁺ was shown to induce the aminating GDH pathway.31,62 In our case, the induction by root hypoxia of the aminating GDH pathway suggests that GDH may be involved in vivo in the NH₄⁺ detoxification and in the replenishment of glutamate pool, which is highly required to produce protective metabolites (proline, phytochelatines, etc.).

The higher increase in the NH₄⁺ content (Fig. 1C) and in the protease activity (Fig. 2C) in roots and leaves of HT plants coincide with a greater decrease in soluble protein content (Fig. 1D). These results suggest that root hypoxia leaded to higher protein degradation. Such a process was observed in maize root tips,^43,63 and in tomato fruits64 and roots65 under sugar starvation or extended darkness.

In conclusion, this study demonstrates that, in tomato plants, root hypoxia (1) decreased root and leaf NO₃⁻ content and enhanced NR activity in the two organs. This process could be an adaptation of plant roots to transient hypoxic conditions.66 (2) leaded to protein degradation via the induction of the protease activity. (3) accelerated N compounds synthesis and accumulation via the stimulation of NiR, GS and GDH activities. The hypoxia-induced increase in the aminating GDH activity may be considered as an alternative N assimilation pathway involved in the NH₄⁺ detoxification and glutamate synthesis. However, the other pathways of glutamate synthesis cannot be excluded. More work at the molecular level will improve our understanding of nitrogen enzyme expressions and activities under hypoxic stress.

Material and Methods

Plant material and growth conditions.

Tomato (Solanum lycopersicum L. cv. Micro-Tom) seeds were germinated on filter paper moistened with distilled water for 1 week at 23°C in the dark, and then grown hydroponically in a growth chamber (16 h light at 23°C/8 h dark at 18°C with an irradiance of 350 µmolm⁻²s⁻¹ and 75–80% relative humidity). Each seedling was placed in a 25 ml vermiculite plug on a polystyrene tray floating on the nutrient solution, with 6 plants per 10 l tank. The nutrient solution, pH 5.8, contained macronutrients: 3 mM KNO₃, 1 mM Ca(NO₃)₂, 2 mM KH₂PO₄, 0.5 mM MgSO₄, 32.9 µM EDTA-Fe, and micronutrients: 30 µM H₃BO₄, 5 µM MnSO₄, 1 µM CuSO₄, 1 µM ZnSO₄, 1 µM (NH₄)₆Mo₇O₂₄. Hypoxic treatment was applied at the second leaf stage (one week after transplanting) for three weeks by stopping air bubbling. Oxygen shortage was imposed progressively by root oxygen consumption as in Horchani et al.36,37 except that before weekly medium renewal, the new nutrient solution was flushed with nitrogen down to about 2% O₂ in order to maintain a low oxygen pressure without re-oxygenation of the roots. Control plants were continuously bubbled with air.

Vegetative growth analysis.

Three weeks after root hypoxia application (at the sixth leaf stage), plants were harvested and divided into roots and shoots. Roots were washed in distilled water. Fresh weights (FW) were immediately determined for roots and shoots. Dry weights (DW) were obtained by weighing the plant material after drying at 80°C until a constant mass was reached. DW production was estimated as the difference between the DW_f obtained at the end of the experiment and the DW₀ measured just before root hypoxia application. Leaf area (LA) and chlorophyll content were measured as in Horchani et al.37

Nitrate, nitrite, ammonium and soluble protein assay.

For NO₃⁻, NO₂⁻ and protein determination, leaf and root samples of three-week-old plants were ground thoroughly with a pestle and mortar in 500 mM Tris-HCl pH 7.5, and centrifuged at 20,000 g for 15 min. NO₃⁻ and NO₂⁻ were assayed in plant tissues using the salicylic acid-sulfuric acid method,38 and the Griess reagent method,39 respectively. Total soluble proteins were assayed according to Bradford40 using γ-globulin as a standard. NH₄⁺ was extracted using 6% (w/v) TCA as in Horchani et al.41 and assayed spectrophotometrically in sample extracts using the phenol-hypochlorite method.42

Enzyme assays.

Nitrate and nitrite reductase. Tissue samples were ground at 4°C using an extraction buffer containing 25 mM Tris-HCl pH 8.5, 1 mM EDTA, 20 µM FAD, 1 mM dithiothreitol (DTT), 1% (w/v) bovine serum albumin (BSA), 10 mM cystein, 20 µM E64 and 2 mM phenyl-methyl-sulphonyl-fluoride (PMSF). The crude extracts were centrifuged at 20,000 g for 15 min and the supernatants were assayed for nitrate and nitrite reductase activities.

Nitrate reductase activity assay was adopted from Miranda et al.39 The reaction medium (1 ml) contained 0.2 M Hepes pH 7.0, 15 mM KNO₃ and 250 µM NADH. Reaction was initiated by NO₃⁻ addition, run at 28°C and then stopped after 30 min by adding 10 µl ADH (180 units/ml) and 10 µl acetaldehyde (4% v/v). The coloured reaction was produced by adding 480 µl of nitrite reagent (240 µl of 1% (w/v) sulphanilamide in 1 N HCl, plus 240 µl of 0.01% (w/v) N-1-naphtylenediamine dihydrochloride in water) and incubating for 30 min at room temperature. The absorbance was read at 540 nm. Nitrite reductase (NiR) activity was assayed by following NO₂⁻ consumption from the assay mixture using the dithionite-methyl viologen method.⁴³ The reaction medium (1 ml) consisted of 20 mM potassium phosphate (pH 7.3), 1 mM NaNO₂, 40 µM methyl viologen and the sample to be assayed. The reaction was started by addition of 10 µl of 100 mM sodium dithionite in 200 mM sodium bicarbonate and run at 28°C. Samples were maintained under anaerobiosis during the incubation. After 30 min, 20 µl aliquot fractions were sampled, diluted in 480 µl H₂O, and shaken energetically for 30 s. A 500 µl aliquot of the NO₂⁻ reagent39 was then added, and the absorbance measured at 540 nm after 30 min. Assay blanks contained enzymic extract plus reagents except dithionite.

Glutamine synthetase. Glutamine synthetase (GS) was extracted in a medium containing 0.1 M Tricine (pH 8.0), 20 mM MgSO₄, 10 µM chymostatin and 1 mM EDTA. The supernatant of the crude extract was used for enzymic measurement. GS was determined according to a modified version of the method of O'Neal and Joy.44 The assay mixture (1 ml) consisted of 0.1 M Tricine (pH 7.8), 4 mM MgSO₄, 0.2 mM EDTA, 80 mM glutamate, 6 mM NH₂OH, 8 mM ATP and enzyme extract. The reaction was started by addition of NH₂OH and terminated, after 10 min at 37°C, with 1 ml FeC1₃ reagent. Extracts were then centrifuged and the absorbance of the γ-glutamyl hydroxamate (GHM) formed was measured at 540 nm. Assay blanks contained all the reagents except ATP.

Glutamate dehydrogenase. Glutamate dehydrogenase (GDH) extraction was performed according to the method described by Magalhaes and Huber.45 Frozen samples were homogenized in a cold mortar and pestle with 100 mM Tris-HCl (pH 7.5), 14 mM β-mercaptoethanol and 1% (w/v) polyvinyl polypyrrolidone (PVP). The extract was centrifuged at 12,000 g for 15 min at 4°C. GDH activity was determined in the aminating direction by following the absorption change at 340 nm by NADH oxidation.46

Protease.

Protease activity was measured by the method of Weckenmann and Martin,47 using azocasein as substrate. Absorbance of the released-azo-dye- was measured at 340 nm and one unit of activity was defined as the activity producing an increase of 0.01 units of absorbance during 1 h incubation.48

Statistics.

Statistical data analysis was made using the Student's t-test. The results are given as means (±SD) of at least six replicates per treatment. The significance of differences between the control and the treatment mean values was determined at the significance level of p < 0.05. Experiments were replicated two to three times.

Figures and Tables

Figure 1 Nitrate (A), nitrite (B), ammonium (C) and soluble protein (D) contents in root and leaf tissues of aerated (□) and hypoxically treated (■) tomato plants for three weeks. Values are the mean of six replicates ± SD. *The significance of differences between the control and the treatment mean values was determined by the Student's t-test at the significance level of p < 0.05.

Figure 2 Glutamine synthetase (A), glutamate dehydrogenase (B) and protease (C) activities in roots and leaves of aerated (□) and hypoxically treated (■) tomato plants for three weeks. Values are the mean of six replicates ± S.D. *The significance of differences between the control and the treatment mean values was determined by the Student's t-test at the significance level of p < 0.05.

Table 1 Root and shoot dry weights (DW), shoot/root ratio, root and leaf water contents, leaf area and leaf chlorophyll content of tomato plants grown in aerated (NT) and hypoxic (HT) nutrient solution

	NT	HT
Root DW (g plant^−1)	0.32 ± 0.04	0.21 ± 0.03*
Shoot DW (g plant^−1)	1.87 ± 0.08	1.53 ± 0.05*
Shoot/root ratio	5.8	7.3*
Root water content (mlg^−1 DW)	17.2 ± 1.4	12.5 ± 0.9*
Leaf water content (mlg^−1 DW)	14.1 ± 1.2	11.6 ± 1.1*
Leaf area (cm^2 plant^−1)	128 ± 5.7	133 ± 6.2
Chlorophyll content (mgg^−1 FW)	1.22 ± 0.08	0.84 ± 0.61*

Samples were taken from plants after three weeks of root hypoxia treatment. Values are the mean of six replicates ± S.D.

* The significance of differences between the control and the treatment mean values was determined by the Student's t-test at the significance level of p < 0.05.

Table 2 Changes in nitrate reductase (NR) and nitrite reductase (NiR) activities, and NiR/NR ratio in roots and leaves of aerated (NT) and hypoxically treated (HT) tomato plants for three weeks

	Plant organ	NT	HT
NR (nmol NO2 formed min^−1g^−1 FW)	Root	3.2 ± 0.7	25.5 ± 2.4*
	Leaf	2.4 ± 0.4	5.1 ± 1.2*
NiR (nmol NO2 reduced min^−1g^−1 FW)	Root	22.5 ± 2.7	51.2 ± 5.3*
	Leaf	12.3 ± 2.1	35.9 ± 2.4*
NiR/NR ratio	Root	7.0	2.0*
	Leaf	5.1	7.0*

Values are the mean of six replicates ± S.D.

* The significance of differences between the control and the treatment mean values was determined by the Student's t-test at the significance level of p < 0.05.