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Identification of Differentially Expressed Genes Induced by Ammonium Nitrogen in Rice Using mRNA Differential Display
Rice Science, 2008, 15(3): 247–250 Copyright © 2008, China National Rice Research Institute. Published by Elsevier BV. All rights reserved
Identification of Differentially Expressed Genes Induced by Ammonium Nitrogen in Rice Using mRNA Differential Display ZHU Guo-hui, HUANG Zhuo-lie (College of Life Sciences, South China Agricultural University, Guangzhou 510642, China)
Abstract: RNAs isolated from ammonium- and nitrate-treated rice leaves were used to screen differentially expressed genes through mRNA differential display. A total of 72 bands appeared significant differences and some of them were further confirmed by reverse Northern and Northern blot. The results showed that two genes, A-02 (Oryza sativa drought stress related mRNA) and A-03 (Zea mays partial mRNA for TFIIB-related protein) were highly up-regulated in the ammonium-fed rice leaves. The enzyme assays showed that the activities of the two anti-oxidative enzymes, catalase and peroxidase, and the content of a non-enzymic antioxidant, glutathione, were significantly higher in the ammonium-fed rice leaves than those in the nitrate-fed ones, indicating that the ammonium nutrition might be beneficial for rice plants to improve the stress resistance during growth and development. Key words: Oryza sativa; mRNA differential display; ammonium nitrogen; nitrate nitrogen; stress resistance
Nitrogen is an essential nutrient for plant growth and development, comprising most of biomolecules. Ammonium and nitrate are two major nitrogen forms acquired by plants. Studies in recent years have indicated that different nitrogen forms may lead to plant physiological differences, such as photosynthesis, respiration, ions uptake, enzyme metabolism and even stress resistance [1-3]. Generally, rice is considered as an ammonium-preferring plant, which grows better under ammonium than under nitrate nutrition. At present, much work on molecular mechanisms has been done to investigate the reasons of physiological difference under the nutrition of different nitrogen forms. Numerous studies have shown that nitrate serves not only as the nutrient element, but as the signal. Several approaches, such as nitrate reductase (NR)- deficient mutants or short-term treatment with low levels of nitrate, have been employed to screen the specific genes induced by nitrate signal [4-6], and the results show that the nitrate signal plays important roles in regulating plant root development, nutrient uptake, nitrogen and organic acid metabolism, etc [7-8]. However, among most reported nitrate signaling responses, it has not been identified that which is directly aroused by nitrate itself and which is influenced indirectly as a result of nitrate metabolism to ammonia. Compared with the interests of nitrate signal, considerably less attention have been paid to the ammonium-induced genes. In this study, mRNA differential display was applied to identify the differential expressions of genes under different nitrogen nutrition conditions by contrasting ammonium-fed plants to nitrate-fed ones, and some ammonium-inducible genes were gained. The study also tried to clarify the molecular mechanisms of rice physiological differences under ammonium Received: 10 March 2008; Accepted: 14 May 2008 Corresponding author: ZHU Guo-hui ([email protected]) This is an English version of the paper published in Chinese in Chinese Journal of Rice Science, Vol. 22, No. 3, 2008, Pages 261–265.
and nitrate nutrition.
MATERIALS AND METHODS Plant materials Seeds of rice (Oryza sativa L. cv. Xiangzhongxian 2) were sterilized with 3% NaClO for 20 min and then germinated in an incubator at 28ºC. When the lengths of radical roots were approximately 1.0 cm, seedlings were transferred to the greenhouse at day/night temperature of 29ºC/24ºC with a 14-h light and 10-h dark cycle in normal culture solution [9]. After 10 days culture, seedlings were divided into three groups to conduct the treatments by replacing the nitrogen element with various nitrogen forms: 1) 3.0 mmol/L nitrate exclusively (in the form of calcium nitrate); 2) 3.0 mmol/L ammonium exclusively (in the form of ammonium sulfate); 3) 3.0 mmol/L mixed nitrogen (1.5 mmol/L nitrate and 1.5 mmol/L ammonium). Leaves were sampled at 4, 12 and 36 h after the treatments. All the samples for RNA extraction were immediately frozen in liquid nitrogen and stored at –75ºC. In addition, Leaves under different nitrogen treatments for 3 days were collected for enzyme assays. mRNA differential display analysis Leaves treated with ammonium and nitrate respectively for 4 h were sampled for RNA extraction. Total RNA was extracted using the Trizol Reagent (Qiagen, Hamburg, Germany) and digested with DNase I (TaKaRa, Tokyo, Japan) to remove the residual DNA contamination. DNase-treated RNA samples were reverse transcribed in the presence of the anchored primers [10] as follows: H-T11A (5ƍ-AAGCTTTTTTTTTTTA-3ƍ), H-T11G (5ƍ-AAGCTTTTTTTTTTTG-3ƍ) and H-T11C (5ƍ-AAGCTTTT TTTTTTTC-3ƍ), respectively. Second strand synthesis and PCR
Rice Science, Vol. 15, No. 3, 2008
248 amplification were performed in a total volume of 20 μL. The reaction mixture contained 0.2 mmol/L dNTPs, 1 U of Taq polymerase (TaKaRa, Tokyo, Japan), 1 μmol/L anchored primer, 1 μmol/L random primer (Table 1) and 1 μL cDNA solution. PCR amplification was carried out with one cycle of 94ºC for 2 min, 40 cycles of 94ºC for 30 s, 40ºC for 2 min, 72ºC for 30 s, and one cycle of 72ºC for 5 min. PCR products were separated on a 6% denatured polyacrylamide gel with silver staining [11]. Selected bands were purified, precipitated and then used as the templates for the second PCR amplification. Each sample was re-amplified with the respective primers as described earlier. Positive analysis and gene cloning All the selected bands were further identified by reverse Northern blot [12-13]. The second PCR products were transferred to Hybond-N+ nylon membrane (Amersham, Piscataway, NJ, USA) after electrophoresis. The 5 μg RNA of ammonium-fed and nitrate-fed leaves were reversely transcribed respectively and used as probes. The probes were labeled with the random primer DNA labeling kit (TaKaRa, Tokyo, Japan) and hybridized with the membrane referred above. The positive bands proved by reverse Northern blot were subcloned into the pGEM-T easy vector (Promega, USA) and transformed to competent cells of DH10B. Positive recombinant clones were further sequenced, and DNA sequences were analyzed at the National Center for Biotechnology Information (NCBI; NIH, Bethesda, USA) using the BLASTN and BLASTX algorithms. Northern blot was carried out according to the methods of Sambrook et al [14]. Total RNA of rice leaves treated for 4, 12 and 36 h were subjected to electrophoresis on 1.2 % formaldehyde agarose gels and blotted to a nylon membrane (Hybond-N+, Amersham, Piscataway, NJ, USA). Hybridizations were performed overnight at 42ºC with the hybridization buffer containing 50% formamide, 5×SSC, 1×Denhardt’s solution, 1% SDS and 100 μg/ mL heat-denatured salmon sperm. After hybridization, the blots were washed in 2×SSC, 0.5% SDS solution at 42ºC and 65ºC respectively for 15 min, before in Table 1. Random primers of mRNA differential display. Primer name
Primer sequence
H-AP1 H-AP2 H-AP3 H-AP4 H-AP5 H-AP6 H-AP7 H-AP8 H-AP9 H-AP10 H-AP11 H-AP12 H-AP13 H-AP14 H-AP15 H-AP16
5ƍ-AAGCTTGATTGCC-3ƍ 5ƍ-AAGCTTCGACTGT-3ƍ 5ƍ-AAGCTTTGGTCAG-3ƍ 5ƍ-AAGCTTCTCAACG-3ƍ 5ƍ-AAGCTTAGTAGGC-3ƍ 5ƍ-AAGCTTGCACCAT-3ƍ 5ƍ-AAGCTTAACGAGG-3ƍ 5ƍ-AAGCTTTTACCGC-3ƍ 5ƍ-AAGCTTCATTCCG-3ƍ 5ƍ-AAGCTTCCACGTA-3ƍ 5ƍ-AAGCTTCGGGTAA-3ƍ 5ƍ-AAGCTTGAGTGCT-3ƍ 5ƍ-AAGCTTCGGCATA-3ƍ 5ƍ-AAGCTTGGAGCTT-3ƍ 5ƍ-AAGCTTACGCAAC-3ƍ 5ƍ-AAGCTTTAGAGCG-3ƍ
0.2×SSC, 0.2% SDS solution at 65ºC for 15 min. Hybridization signals were analyzed using the Molecular Imager FX-Plus (Bio-Rad, Ivry sur Seine, France). Assay of enzyme activities Rice leaves were homogenized with 50 mmol/L chilled phosphate buffer (pH 7.0). The homogenate was centrifuged and the supernatants were used for enzyme assays. Superoxide dismutase (SOD) activity was estimated according to the method of Beauchamp and Fridovich [15], and expressed as U/mg (One unit of enzyme is defined as the amount of enzyme which produces a 50% inhibition of NBT reduction under assay conditions). Catalase (CAT) activity was assayed from the rate of H2O2 decomposition following the procedure of Aebi [16], and expressed as ǻA240/(mg·min). Peroxidase (POD) activity was measured according to Chance et al [17] and expressed as ǻA470/(mg·min). To estimate reduced glutathione (GSH) content, the fresh leaves were homogenized in 5% chilled trichloroacetic acid and the supernatants were used for determination according to the method of Chance et al [17].
RESULTS mRNA differential display analysis PCR were performed with 48 primer combinations (16 of random primers vs 3 of anchored primers). Amplified products were analyzed by electrophoresis on 6% denatured polyacrylamide gels (Fig. 1) and each lane had 50–100 bands. Ultimately, 72 bands were shown to have unique expressions responding to each treatment. Positive analysis and gene cloning Reverse Northern was generally used for high throughput screening for positive clones. Here, differentially displayed bands were equally transferred to Hybond-N+ nylon membrane after electrophoresis, and hybridized with cDNA probes reversely transcribed with RNA extracted from ammonium-fed and nitrate-fed leaves, respectively (Fig. 2). As shown in Fig. 2, the majority of bands were considered to be the false positive bands. The positive bands
Fig. 1. Results of mRNA differential display. A, Samples treated with ammonium nitrogen; N, Samples treated with nitrate nitrogen. Differential bands are indicated by arrows.
ZHU Guo-hui et al. Identification of Differentially Expressed Genes Induced by Ammonium Nitrogen in Rice
249
Fig. 3. Northern blot analysis of two differentially expressed genes. A, Ammonium nitrogen; N, Nitrate nitrogen; A/N, 50% ammonium and 50% nitrate nitrogen. Fig. 2. Reverse Northern blot of differential bands. A, Hybridized with ammonium-treated cDNA probe; B, Hybridized with nitrate-treated cDNA probe. Arrows indicate positive clones.
which showed differential hybridization signals between the two membranes were then cloned to the pGEM-T vector. Sequencing results and homologous analysis are shown in Table 2. Two clones strongly expressed in ammonium-fed rice leaves were further confirmed by Northern blot (Fig. 3). Gene of A-01 (Oryza sativa metallothionein-like protein mRNA), which encodes a rice metallothione protein, had been proved to be remarkably induced by ammonium nitrogen in previous study [19]. The hybridization showed that the expression profile of A-02 (Oryza sativa drought stress related gene), regulated by nitrogen forms, was the highest in the ammonium- fed rice leaves, weaker in the mixed nitrogen-fed ones, and the weakest in the nitrate-fed ones, indicating that the expression of A-02 was not only induced by ammonium nitrogen, but had a postive correlation with ammonium content. The expression profile of A-03 (Zea mays partial mRNA for TFIIB-related protein) was also induced by ammonium nitrogen, which increased with the time for ammonium treatment, whereas maitained stable in nitrate-fed rice leaves. Analysis of rice anti-oxidative enzymes under different nitrogen forms Genes of A-01 and A-02, reported as resistance- related genes, were both up-regulated by ammonium nitrogen. To identify whether it can be the implication that rice would have
stronger stress resistant ability under ammonium nutrition than nitrate nutrition, we analyzed the activities of several antioxidative enzymes such as SOD, POD, CAT and the contents of an important antioxidant GSH in both ammonium- and nitrate-fed rice leaves (Fig. 4). The results showed that ammonium nitrogen enhanced the activities of antioxidative enzymes and the contents of nonenzymic antioxidants. The CAT and POD activities and the GSH content were increased by 91.5%, 37.9% and 63.6% under ammonium nutrition compared with under nitrate nutrition, respectively. Whereas it was no obvious differences between ammonium and ammonium-nitrate mixed nutrition.
DISCUSSION Several ammonium-induced genes were screened by mRNA differential display in this study. A-01 was highly homologous to rice metallothionein (MT)-like mRNA encoding metallothionein. MTs are defined as a class of proteins with low molecular mass, high cysteine content and response to various stresses including drought, salt, and cold [20]. Our results also showed that the expression of A-01 was markedly induced by abiotic stresses, such as PEG and NaCl treatments (data not shown), suggesting that A-01 played important roles in scavenging the reactive oxygen species (ROS) and protecting cells against drought stress. EST analysis of A-02 showed that the fragment was a drought stress-related gene,
Table 2. Homology comparison of cloned genes. Sequence No. A-01 A-02 A-03 A-04 A-05 A-06 N-01 N-02 N-03
Length (bp) 226 141 350 327 201 291 159 173 175
Current annotation Oryza sativa metallothionein-like protein mRNA (AF001396.1) Oryza sativa drought stress related mRNA (CB964459.1) Zea mays partial mRNA for TFIIB-related protein (AJ295070.1) 55 Oryza sativa mRNA for calcium dependent protein kinaser 55 (AB078634) Oryza sativa protein kinase mRNA (AF004947) Unknown Oryza sativa early proembryo mRNA (AF454918.1) Unknown Arabidopsis thaliana GCN5-related N-acetyltransferase mRNA (NP 173946.1)
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Fig. 4. Effects of different nitrogen nutritions on anti-oxidative enzyme activities and GSH content. A, Ammonium nitrogen; N, Nitrate nitrogen; A/N, 50% ammonium and 50% nitrate nitrogen.
which was also reported by Babu [21] when separating unique genes from drought-stress rice seedlings. Interestingly, the two resistance-related genes were up-regulated in ammonium-fed rice leaves, indicating that it might be more suitable for rice plants cultivated in ammonium nitrogen than in nitrate nitrogen when being subjected to stresses. In our experiments, several physiological parameters were determined to investigate the resistance difference between ammonium- and nitrate-fed rice leaves. SOD, POD and CAT are the most important anti-oxidative enzymes, and the main function of these enzymes is to scavenge ROS generated in various physiological processes, thus preventing the oxidation of biological molecules. Glutathione is commonly accepted as a key antioxidative metabolite in plants and functions in coping with various stresses by scavenging ROS [22]. The results showed that the activities of CAT and POD and the GSH content were increased in ammonium-fed rice leaves compared with nitrate-fed ones, which might be one of the reasons why rice prefers ammonium nitrogen. This suggests that ammonium nutrition might help to improve the stress resistance of rice plants.
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4 5
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Raab T, Terry N. Nitrogen source regulation of growth and photosynthesis in Beta vulgaris L. Plant Physiol, 1994, 105: 1159í1166. Engels C, Marschner H. Influence of the form nitrogen supply on root uptake and translocation of cations in the xylem exudates of maize (Zea mays L.). J Exp Bot, 1993, 44: 1695í1701. Cramer M D, Lewis O A, Lips S H. Inorganic carbon fixation and metabolism in maize roots as affected by nitrate and ammonium nutrition. Physiol Plant, 1993, 89: 632í639. Stitt M. Nitrate regulation of metabolism and growth. Curr Opin Cell Biol, 1999, 2(3): 178í186. Wang R C, Okamoto M, Xing X, Crawford N M. Microarray analysis of the nitrate response in Arabidopsis roots and shoots over 1000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiol, 2003, 132: 556í567. Wang R C, Tischner R, Gutiérrez RA, Hoffman M, Xing X, Chen M, Coruzzi G, Crawford N M. Genomic analysis of the nitrate
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response using a nitrate reductase-null mutant of Arabidopsis. Plant Physiol, 2004, 136: 2512í2522. Zhang H, Forde B G. An Arabidopsis MADS box gene that controls nutrient induced changes in root architecture. Science, 1998, 179: 407í409. Foyer C H, Parry M, Noctor G. Markers and signals associated with nitrogen assimilation in higher plants. J Exp Bot, 2003, 54: 585–593. Yoshida S, Forno D A, Cock J H, Gomez K A. Laboratory Manual for Physiological Studies of Rice. 3rd edn. Manila: IRRI, 1976. Liang P, Averboukh L, Pardee A. Distribution and cloning of eukaryotic mRNAs by means of differential display: Refine- ments and optimization. Nucl Acids Res, 1993, 21: 3296í3275. Brant J B, Gustavo C A, Peter M G. Fast and sensitive staining of DNA in polyacrylamide gels. Anal Biochem, 1991, 196: 80í83. Zhang H, Zhang R, Liang P. Differential screening of gene expression difference enriched by differential display. Nucl Acids Res, 1996, 24: 2454í2455. Mou L, Miller H, Li J, Wang E, Chalifour L. Improvements to the differential display method for gene analysis. Biochem Biophys Res Comm, 1994, 199: 564í569. Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd edn. New York: Cold Spring Harbor Laboratory Press, 1989. Beauchamp C, Fridovich I. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal Biochem, 1971, 44: 276í286. Aebi H. Catalase in vitro. Methods Enzymol, 1984, 105: 121í126. Chance B, Maehly A C. Assay of catalase and peroxidases. Methods Enzymol, 1955, 2: 764í775. Ellman G L. Tissue sulfhydryl groups. Arch Biochem Biophys, 1959, 82: 70í77. Zhu G H, Zhuang C X, Wang Y Q, Jiang L R, Peng X X. Differential expression of rice genes under different nitrogen forms and their relationship with sulfur metabolism. J Integr Plant Biol, 2006, 48: 1177í1184. Cobbett C S, Goldsbrough P B. Phytochelatins and metallothioneins: Roles in heavy metal detoxification and home- ostasis. Annu Rev Plant Biol, 2002, 53: 159í183. Babu P R, Chandra S, Ithal N, Markandeya G, Reddy A R. Annotation and BAC/PAC localization of nonredundant ESTs from drought-stressed seedlings of an indica rice. J Genet, 2002, 81: 25í44. Alscher R G. Biosynthesis and antioxidant function of glutathione in plants. Physiol Plant, 1989, 77: 457–464.
Identification of Differentially Expressed Genes Induced by Ammonium Nitrogen in Rice Using mRNA Differential Display ZHU Guo-hui, HUANG Zhuo-lie (College of Life Sciences, South China Agricultural University, Guangzhou 510642, China)
Abstract: RNAs isolated from ammonium- and nitrate-treated rice leaves were used to screen differentially expressed genes through mRNA differential display. A total of 72 bands appeared significant differences and some of them were further confirmed by reverse Northern and Northern blot. The results showed that two genes, A-02 (Oryza sativa drought stress related mRNA) and A-03 (Zea mays partial mRNA for TFIIB-related protein) were highly up-regulated in the ammonium-fed rice leaves. The enzyme assays showed that the activities of the two anti-oxidative enzymes, catalase and peroxidase, and the content of a non-enzymic antioxidant, glutathione, were significantly higher in the ammonium-fed rice leaves than those in the nitrate-fed ones, indicating that the ammonium nutrition might be beneficial for rice plants to improve the stress resistance during growth and development. Key words: Oryza sativa; mRNA differential display; ammonium nitrogen; nitrate nitrogen; stress resistance
Nitrogen is an essential nutrient for plant growth and development, comprising most of biomolecules. Ammonium and nitrate are two major nitrogen forms acquired by plants. Studies in recent years have indicated that different nitrogen forms may lead to plant physiological differences, such as photosynthesis, respiration, ions uptake, enzyme metabolism and even stress resistance [1-3]. Generally, rice is considered as an ammonium-preferring plant, which grows better under ammonium than under nitrate nutrition. At present, much work on molecular mechanisms has been done to investigate the reasons of physiological difference under the nutrition of different nitrogen forms. Numerous studies have shown that nitrate serves not only as the nutrient element, but as the signal. Several approaches, such as nitrate reductase (NR)- deficient mutants or short-term treatment with low levels of nitrate, have been employed to screen the specific genes induced by nitrate signal [4-6], and the results show that the nitrate signal plays important roles in regulating plant root development, nutrient uptake, nitrogen and organic acid metabolism, etc [7-8]. However, among most reported nitrate signaling responses, it has not been identified that which is directly aroused by nitrate itself and which is influenced indirectly as a result of nitrate metabolism to ammonia. Compared with the interests of nitrate signal, considerably less attention have been paid to the ammonium-induced genes. In this study, mRNA differential display was applied to identify the differential expressions of genes under different nitrogen nutrition conditions by contrasting ammonium-fed plants to nitrate-fed ones, and some ammonium-inducible genes were gained. The study also tried to clarify the molecular mechanisms of rice physiological differences under ammonium Received: 10 March 2008; Accepted: 14 May 2008 Corresponding author: ZHU Guo-hui ([email protected]) This is an English version of the paper published in Chinese in Chinese Journal of Rice Science, Vol. 22, No. 3, 2008, Pages 261–265.
and nitrate nutrition.
MATERIALS AND METHODS Plant materials Seeds of rice (Oryza sativa L. cv. Xiangzhongxian 2) were sterilized with 3% NaClO for 20 min and then germinated in an incubator at 28ºC. When the lengths of radical roots were approximately 1.0 cm, seedlings were transferred to the greenhouse at day/night temperature of 29ºC/24ºC with a 14-h light and 10-h dark cycle in normal culture solution [9]. After 10 days culture, seedlings were divided into three groups to conduct the treatments by replacing the nitrogen element with various nitrogen forms: 1) 3.0 mmol/L nitrate exclusively (in the form of calcium nitrate); 2) 3.0 mmol/L ammonium exclusively (in the form of ammonium sulfate); 3) 3.0 mmol/L mixed nitrogen (1.5 mmol/L nitrate and 1.5 mmol/L ammonium). Leaves were sampled at 4, 12 and 36 h after the treatments. All the samples for RNA extraction were immediately frozen in liquid nitrogen and stored at –75ºC. In addition, Leaves under different nitrogen treatments for 3 days were collected for enzyme assays. mRNA differential display analysis Leaves treated with ammonium and nitrate respectively for 4 h were sampled for RNA extraction. Total RNA was extracted using the Trizol Reagent (Qiagen, Hamburg, Germany) and digested with DNase I (TaKaRa, Tokyo, Japan) to remove the residual DNA contamination. DNase-treated RNA samples were reverse transcribed in the presence of the anchored primers [10] as follows: H-T11A (5ƍ-AAGCTTTTTTTTTTTA-3ƍ), H-T11G (5ƍ-AAGCTTTTTTTTTTTG-3ƍ) and H-T11C (5ƍ-AAGCTTTT TTTTTTTC-3ƍ), respectively. Second strand synthesis and PCR
Rice Science, Vol. 15, No. 3, 2008
248 amplification were performed in a total volume of 20 μL. The reaction mixture contained 0.2 mmol/L dNTPs, 1 U of Taq polymerase (TaKaRa, Tokyo, Japan), 1 μmol/L anchored primer, 1 μmol/L random primer (Table 1) and 1 μL cDNA solution. PCR amplification was carried out with one cycle of 94ºC for 2 min, 40 cycles of 94ºC for 30 s, 40ºC for 2 min, 72ºC for 30 s, and one cycle of 72ºC for 5 min. PCR products were separated on a 6% denatured polyacrylamide gel with silver staining [11]. Selected bands were purified, precipitated and then used as the templates for the second PCR amplification. Each sample was re-amplified with the respective primers as described earlier. Positive analysis and gene cloning All the selected bands were further identified by reverse Northern blot [12-13]. The second PCR products were transferred to Hybond-N+ nylon membrane (Amersham, Piscataway, NJ, USA) after electrophoresis. The 5 μg RNA of ammonium-fed and nitrate-fed leaves were reversely transcribed respectively and used as probes. The probes were labeled with the random primer DNA labeling kit (TaKaRa, Tokyo, Japan) and hybridized with the membrane referred above. The positive bands proved by reverse Northern blot were subcloned into the pGEM-T easy vector (Promega, USA) and transformed to competent cells of DH10B. Positive recombinant clones were further sequenced, and DNA sequences were analyzed at the National Center for Biotechnology Information (NCBI; NIH, Bethesda, USA) using the BLASTN and BLASTX algorithms. Northern blot was carried out according to the methods of Sambrook et al [14]. Total RNA of rice leaves treated for 4, 12 and 36 h were subjected to electrophoresis on 1.2 % formaldehyde agarose gels and blotted to a nylon membrane (Hybond-N+, Amersham, Piscataway, NJ, USA). Hybridizations were performed overnight at 42ºC with the hybridization buffer containing 50% formamide, 5×SSC, 1×Denhardt’s solution, 1% SDS and 100 μg/ mL heat-denatured salmon sperm. After hybridization, the blots were washed in 2×SSC, 0.5% SDS solution at 42ºC and 65ºC respectively for 15 min, before in Table 1. Random primers of mRNA differential display. Primer name
Primer sequence
H-AP1 H-AP2 H-AP3 H-AP4 H-AP5 H-AP6 H-AP7 H-AP8 H-AP9 H-AP10 H-AP11 H-AP12 H-AP13 H-AP14 H-AP15 H-AP16
5ƍ-AAGCTTGATTGCC-3ƍ 5ƍ-AAGCTTCGACTGT-3ƍ 5ƍ-AAGCTTTGGTCAG-3ƍ 5ƍ-AAGCTTCTCAACG-3ƍ 5ƍ-AAGCTTAGTAGGC-3ƍ 5ƍ-AAGCTTGCACCAT-3ƍ 5ƍ-AAGCTTAACGAGG-3ƍ 5ƍ-AAGCTTTTACCGC-3ƍ 5ƍ-AAGCTTCATTCCG-3ƍ 5ƍ-AAGCTTCCACGTA-3ƍ 5ƍ-AAGCTTCGGGTAA-3ƍ 5ƍ-AAGCTTGAGTGCT-3ƍ 5ƍ-AAGCTTCGGCATA-3ƍ 5ƍ-AAGCTTGGAGCTT-3ƍ 5ƍ-AAGCTTACGCAAC-3ƍ 5ƍ-AAGCTTTAGAGCG-3ƍ
0.2×SSC, 0.2% SDS solution at 65ºC for 15 min. Hybridization signals were analyzed using the Molecular Imager FX-Plus (Bio-Rad, Ivry sur Seine, France). Assay of enzyme activities Rice leaves were homogenized with 50 mmol/L chilled phosphate buffer (pH 7.0). The homogenate was centrifuged and the supernatants were used for enzyme assays. Superoxide dismutase (SOD) activity was estimated according to the method of Beauchamp and Fridovich [15], and expressed as U/mg (One unit of enzyme is defined as the amount of enzyme which produces a 50% inhibition of NBT reduction under assay conditions). Catalase (CAT) activity was assayed from the rate of H2O2 decomposition following the procedure of Aebi [16], and expressed as ǻA240/(mg·min). Peroxidase (POD) activity was measured according to Chance et al [17] and expressed as ǻA470/(mg·min). To estimate reduced glutathione (GSH) content, the fresh leaves were homogenized in 5% chilled trichloroacetic acid and the supernatants were used for determination according to the method of Chance et al [17].
RESULTS mRNA differential display analysis PCR were performed with 48 primer combinations (16 of random primers vs 3 of anchored primers). Amplified products were analyzed by electrophoresis on 6% denatured polyacrylamide gels (Fig. 1) and each lane had 50–100 bands. Ultimately, 72 bands were shown to have unique expressions responding to each treatment. Positive analysis and gene cloning Reverse Northern was generally used for high throughput screening for positive clones. Here, differentially displayed bands were equally transferred to Hybond-N+ nylon membrane after electrophoresis, and hybridized with cDNA probes reversely transcribed with RNA extracted from ammonium-fed and nitrate-fed leaves, respectively (Fig. 2). As shown in Fig. 2, the majority of bands were considered to be the false positive bands. The positive bands
Fig. 1. Results of mRNA differential display. A, Samples treated with ammonium nitrogen; N, Samples treated with nitrate nitrogen. Differential bands are indicated by arrows.
ZHU Guo-hui et al. Identification of Differentially Expressed Genes Induced by Ammonium Nitrogen in Rice
249
Fig. 3. Northern blot analysis of two differentially expressed genes. A, Ammonium nitrogen; N, Nitrate nitrogen; A/N, 50% ammonium and 50% nitrate nitrogen. Fig. 2. Reverse Northern blot of differential bands. A, Hybridized with ammonium-treated cDNA probe; B, Hybridized with nitrate-treated cDNA probe. Arrows indicate positive clones.
which showed differential hybridization signals between the two membranes were then cloned to the pGEM-T vector. Sequencing results and homologous analysis are shown in Table 2. Two clones strongly expressed in ammonium-fed rice leaves were further confirmed by Northern blot (Fig. 3). Gene of A-01 (Oryza sativa metallothionein-like protein mRNA), which encodes a rice metallothione protein, had been proved to be remarkably induced by ammonium nitrogen in previous study [19]. The hybridization showed that the expression profile of A-02 (Oryza sativa drought stress related gene), regulated by nitrogen forms, was the highest in the ammonium- fed rice leaves, weaker in the mixed nitrogen-fed ones, and the weakest in the nitrate-fed ones, indicating that the expression of A-02 was not only induced by ammonium nitrogen, but had a postive correlation with ammonium content. The expression profile of A-03 (Zea mays partial mRNA for TFIIB-related protein) was also induced by ammonium nitrogen, which increased with the time for ammonium treatment, whereas maitained stable in nitrate-fed rice leaves. Analysis of rice anti-oxidative enzymes under different nitrogen forms Genes of A-01 and A-02, reported as resistance- related genes, were both up-regulated by ammonium nitrogen. To identify whether it can be the implication that rice would have
stronger stress resistant ability under ammonium nutrition than nitrate nutrition, we analyzed the activities of several antioxidative enzymes such as SOD, POD, CAT and the contents of an important antioxidant GSH in both ammonium- and nitrate-fed rice leaves (Fig. 4). The results showed that ammonium nitrogen enhanced the activities of antioxidative enzymes and the contents of nonenzymic antioxidants. The CAT and POD activities and the GSH content were increased by 91.5%, 37.9% and 63.6% under ammonium nutrition compared with under nitrate nutrition, respectively. Whereas it was no obvious differences between ammonium and ammonium-nitrate mixed nutrition.
DISCUSSION Several ammonium-induced genes were screened by mRNA differential display in this study. A-01 was highly homologous to rice metallothionein (MT)-like mRNA encoding metallothionein. MTs are defined as a class of proteins with low molecular mass, high cysteine content and response to various stresses including drought, salt, and cold [20]. Our results also showed that the expression of A-01 was markedly induced by abiotic stresses, such as PEG and NaCl treatments (data not shown), suggesting that A-01 played important roles in scavenging the reactive oxygen species (ROS) and protecting cells against drought stress. EST analysis of A-02 showed that the fragment was a drought stress-related gene,
Table 2. Homology comparison of cloned genes. Sequence No. A-01 A-02 A-03 A-04 A-05 A-06 N-01 N-02 N-03
Length (bp) 226 141 350 327 201 291 159 173 175
Current annotation Oryza sativa metallothionein-like protein mRNA (AF001396.1) Oryza sativa drought stress related mRNA (CB964459.1) Zea mays partial mRNA for TFIIB-related protein (AJ295070.1) 55 Oryza sativa mRNA for calcium dependent protein kinaser 55 (AB078634) Oryza sativa protein kinase mRNA (AF004947) Unknown Oryza sativa early proembryo mRNA (AF454918.1) Unknown Arabidopsis thaliana GCN5-related N-acetyltransferase mRNA (NP 173946.1)
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Fig. 4. Effects of different nitrogen nutritions on anti-oxidative enzyme activities and GSH content. A, Ammonium nitrogen; N, Nitrate nitrogen; A/N, 50% ammonium and 50% nitrate nitrogen.
which was also reported by Babu [21] when separating unique genes from drought-stress rice seedlings. Interestingly, the two resistance-related genes were up-regulated in ammonium-fed rice leaves, indicating that it might be more suitable for rice plants cultivated in ammonium nitrogen than in nitrate nitrogen when being subjected to stresses. In our experiments, several physiological parameters were determined to investigate the resistance difference between ammonium- and nitrate-fed rice leaves. SOD, POD and CAT are the most important anti-oxidative enzymes, and the main function of these enzymes is to scavenge ROS generated in various physiological processes, thus preventing the oxidation of biological molecules. Glutathione is commonly accepted as a key antioxidative metabolite in plants and functions in coping with various stresses by scavenging ROS [22]. The results showed that the activities of CAT and POD and the GSH content were increased in ammonium-fed rice leaves compared with nitrate-fed ones, which might be one of the reasons why rice prefers ammonium nitrogen. This suggests that ammonium nutrition might help to improve the stress resistance of rice plants.
REFERENCES 1
2
3
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