Nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedlings
Sadegh Balotf1, Gholamreza Kavoosi1*, Bahman Kholdebarin2 1- Institute of Biotechnology, Shiraz University, Shiraz, 71441-65186, Iran 2- Department of Biology, Faculty of Sciences, Shiraz University, Shiraz,Iran *Corresponding author: [email protected], [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bab.1362. This article is protected by copyright. All rights reserved.
1
ABSTRACT The objective of this study was to examine the expression and activity of nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase in response to potassium nitrate, ammonium chloride and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for four days and the leaves were harvested. The relative expression of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.7.1) as well as the enzymes activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase activities in wheat seedlings while potassium nitrate being more effective.
Keywords: Wheat seedlings, Nitrate reductase, Nitrite reductase, Glutamine synthetase, Glutamate synthase
1. Introduction
This article is protected by copyright. All rights reserved.
2
Nitrate and ammonium are the major sources of nitrogen for plant growth and development while nitrate being more important for such processes. In plants, nitrate is readily mobile and can also be stored in vacuoles. However, for nitrate to be used in the biosynthesis of amino acids, proteins and other nitrogenous compounds, it must be reduced to ammonium. In leaves, nitrate reduction to nitrite followed by nitrite reduction to ammonium is catalyzed by nitrate reductase (NR, EC 1.7.1.1) and nitrite reductase (NiR, EC 1.7.2.2) which are located in cytosol and chloroplast, respectively[1]. Ammonium is assimilated by the biosynthetic pathways taking place in plant cells to produce different amino acids by the combined action of glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1) or glutamine 2-oxoglutarate aminotransferase (GOGAT, EC 1.4.7.1) in a cyclic manner[2,3]. In plants, NR is present in cytosol which catalyzes the reduction of nitrate to nitrite. Several studies have shown that NR is regulated by nitrate ions, light, growth conditions, hormones, reduced nitrogen metabolites as well as by phosphorylation [4-6]. Nitrate can be stored in plant cells without toxic effects, but nitrite is toxic and must be metabolized. NiR is a plastidic enzyme which catalyzes a six-electron reduction of nitrite to ammonium supplied by reduced ferredoxin. The rate of nitrite conversion to ammonium depends on the suppressive effects exerted by reduced nitrogen metabolites such as ammonium and amino acids [7-9]. Ammonium, even at low concentrations is very toxic and must be assimilated and detoxified into organic nitrogenous compounds. The metabolism of ammonium to amino acids and amides is the main mechanism of ammonium detoxification. In the leaves of higher plant, GS and GOGAT are involved in ammonium assimilation [10]. GS has a high affinity for ammonium and thus can assimilate ammonium at low concentrations to avoid ammonium accumulation to phytotoxic levels. The action of this enzyme is crucial in the assimilation of ammonium, which catalyzes the fixation of γ-carboxyl group of glutamate to form glutamine [11-13]. GOGAT is located in the leaf chloroplasts and is involved in ammonium assimilation [14, 15]. Glutamic acid formation is a process by which ammonium enters nitrogenous compounds catalyzed by GOGAT. Ammonium ions assimilated into glutamate can be transferred by aminotransferases to an appropriate α-ketoacids to form α-amino This article is protected by copyright. All rights reserved.
3
acids. Amino acids in turn are assimilated into proteins and other nitrogenous compounds such as nucleic acids [16]. Wheat (Triticum aestivum L.) is an important strategic cereal grain cultivated and consumed world-wide. Although there is abundant information in literatures showing the inhibition of NR activity in plant cells by reduced nitrogen metabolites under hydroponic conditions, there are relatively little reports on the activity of NR, NiR, GS and GOGAT at transcriptional levels in Iranian wheat leaves cultivated in soil in the presence of potassium nitrate, ammonium nitrate and ammonium chloride. Thus the novelty of this study is the analysis of NR, NiR, GS and GOGAT at transcript level and enzyme activity under more natural condition.
This article is protected by copyright. All rights reserved.
4
2. Materials and Methods
2.1. Plant treatments and sample preparation Wheat (Triticum aestivum L. var. Shiraz) seedlings were grown in Hoagland’s solution at 26/20°C day/light temperature under a 16/8 h light/dark regime and 55-60% relative humidity for 17 days. Plants were then divided into two groups: (a) control plants that were allowed to continue to grow in Hoagland’s solution, (b) N-starved plants that were grown in media without nitrogen for seven days. After this period, N-starved plants were exposed to different salt solutions (50 mM) of potassium nitrate, ammonium nitrate and ammonium chloride, for three days. One day after nitrogen treatment, leaves were harvested and frozen at -20 for future use. The wheat leaves (20 g) were ground in a mortar with pestle in 100 mL of 10 mM Tris buffer (pH 7.4) containing 2% (w/v) polyvinylpyrrolidone, 2 mM EDTA, 10% (v/v) glycerol and 1 mM DTT at 4 °C. The homogenate was centrifuged at 12000 g for 15 min. The supernatants were stored at -70 °C until use. The supernatants were assayed for NR, NiR, GS and GOGAT activities as well as for nitrate, nitrite, ammonium, total amino acid and total protein contents.
2.2. RNA extraction and real-time PCR Total RNA was extracted using RNA-plus buffer supplied by Cinagen (RNX-plus, Iran) according to manufacturer’s protocol. The concentration of purified total RNA was determined using a Nano-Drop (ND) 1000 spectrophotometer (Wilmington, USA). Agarose gel electrophoresis was used for the total RNA integrity determination prior to first-strand cDNA synthesis. Total RNA was determined by DNase kit (Fermentase, Hanover, MD) according to manufacturer’s protocol. Fisrt-strand cDNA synthesis was performed using DNase-treated total RNA (5 µg) using the Revert Aid First Strand This article is protected by copyright. All rights reserved.
5
cDNA Synthesis Kit (Thermo Scientific, Fermentas) according to manufacturer’s protocol. Primers were designed using Allele ID 7 software (Premier Biosoft Intl, Palo Alto, CA, USA) for 18SrRNA (AJ272181) as internal control and NR (X57844), NiR (FJ527909), GS (AY491968) and GOGAT (primer for GOGAT gene designed based on the aligned nucleotide file) genes (Table 1). Relative real-time PCR was performed in 20 µL solution containing 4 pM of each primers, 1 µL of the firststrand cDNA and 1x SYBR Premix Ex TaqTM Π (Takara, Japan). The amplification reactions were carried out in a line Gene K Thermal cycler (Bioer Technology Co, Hangzhou, China) with initial denaturing temperature of 94 °C for 10 min, followed by 40 cycles of 94 °C for 10 s, annealing temperatures of each primer 15 s and the extension at 72 °C for 30 s. The specificity of the amplifications was checked based on the melting curves resulting from heating the amplicons from 50 to 95 °C. All amplification reactions were repeated twice under identical conditions. To ensure that the PCR was generated from cDNA but not from genomic DNA, proper control reaction was carried out without the reverse transcriptase treatment. Analysis of the relative gene expression was done based on the comparative Cycle Threshold (CT) method as described by Livak [17].The values of relative mRNA accumulation were means of three independent experiment ± SD. The final results of NR, NiR, GS and GOGAT mRNA accumulation were reported as percent of those found in control plants.
2.3. Enzymes activity assay NR activity in supernatants was determined by the reduction of nitrate to nitrite as described by Krishna Rao et al. [18] in a reaction mixture containing 100 mM KNO3 and 0.25 mM NADH. One unit of the NR was defined as the amount of enzyme required for production of 1µmol nitrite per min.The NR activity was expressed as NR units per gram of fresh weight. NiR activity in the supernatants was assayed by the reduction of nitrite to ammonium as described by Krishna Rao et al. [18] in a reaction mixture containing 50 mM NaNO2 as substrate and 5 mM methyl viologen as electron donor. One unit of the NiR is defined as the
This article is protected by copyright. All rights reserved.
6
amount of enzymes required for conversion of 1µmol nitrite to ammonium per min. The NiR activity was expressed as NiR units per gram of fresh weight. GS activity in the supernatants was determined by the methods described by Nagy et al.[11] in a reaction mixture containing 50 mM immidzole, 30 mM MgCl2, 25 mM hydroxylamine and 100 mM L-glutamate. One unit of GS is the amount of enzyme catalyzing the formation of 1 µmol of glutamylmonohydroxamate per min. The GS activity was expressed as GS units per gram of fresh weight. GOGAT activity in the supernatants was determined by the conversion of 2ketoglutarate to glutamate in a reaction mixture containing 200 mM KH2PO-KOH pH 7.5, 10 mM glutamine (Gln), 10 mM 2-ketoglutarate, 15 mM methyl viologen and 1 mM aminooxyacetic acid [19]. One unit of the GOGAT is defined as the amount of enzyme required for the conversion of 1µmol2-ketoglutarate per min. The GOGAT activity was expressed as GOGAT units per gram of fresh weight. The values of NR, NiR, GS and GOGAT activities were the means of three independent experiment ± SD.
2.4. Biochemical analysis The amount of nitrate in the supernatants was determined colorimetrically using salicylic acid methods [20]. Potassium nitrate was used as standard and nitrate content was expressed as µmol nitrate per gram fresh weight. Nitrite content in the supernatants was analyzed by Griess reagent [0.1% naphthylethylenediaminedihydrochloride in H2O + 1% sulphanilamide in 2.5% H3PO4]. Sodium nitrite was used as standard and nitrite content was expressed as µmol nitrite per gram fresh weight [20]. Supernatants ammonium content was determined by spectrophotometric method according to ISO [21]. Ammonium chloride was used as standard and ammonium content was expressed as µmol ammonium per gram fresh weight. The supernatants were also assayed for total amino acids by ninhydrin using glycine as standard [22]. Total amino acid content was expressed as µg glycine per gram fresh weight. Protein
This article is protected by copyright. All rights reserved.
7
content in the supernatants were determined using coomassie brilliant blue G-250 according to the Bradford method using bovine serum albumin as standard [23]. Total protein content was expressed as mg protein per gram fresh weight. The values of nitrate, nitrite, ammonium, amino acid and protein content were at least means of three independent experiment ± SD.
2.5. Statistical analysis The SPSS statistical package (SPSS, Abaus Concepts, Berkeley, CA) was used for statistical analysis. The data presented here were analyzed as a completely randomized design with three replications and expressed as means ± standard deviation. The significant differences between treatments were analyzed by Duncan and Turkey’s multiple range tests at P
Sadegh Balotf1, Gholamreza Kavoosi1*, Bahman Kholdebarin2 1- Institute of Biotechnology, Shiraz University, Shiraz, 71441-65186, Iran 2- Department of Biology, Faculty of Sciences, Shiraz University, Shiraz,Iran *Corresponding author: [email protected], [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bab.1362. This article is protected by copyright. All rights reserved.
1
ABSTRACT The objective of this study was to examine the expression and activity of nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase in response to potassium nitrate, ammonium chloride and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for four days and the leaves were harvested. The relative expression of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.7.1) as well as the enzymes activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase activities in wheat seedlings while potassium nitrate being more effective.
Keywords: Wheat seedlings, Nitrate reductase, Nitrite reductase, Glutamine synthetase, Glutamate synthase
1. Introduction
This article is protected by copyright. All rights reserved.
2
Nitrate and ammonium are the major sources of nitrogen for plant growth and development while nitrate being more important for such processes. In plants, nitrate is readily mobile and can also be stored in vacuoles. However, for nitrate to be used in the biosynthesis of amino acids, proteins and other nitrogenous compounds, it must be reduced to ammonium. In leaves, nitrate reduction to nitrite followed by nitrite reduction to ammonium is catalyzed by nitrate reductase (NR, EC 1.7.1.1) and nitrite reductase (NiR, EC 1.7.2.2) which are located in cytosol and chloroplast, respectively[1]. Ammonium is assimilated by the biosynthetic pathways taking place in plant cells to produce different amino acids by the combined action of glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1) or glutamine 2-oxoglutarate aminotransferase (GOGAT, EC 1.4.7.1) in a cyclic manner[2,3]. In plants, NR is present in cytosol which catalyzes the reduction of nitrate to nitrite. Several studies have shown that NR is regulated by nitrate ions, light, growth conditions, hormones, reduced nitrogen metabolites as well as by phosphorylation [4-6]. Nitrate can be stored in plant cells without toxic effects, but nitrite is toxic and must be metabolized. NiR is a plastidic enzyme which catalyzes a six-electron reduction of nitrite to ammonium supplied by reduced ferredoxin. The rate of nitrite conversion to ammonium depends on the suppressive effects exerted by reduced nitrogen metabolites such as ammonium and amino acids [7-9]. Ammonium, even at low concentrations is very toxic and must be assimilated and detoxified into organic nitrogenous compounds. The metabolism of ammonium to amino acids and amides is the main mechanism of ammonium detoxification. In the leaves of higher plant, GS and GOGAT are involved in ammonium assimilation [10]. GS has a high affinity for ammonium and thus can assimilate ammonium at low concentrations to avoid ammonium accumulation to phytotoxic levels. The action of this enzyme is crucial in the assimilation of ammonium, which catalyzes the fixation of γ-carboxyl group of glutamate to form glutamine [11-13]. GOGAT is located in the leaf chloroplasts and is involved in ammonium assimilation [14, 15]. Glutamic acid formation is a process by which ammonium enters nitrogenous compounds catalyzed by GOGAT. Ammonium ions assimilated into glutamate can be transferred by aminotransferases to an appropriate α-ketoacids to form α-amino This article is protected by copyright. All rights reserved.
3
acids. Amino acids in turn are assimilated into proteins and other nitrogenous compounds such as nucleic acids [16]. Wheat (Triticum aestivum L.) is an important strategic cereal grain cultivated and consumed world-wide. Although there is abundant information in literatures showing the inhibition of NR activity in plant cells by reduced nitrogen metabolites under hydroponic conditions, there are relatively little reports on the activity of NR, NiR, GS and GOGAT at transcriptional levels in Iranian wheat leaves cultivated in soil in the presence of potassium nitrate, ammonium nitrate and ammonium chloride. Thus the novelty of this study is the analysis of NR, NiR, GS and GOGAT at transcript level and enzyme activity under more natural condition.
This article is protected by copyright. All rights reserved.
4
2. Materials and Methods
2.1. Plant treatments and sample preparation Wheat (Triticum aestivum L. var. Shiraz) seedlings were grown in Hoagland’s solution at 26/20°C day/light temperature under a 16/8 h light/dark regime and 55-60% relative humidity for 17 days. Plants were then divided into two groups: (a) control plants that were allowed to continue to grow in Hoagland’s solution, (b) N-starved plants that were grown in media without nitrogen for seven days. After this period, N-starved plants were exposed to different salt solutions (50 mM) of potassium nitrate, ammonium nitrate and ammonium chloride, for three days. One day after nitrogen treatment, leaves were harvested and frozen at -20 for future use. The wheat leaves (20 g) were ground in a mortar with pestle in 100 mL of 10 mM Tris buffer (pH 7.4) containing 2% (w/v) polyvinylpyrrolidone, 2 mM EDTA, 10% (v/v) glycerol and 1 mM DTT at 4 °C. The homogenate was centrifuged at 12000 g for 15 min. The supernatants were stored at -70 °C until use. The supernatants were assayed for NR, NiR, GS and GOGAT activities as well as for nitrate, nitrite, ammonium, total amino acid and total protein contents.
2.2. RNA extraction and real-time PCR Total RNA was extracted using RNA-plus buffer supplied by Cinagen (RNX-plus, Iran) according to manufacturer’s protocol. The concentration of purified total RNA was determined using a Nano-Drop (ND) 1000 spectrophotometer (Wilmington, USA). Agarose gel electrophoresis was used for the total RNA integrity determination prior to first-strand cDNA synthesis. Total RNA was determined by DNase kit (Fermentase, Hanover, MD) according to manufacturer’s protocol. Fisrt-strand cDNA synthesis was performed using DNase-treated total RNA (5 µg) using the Revert Aid First Strand This article is protected by copyright. All rights reserved.
5
cDNA Synthesis Kit (Thermo Scientific, Fermentas) according to manufacturer’s protocol. Primers were designed using Allele ID 7 software (Premier Biosoft Intl, Palo Alto, CA, USA) for 18SrRNA (AJ272181) as internal control and NR (X57844), NiR (FJ527909), GS (AY491968) and GOGAT (primer for GOGAT gene designed based on the aligned nucleotide file) genes (Table 1). Relative real-time PCR was performed in 20 µL solution containing 4 pM of each primers, 1 µL of the firststrand cDNA and 1x SYBR Premix Ex TaqTM Π (Takara, Japan). The amplification reactions were carried out in a line Gene K Thermal cycler (Bioer Technology Co, Hangzhou, China) with initial denaturing temperature of 94 °C for 10 min, followed by 40 cycles of 94 °C for 10 s, annealing temperatures of each primer 15 s and the extension at 72 °C for 30 s. The specificity of the amplifications was checked based on the melting curves resulting from heating the amplicons from 50 to 95 °C. All amplification reactions were repeated twice under identical conditions. To ensure that the PCR was generated from cDNA but not from genomic DNA, proper control reaction was carried out without the reverse transcriptase treatment. Analysis of the relative gene expression was done based on the comparative Cycle Threshold (CT) method as described by Livak [17].The values of relative mRNA accumulation were means of three independent experiment ± SD. The final results of NR, NiR, GS and GOGAT mRNA accumulation were reported as percent of those found in control plants.
2.3. Enzymes activity assay NR activity in supernatants was determined by the reduction of nitrate to nitrite as described by Krishna Rao et al. [18] in a reaction mixture containing 100 mM KNO3 and 0.25 mM NADH. One unit of the NR was defined as the amount of enzyme required for production of 1µmol nitrite per min.The NR activity was expressed as NR units per gram of fresh weight. NiR activity in the supernatants was assayed by the reduction of nitrite to ammonium as described by Krishna Rao et al. [18] in a reaction mixture containing 50 mM NaNO2 as substrate and 5 mM methyl viologen as electron donor. One unit of the NiR is defined as the
This article is protected by copyright. All rights reserved.
6
amount of enzymes required for conversion of 1µmol nitrite to ammonium per min. The NiR activity was expressed as NiR units per gram of fresh weight. GS activity in the supernatants was determined by the methods described by Nagy et al.[11] in a reaction mixture containing 50 mM immidzole, 30 mM MgCl2, 25 mM hydroxylamine and 100 mM L-glutamate. One unit of GS is the amount of enzyme catalyzing the formation of 1 µmol of glutamylmonohydroxamate per min. The GS activity was expressed as GS units per gram of fresh weight. GOGAT activity in the supernatants was determined by the conversion of 2ketoglutarate to glutamate in a reaction mixture containing 200 mM KH2PO-KOH pH 7.5, 10 mM glutamine (Gln), 10 mM 2-ketoglutarate, 15 mM methyl viologen and 1 mM aminooxyacetic acid [19]. One unit of the GOGAT is defined as the amount of enzyme required for the conversion of 1µmol2-ketoglutarate per min. The GOGAT activity was expressed as GOGAT units per gram of fresh weight. The values of NR, NiR, GS and GOGAT activities were the means of three independent experiment ± SD.
2.4. Biochemical analysis The amount of nitrate in the supernatants was determined colorimetrically using salicylic acid methods [20]. Potassium nitrate was used as standard and nitrate content was expressed as µmol nitrate per gram fresh weight. Nitrite content in the supernatants was analyzed by Griess reagent [0.1% naphthylethylenediaminedihydrochloride in H2O + 1% sulphanilamide in 2.5% H3PO4]. Sodium nitrite was used as standard and nitrite content was expressed as µmol nitrite per gram fresh weight [20]. Supernatants ammonium content was determined by spectrophotometric method according to ISO [21]. Ammonium chloride was used as standard and ammonium content was expressed as µmol ammonium per gram fresh weight. The supernatants were also assayed for total amino acids by ninhydrin using glycine as standard [22]. Total amino acid content was expressed as µg glycine per gram fresh weight. Protein
This article is protected by copyright. All rights reserved.
7
content in the supernatants were determined using coomassie brilliant blue G-250 according to the Bradford method using bovine serum albumin as standard [23]. Total protein content was expressed as mg protein per gram fresh weight. The values of nitrate, nitrite, ammonium, amino acid and protein content were at least means of three independent experiment ± SD.
2.5. Statistical analysis The SPSS statistical package (SPSS, Abaus Concepts, Berkeley, CA) was used for statistical analysis. The data presented here were analyzed as a completely randomized design with three replications and expressed as means ± standard deviation. The significant differences between treatments were analyzed by Duncan and Turkey’s multiple range tests at P
