Activation of nitrate reductase of cashew leaf by exogenous nitrite

Ativação de redutase de nitrato de folhas de cajueiro por NO2- exogéno

Abstracts

Nitrate reductase (NR) plays a central role in plant primary metabolism and exhibits complex regulation mechanisms for its catalytic activity. There is controversy in the literature concerning the possible direct effect of NO2- on the stimulation and/or activation of NR activity. The influence of NO2- was studied on the NR activity in the leaves of 30-day-old cashew (Anacardium occidentale L.) seedlings. Addition of NO2- to the reaction mixture containing leaf enzymatic extract resulted in a remarkable increase in NR activity. A trace amount (5 mumol.L-1) of NO2- was required to achieve full NR activity. The in vitro NR-activity showed a steady time-dependent increase when incubated in the presence of only NO3- + NO2-. In contrast, in vitro NR activity was practically absent in a NO2- -free reaction medium, even in the presence of a saturating NO3- concentration. No oxidation of NO2- to NO3- was detected during the experiment. Although NO2- clearly activated the in vitro NR activity, it had no effect on the in vivo leaf-NR activity determined under absence of oxygen. NADH at concentrations greater than 0.5 mmol.L-1 decreased the rates of in vitro NR activity. These results indicated, at least partially, a strong cashew leaf NO2- dependency of NR activation and/or activity. Finally, based on these results, it is suggested that this singular NR activity model induced by exogenous NO2- in cashew leaf extracts is caused by changes in the NR activation state by NO2- itself.

Anacardium occidentale; enzymes; NADH; nitrate metabolism; nitrate reductase

O efeito do NO2- sobre a atividade de RN foi estudado em folhas de plantas jovens de cajueiro, com 30 dias de idade, cultivadas em solução nutritiva. A atividade in vitro de NADH-RN aumentou com a presença de NO2- no meio de reação. Este aumento foi máximo em presença de 5,0 mimol.L-1 de NO2-. Com a remoção de NO2-, do meio de reação, a atividade de RN foi praticamente suprimida, apesar da presença de NO3- em concentração em nível de saturação da enzima. Este fato, ao menos em parte, sugere uma certa dependência da RN, por NO2-, para redução de NO3-. Muito embora o NO2- tenha sido necessário para a ativação in vitro de RN, este íon não foi capaz de produzir o mesmo efeito sobre a atividade de RN in vivo, quando o mesmo foi acrescido ao meio de reação contendo discos de folhas sob anaerobiose. A atividade de RN in vitro foi proporcionalmente dependente do tempo de incubação na presença de NO3- + NO2-. Com relação à concentração de NADH, a atividade de RN apresentou saturação com 0,50 mmol.L-1 de NADH e um discreto decréscimo a partir dessa concentração. Finalmente, os resultados do presente estudo sugerem que o NO2- induziu a atividade de RN, em extratos de folhas de plantas de cajueiro, possivelmente, por causar mudanças no estado de ativação da enzima.

Anacardium occidentale L.; assimilação do nitrato; NADH; enzima; redutase do nitrato

Activation of nitrate reductase of cashew leaf by exogenous nitrite

Ricardo Almeida Viégas¹ and Joaquim Albenísio Gomes Silveira^2,*

¹Departamento. de Engenharia Florestal, Universidade Federal da Paraíba, Campus VII, CP 64, CEP 58700-970, Patos, PB, Brasil;

2Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, CE, Brasil;

* Corresponding author: [e-mail]

Nitrate reductase (NR) plays a central role in plant primary metabolism and exhibits complex regulation mechanisms for its catalytic activity. There is controversy in the literature concerning the possible direct effect of NO₂^- on the stimulation and/or activation of NR activity. The influence of NO₂^- was studied on the NR activity in the leaves of 30-day-old cashew (Anacardium occidentale L.) seedlings. Addition of NO₂^- to the reaction mixture containing leaf enzymatic extract resulted in a remarkable increase in NR activity. A trace amount (5 mmol.L^-1) of NO₂^- was required to achieve full NR activity. The in vitro NR-activity showed a steady time-dependent increase when incubated in the presence of only NO₃^- + NO₂^-_. In contrast, in vitro NR activity was practically absent in a NO₂^- -free reaction medium, even in the presence of a saturating NO₃^- concentration. No oxidation of NO₂^- to NO₃^- was detected during the experiment. Although NO₂^- clearly activated the in vitro NR activity, it had no effect on the in vivo leaf-NR activity determined under absence of oxygen. NADH at concentrations greater than 0.5 mmol.L^-1 decreased the rates of in vitro NR activity. These results indicated, at least partially, a strong cashew leaf NO₂^- dependency of NR activation and/or activity. Finally, based on these results, it is suggested that this singular NR activity model induced by exogenous NO₂^- in cashew leaf extracts is caused by changes in the NR activation state by NO₂^- itself.

Key words:Anacardium occidentale, enzymes, NADH, nitrate metabolism, nitrate reductase.

Ativação de redutase de nitrato de folhas de cajueiro por NO₂^- exogéno: O efeito do NO₂^- sobre a atividade de RN foi estudado em folhas de plantas jovens de cajueiro, com 30 dias de idade, cultivadas em solução nutritiva. A atividade in vitro de NADH-RN aumentou com a presença de NO₂^- no meio de reação. Este aumento foi máximo em presença de 5,0 mmol.L^-1 de NO₂^-. Com a remoção de NO₂^-,do meio de reação, a atividade de RN foi praticamente suprimida, apesar da presença de NO₃^- em concentração em nível de saturação da enzima. Este fato, ao menos em parte, sugere uma certa dependência da RN, por NO₂^-, para redução de NO₃^-. Muito embora o NO₂^- tenha sido necessário para a ativação in vitro de RN, este íon não foi capaz de produzir o mesmo efeito sobre a atividade de RN in vivo, quando o mesmo foi acrescido ao meio de reação contendo discos de folhas sob anaerobiose. A atividade de RN in vitro foi proporcionalmente dependente do tempo de incubação na presença de NO₃^- + NO₂^-. Com relação à concentração de NADH, a atividade de RN apresentou saturação com 0,50 mmol.L^-1 de NADH e um discreto decréscimo a partir dessa concentração. Finalmente, os resultados do presente estudo sugerem que o NO₂^- induziu a atividade de RN, em extratos de folhas de plantas de cajueiro, possivelmente, por causar mudanças no estado de ativação da enzima.

Palavras-chave: Anacardium occidentale L., assimilação do nitrato, NADH, enzima, redutase do nitrato.

INTRODUCTION

Nitrate is the major N-source for cultivated plants and is an important signaling ion that influences plant growth and differentiation (Kaiser and Huber, 2001). Nitrate assimilation represents a central point in the growth of plants and other organisms (Silveira et al., 2001a). The utilization of NO₃^- by higher plants involves its reduction to NH₄⁺ in a two-step pathway and the incorporation of NH₄⁺ to skeletons of carbon to produce amino acids (Galván et al., 1992; Viégas et al., 1999). The reduction of NO₃^- to NO₂^- is a limiting step catalyzed by nitrate reductase -NR (Silveira et al., 2001b). NR is a most interesting enzyme especially from the aspect of its complex regulation expression and control of catalytic activity. NR requires NADH as electron donor in most higher plant species (Kaiser and Huber, 2001).

NR enzyme is a complex molecule containing FAD, Cyt b 557, and a Mo-Co as prosthetic groups. In higher plants, NR exists as a homodimer with a subunit molecular mass of 100 to 120 kDa (Campbell, 1999). In addition, NR is regarded as one of the best-characterized examples of substrate-induced enzyme in higher plants (Adrews et al., 1990). NR is subject to mechanisms controlling its gene induction and expression and catalytic activity (Haba et al., 2001). In most plant species, for instance, NR is specifically induced by NO₃^- while nitrite reductase (NiR) synthesis is enhanced by both NO₃^- and NO₂^- (Aguera et al., 1990).

Aslam et al. (1987) reported that barley leaf NR activity was induced by nutrient solution-NO₂^-, but a much higher concentration of external NO₂^- than NO₃^- was required for induction. According to these authors, there was no induction with NO₂^- until tissue-NO₃^- appeared, apparently from the internal oxidation of NO₂^-. In contrast, Siddiqui et al. (1992) reported that external-NO₂^- pretreatment was not able to induce NR activity in barley seedlings. The appearance of NO₃^- from the oxidation of NO₂^- into tissue is well established (Lips et al., 1973; Kaplan et al., 1974; Sahulka and Lisa, 1978; Aslam and Huffaker, 1989; Aslam et al., 1993). However, it is still uncertain whether NO₂^- oxidation in the cell is a chemical or biochemical process. Aslam et al. (1993) observed that NR induction by both NO₂^- and NO₃^- was inhibited by cycloheximide, indicating that the increase in NR activity was a result of increased NR synthesis rather than the activation of preexisting NR protein.

Since NO₂^- is oxidized to NO₃^- in the cell and the latter is an NR substrate, it is difficult to determine whether NO₂^- plays any role directly in NR activation, i.e., before it has been oxidized to NO₃^-_. In vitro studies involving NR enzyme should be carried out to overcome this difficulty. Aslam et al. (1993), for instance, could not detect any in vitro activation of NR activity by NO₂^-. To our knowledge, only Kaplan et al. (1978) reported in vitro activation of NR activity by NO₂^-. They found that the NR induced by NO₃^- and the NR activated by NO₂^- are different and the molecular mass of the NO₂^- activated-NR is lower than that of NO₃^- NR. They also postulated that the potential NR-component that could be activated by NO₂^- is a constitutive subunit that becomes incorporated into the NR protein during the Cyt c synthesis.

In the present study, it was shown that cashew seedling leaf NADH-NR was directly activated by exogenous NO₂^-. It is suggested that the changes in the NR activation state in cashew leaves are rapidly and effectively controlled by NO₂^- at very low tissue concentrations.

MATERIAL AND METHODS

Plant growth conditions: Dwarf cashew seeds (Anacardium occidentale L.) supplied by Embrapa, Fortaleza, Brazil, were surface-sterilized in 1 % (v/v) hypochlorite solution for 15 min, rinsed in distilled water and imbibed for 12 h. The seeds were subsequently sown in vermiculite and watered daily with a solution containing 1.0 mmol.L^-1 CaSO₄ (Viégas and Silveira, 1999). Ten days after sowing, seedlings selected by similar height were placed in plastic pots (1.0 dm³ capacity) filled with continuously air bubbled modified half-strength Hoagland and Arnon nutrient solution with the following composition (mmol.L^-1): 2.0, KNO₃; 1.5, Ca (NO₃) ₂; 0.5, CaCl₂; 0.5, MgSO₄; 0.5, K₂HPO₄; and micronutrients. After 10 days, the ionic strength of the nutrient solution was twofold increased. The plants were grown under greenhouse conditions, exposed to natural sunlight and day/night temperature means of 32/23 ^oC and 60/80 % relative humidity. The nutrient solution was completely changed every day and the pH maintained at 5.5 ± 0.5. Leaf discs were collected from the second youngest fully expanded leaves, as described by Viégas et al. (1999), to evaluate the in vivo NR activity when the homogeneous plant population was 30 days old. At this time, other second youngest fully expanded leaves were excised, immediately frozen in liquid N₂, ground to a fine powder, and stored at ¾80 ^oC until determination of the in vitro NR activity.

In vitro NR assay: Frozen leaf was homogenized in an extraction medium (6.0 ml.g^-1 fresh mass, at 4 ^oC, for 5 min) with a pestle and mortar. The extraction medium consisted of 100 mmol.L^-1 Tris-HCl buffer (pH 7.6) containing EDTA-Na⁺ (5 mmol.L^-1), polivinypolypyrrolidone (5 %, w/v), casein (1.5 %, w/v) and b-mercaptoethanol (5 mmol.L^-1). The homogenate was centrifuged at 20,000 g_n, for 20 min at 4 ^oC, and then filtered through two layers of cheesecloth. The supernatant was collected for evaluation of the in vitro leaf¾NR activity as previously described (Viégas et al., 1999). The reaction mixture contained 100 mmol.L^-1 Tris-HCl buffer pH 7.5; NADH was supplied at 0.5 mmol.L^-1, 40 mmol.L^-1 KNO₃; and the presence (+ NO₂^-) or absence (- NO₂^-)of 5.0 mmol.L^-1 KNO₂in a final volume of 1.2 mL. Then 0.25 mL of leaf crude extract was added to the reaction mixture and incubated at 35 ^oC for 15 min. The mixtures were previously and vigorously shaken to ensure thorough mixing. After centrifuging, a supernatant sample was taken to determine NO₂^- concentration.

In vivo NR assay: The in vivo NR activity was assessed by the "anaerobic tissue slice method" described by Silveira et al. (1998). Leaf discs from the second youngest fully expanded leaves (200 mg fresh mass, 10 mm diameter) were collected 4 h after the beginning of the natural light period (10:00 a.m.) and infiltrated twice for 2 min (¾67 kPa vacuum) with 5 mL of a reaction mixture containing 100 mmol.L^-1 potassium phosphate buffer pH 7.5; 25 mmol.L^-1 KNO₃; and 1 % iso-propanol. The reaction mixture was incubated at 35 ^oC for 30 min in the dark. NR activity was estimated from the amount of NO₂^- formed during the incubation period and released from the leaf discs to the medium after boiling for 5 min. Aliquots were mixed with 2 mL of (1:1) 1 % sulfanilamide in HCl 2.4 mol.L^-1: 0.02 % N-1-naphtyl-ethylenediamine and the absorbance determined at 540 nm. Five independent replications were used in all the enzymatic assays.

RESULTS AND DISCUSSION

Figure 1 shows that in vitro NR activity measured in crude leaf extract exposed to 25 mmol.L^-1 NO₃^- in absence of NO₂^- continued unchanged at the indicated times. In contrast, when the same crude leaf extracts were measured in the presence of both 5 mmol.L^-1 NO₂^- and 25 mmol.L^-1 NO₃^-,the NR activity was greatly increased in relation to the reaction time. It is suggested that the joint action of NO₂^- and NO₃^- on NR activity may represent a specific rather than a general rule. NR activity assayed in the presence of 5 mmol.L^-1 NO₂^- reflected the maximum activity observed in the assay (data not shown). The absence of any lag period in the NR activity along the course of the assay (figure 1) suggests that NO₂^- directly activated NR. In addition, the linear rate (r² = 0,9917**) of NO₂^- formation during the NR assay, in the presence of NO₂^-_, ensured a sufficient supply of NADH to the reaction. In fact, our experiments had shown that 0.5 mmol.L^-1 NADH was required to achieve the maximum effect of NO₂^- on the NR activity (figure 2). The overall results showed, initially, a clear stimulating effect of the short-term treatment with NO₂^- on the in vitro NR activity.

There are conflicting reports in the literature on the induction and activation of NR by NO₂^-. The fact that NO₃^- appears in the tissues of a number of plant species through internal NO₂^- oxidation, when the latter is the N-sole source in the root growth medium (Lips et al., 1973; Kaplan et al., 1974; Aslam et al., 1987; Aslam and Huffaker, 1989; Aslam et al., 1993), strongly limits any conclusion on the direct induction and/or activation of the NR enzyme by NO₂^-. In the current study, however, it was observed that leaf-NR activity in cashew plants was directly dependent on the presence of NO₂^- in the reaction medium (figure 1). This result provides evidence for the possible occurrence of a NO₂^--actived NR state. Conversely, Aslam et al. (1993) were not able to detect any NO₂^- activated barley NR under in vitro conditions. On the other hand, Kaplan et al. (1978) observed an in vitro activation of NR by exogenous NO₂^-. To our knowledge, cashew is the first plant species in which the presence of NO₂^- in the reaction medium was required to in vitro activate NR. It seems that NO₂^- and NO₃^- ions share different binding sites of leaf NR in cashew, but it is acknowledged that further study is necessary to confirm this hypothesis. It is suggested that NO₂^- might change NR to a more active state.

NR is subjected to several mechanisms controlling both its synthesis and catalytic activity (Campbell, 1999). Some of the post-translational mechanisms are very well and/or still poorly established. Furthermore, besides NO^-3 (NR substrate), NO₂^- and several organic nitro-compounds have been shown to induce NR in rice seedlings (Shen, 1972), but the inherent mechanisms are not clearly understood. Thus, NO₂^- might be acting as a positive modulator, changing the NR activation state. It is also possible that NO^-2 activates in vitro NR by changing its posphorylation state. One line of evidence in support of this suggestion is that cashew leaf activity leveled off in the absence of NO₂^- NR (figure 1). In cucumber, for instance, anoxia, uncouplers, and mannose feeding activate NR, at least by enhancing NR dephosphorylation via a decrease in the internal level of adenine nucleotides and a concomitant cellular acidification (Haba et al., 2001).

According to Kaplan et al. (1979), who observed direct NR activation by NO₂^-, there is an NO₂^--actived component of NR that is a constitutive subunit that becomes incorporated into the NR protein during the synthesis of the Cyt c component. Additionally, there are some reports that showed the occurrence of an unidentified NR activated component (Kaplan et al., 1978). Shen (1972) proposed that NR-RNA and protein synthesis, which was associated with NR induction in radish cotyledons, might be required for the synthesis of an signal molecule necessary for NR activity. On the other hand, it was observed that very small changes of cytosol pH might induce changes in the NR activation state by mechanisms yet unknown (Kaiser and Huber, 2001).

The concentration of NO₂^- that directly activates cashew leaf NR (figure 1) was substantially lower (by about 100 fold) than that required in the nutrient solution (Aslam et al., 1987). These authors reported that NR induction by environmental NO₂^- was not observed until NO₃^- was detected in the leaves of plants grown in 1.0 mmol.L^-1 NO₂^-. They also showed that the quantitative relationship between internal NO₃^- concentration and the induction of NR was maintained irrespective of whether N was supplied as NO₃^- or NO₂^-_. According to Aslam et al. (1987), it can be concluded that NO₂^-, due its internal oxidation to NO₃^-, indirectly induced de novo synthesis of NR protein rather than the activation of the preexisting protein.

Evidence that the induction of NR activity by NO₃^- is regulated by enzyme synthesis rather than by an activation mechanism was also obtained by Somers et al (1983) and, more recently, by Kaiser and Huber (2001). According to Kaplan et al. (1978), however, the NR induced by NO₂^- and NR activated by NO₂^- are different, the latter protein presenting a comparatively lower molecular mass.

Since NO₂^- oxidation to NO₃^- proceeds rapidly into the cell, there is no available information on a direct activation of NR after plants have been grown in the presence of external NO₂^-. For instance, while NO₂^- was not detected in detached leaves of 8-day-old barley seedlings placed in 1 mmol.L^-1 NO₂^-, 0.107 mmol.kg^-1 NO₃^- appeared in leaf tissue (Aslam et al., 1987). In the present study, analysis of the in vivo NR activity over time showed no NO₂^--dependent variations (figure 3). Similarly, in the study by Siddiqui et al. (1992), the in vivo NR activity was almost absent (0.003 mmol NO₂^-.g^-1 fresh wt.h^-1) in the leaves of barley plants supplied with 0.1 mmol.L^-1 NO₂^- for one day. They pointed out that NO₂^- was unable to induce in vitro leaf NR activity, although NO₃^- accumulation in the leaf tissue (2.70 mmol.kg^-1 fresh wt) was higher than in the study by Aslam et al (1987) (0.107 mmol.kg^-1 fresh wt), who observed an in vitro induction of NR by environmental NO₂^-.

These conflicting results on the in vitro induction of NR by external NO₂^- in the studies of Aslam et al. (1987) and by Siddiqui et al. (1992) could be partially explained by the procedures they used in relation to NADH. Aslam and co-workers oxidized the NADH excess following the NR assay with phenazine methosulfate. In the present study, we observed that NADH interferes in the color formation in the NO₂^- determination method (Figure 4). Thus, NADH concentration of 1.0 mmol.L^-1 caused an overall 30 % reduction in the NO₂^- concentration. Similar results were also described by Shen (1972). There was no attempt in this work to clarify the chemical mechanisms related to NADH interference on the reaction of the colored-complex formation after reaction between sulfanilamide, N-1-naphtyl-ethylenediamine and NO₂^-.

The results shown in the present work are particularly interesting because they suggest that very low NO₂^- concentrations like those found in the plant cytosol might be able to activate NR, at least in cashew leaf tissue. Apparently, it is the first time that NO₂^- has been shown to directly activate NR, suggesting that NO₂^- may play a crucial role in the modulation state of NR in cashew leaf. Nevertheless, proper interpretation of these results may be somewhat masked by the occurrence of no effect of NO₂^- on the in vivo NR activity, demanding further studies.

Acknowledgements: The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Cearense de Amparo à Pesquisa (FUNCAP) for funding this research.

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Publication Dates

• Publication in this collection
  30 Oct 2002
• Date of issue
  Jan 2002