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Expression analysis of the glutamine synthetase and glutamate synthase gene families in young rice (Oryza sativa) seedlings
Plant Science 170 (2006) 748–754 www.elsevier.com/locate/plantsci
Expression analysis of the glutamine synthetase and glutamate synthase gene families in young rice (Oryza sativa) seedlings Xue-Qiang Zhao a,b, Wei-Ming Shi a,* a
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China b Graduate School of Chinese Academy of Sciences, Beijing 100081, China Received 10 August 2005; received in revised form 2 November 2005; accepted 12 November 2005 Available online 5 December 2005
Abstract Glutamine synthetase (GS) and glutamate synthase (GOGAT) serve for primary assimilation of N in higher plants. When NH4+ is the major inorganic N source, rice plants must assimilate NH4+ quickly in roots through the GS/GOGAT cycle to ameliorate the toxic effect of excess NH4+. The sequence of the rice genome is almost complete, facilitating the identification of the GS and GOGAT gene families in this species. Thus, we investigated the different members of GS and GOGAT genes, and analyzed the pattern of expression of each gene in young rice seedlings by quantitative real-time PCR, revealing a distinct expression pattern for these genes. OsGln1;1 and OsGln1;2 mainly functions in roots, and OsGln2 and OsGlu1 are preferentially expressed in leaves. However, transcriptions of OsGln1;1, OsGln1;2, OsGln2 and OsGlu1 in leaves are all increased by increased N level while those in roots not influenced or even decreased. OsGlt1 and OsGlt2 are expressed primarily in roots when N is limiting, but in leaves when non-limiting. Transcription of OsGlt1 is decreased, but OsGlt2 is increased by increased N level in roots and leaves. When rice roots are exposed to NO3 and NH4+ for 2 h after N-starvation, transcriptions of OsGln1;1, OsGlt1, OsGlt2 and OsGlu1 are all repressed by NO3 and NH4+. OsGln1;2 expression shows significant up-regulation by NH4+ and down-regulation by NO3 while OsGln2 down-regulation by NH4+ and up-regulation by NO3. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Gene expression; Glutamate synthase; Glutamine synthetase; Real-time PCR; Rice seedlings
1. Introduction Nitrogen is an essential element for plant growth. NH4+ and NO3 are initially taken up by root cells from the soil solution in processes mediated by AMT (ammonium transporter) and NRT (nitrate transporter), and previous reports have described AMT and NRT in rice [1–4]. Then plants assimilate these inorganic N forms into amino acids through the GS/ GOGAT cycle. Either from a direct uptake by plant roots or produced by reduction of nitrate, NH4+ is first assimilated through the GS/ GOGAT cycle defined by Lea and Miflin [5]. GS catalyzes synthesis of Gln from NH4+ and Glu in an ATP-dependent
Abbreviations: GS, glutamine synthetase; GOGAT, glutamate synthase * Corresponding author at: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, East Beijing Road, No. 71, Nanjing 210008, China. Tel.: +86 25 86881566; fax: +86 25 86881000. E-mail address: [email protected] (W.-M. Shi). 0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.11.006
manner. GOGAT catalyzes reductive transfer of the amide group of Gln to 2-oxogluarate to form two Glu molecules [6]. In higher plants, there are two major isoforms of GS: cytosolic GS (GS1), which occurs in the cytosol, and chloroplastic GS (GS2), which, although nuclear encoded, is located in the chloroplasts or plastids [7–9]. In all plants studied, GS2 is encoded by a single gene, whereas GS1 belongs to a small gene family. Distinct roles for these two enzymes have been suggested by a number of studies on organ, tissue, and development [7–10]. Similarly, Fd-GOGAT and NADHGOGAT are two major forms in most species based on whether they use NADH (NADH-GOGAT) or reduced ferredoxin (FdGOGAT) as the electron donor for the (two-electron) conversion of L-glutamine plus 2-oxoglutarate to L-glutamate [7,9–13]. These observations provided the rationale for us to study the role of the GS/GOGAT cycle in rice. Immunocytochemical localization of GS and GOGAT has been studied extensively in most plant species, especially in alfalfa [14], rice [15–17] and Arabidopsis [18]. NADH-
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GOGAT is responsible for synthesis of glutamate from glutamine that is transported from senescing tissues to the spikelets in rice [19]. Transgenic tobacco plants that overexpress alfalfa NADH-GOGAT have increased carbon and N content [20]. Over-production of NADH-GOGAT in transgenic rice advocates that NADH-GOGAT is indeed a key step for N utilization and grain filling in rice [21]. Modification of N assimilation efficiency has been approached recently in trees by overexpression of a cytosolic pine GS in poplar [22]. Plastidic GS2 encoded by the GLN2 gene facilitates NH4+ recovery during photorespiration in both leaf mitochondria and chloroplasts [7,8]. Although these biochemical and molecular biological studies provide a solid groundwork in most plant species, a systematic picture of the N-assimilation process and its regulation in a single plant is still in need. Rice, growing in paddy fields, uses NH4+ as its major source of N. Because of the toxicity of NH4+ in plants, NH4+ must be assimilated quickly in rice roots [15]. Therefore, NH4+ uptake and metabolism in plants must be tightly regulated. The GS/ GOGAT cycle plays an important role in anaerobic amino acid accumulation in rice roots [23]. Although good progress has been made to dissect and better understand both the major steps and the regulation of inorganic N assimilation in higher plants, the role of alternative metabolic pathways that are potentially able to incorporate NH4+ into organic molecules is still not fully understood. Moreover, regulation and control of NH4+ uptake and assimilation by the GS and GOGAT gene families remains perplexing to this day. Furthermore, much less is known about how the expression of these genes is regulated and under what conditions they execute their functions. Considering great amount of basal N fertilizer but lower uptake ability at young rice seedling stage, it is essential for us to explore the molecular basis of N efficiency of young rice seedlings in order to improve N use efficiency of basal N fertilizer and reduce N pollution. Therefore, the expression patterns of GS and GOGAT gene families were studied mainly at young rice seedling stage in the present study. To better understand and to systematically clarify the role and regulation of the GS and GOGAT gene families in young rice seedlings, we investigated different members of the GS and GOGAT genes, and analyzed the pattern of expression of each gene in different plant organs and in plants grown with different sources of N by quantitative real-time PCR.
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2. Materials and methods 2.1. Plant materials and growth conditions Rice (Oryza sativa) plants were cultivated in a growth chamber controlled at 25 8C with 70% relative humidity under a 14 h light and 10 h dark cycle. The light intensity was 300 mmol photon m2 s1. Rice plants were first grown hydroponically in a modified Kimura B nutrient solution [24] before treated, which contained NH4NO3 (0.5 mM), NaH2PO42H2O (0.18 mM), KCl (0.55 mM), CaCl22H2O (0.36 mM), MgSO47H2O (0.6 mM), FeSO47H2O-EDTA (20 mM), H3BO3 (50 mM), MnCl24H2O (9 mM), CuSO45H2O 5H2O (0.3 mM), ZnSO47H2O (0.7 mM), and H2MoO44H2O (0.1 mM). Because co-provision of NH4+ and NO3 could improve rice growth [25,26], NH4NO3 was used for the N form in the pre-culture cultivated stage. Nitrification was inhibited by 5.89 mg L1 C2H4N4 (Shanghai Chemical Co. www.reagent.com.cn). The pH of growth media was maintained at 5.5 by adding diluted NaOH or HCl daily. Three replicates were carried out for each procedure. For RNA extraction, plant materials were harvested, frozen quickly in liquid nitrogen, and stored at 80 8C. 2.2. Total RNA extraction and cDNA synthesis Total RNA was extracted by Guanidine Isothiocyanate (Nanjing Sunshine Biotechnology Co. Ltd.) methods [27]. Five micrograms total RNA was used to synthesize cDNA by reverse transcriptase powerscriptTM (BD Bioscience Clontech www.bdbiosciences.com) following the manufacturer’s protocol. The cDNA samples were used as templates to quantify target gene expression levels. 2.3. Quantitative real-time PCR Quantitative real-time PCR analysis was carried out using gene-specific primers (Table 1). Primers were designed according to the sequences of BAC clones (Table 2). Constitutive expression of OsActin (NCBI/GenBank accession number XM_469569) was determined to confirm the equality of molecular copies. The PCR products were detected as SYBR Green I fluorescence using the DNA Engine Opticon 2 System (MJ,
Table 1 Gene-specific primers used for the real-time PCR analysis Gene
Forward primer
Reverse primer
OsGln1;1 OsGln1;2 OsGln1;3 OsGln2 OsGlt1 OsGlt2 OsGlu1 OsGlu2 OsGlu2 OsActin
50 -CAAGTCCGCCATTGAGAAGC-30 50 -GGTTGGAGGATCGGGCATAG-30 50 -AGCCGATTCCGACGAACAAC-30 50 -ACCAAGAGTATGCGTGAAGA-30 50 -GGAGGGAAATCTAATACAGG-30 50 -AGACAAACAATTTCCCTGAG-30 50 -AAACAGGCAGCGAGAAAGGT-30 50 -TCCAATAGGACCAATACAGA-30 50 -CAAACCGTCCTGTCAATGTG-30 50 -CTTCATAGGAATGGAAGCTGCGGGTA-30
50 -CTTGCCGTTCTGCTCCGTCT-30 50 -TCACCTTGTGGCGTGTAGCA-30 50 -GTAGCGTGCCACCCAGACAT-30 50 -AACCTGTCAACCTCCTTTCA-30 50 -AGTTCATCAGCGTTAGTCAG-30 50 -TAAAGGGTCACTTCCAACAT-30 50 -ACTCGTTCAAACTCGGCACA-30 50 -ATCAACAACATCTCACCACC-30 50 -TGGAACTTGTGGCAGCGTCT-30 50 -CGACCACCTTGATCTTCATGCTGCTA-30
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Table 2 Summary of GS and GOGAT gene families in rice Gene name
Product
BAC clone accession number
Chromosome location
OsGln1;1 OsGln1;2 OsGln1;3 OsGln2 OsGlt1 OsGlt2 OsGlu1 OsGlu2
GS1 GS1 GS1 GS2 NADH-GOGAT NADH-GOGAT Fd-GOGAT Fd-GOGAT
AP004880 AC105364 AC082645 AL662953 AP004363 AC104709 AP003833 AP004343
chr02 chr03 chr03 chr04 chr01 chr05 chr07 chr07
USA, www.mjr.com) for continuous fluorescence detection. Gene copies were determined quantitatively by using a purified cDNA clone as standard.
NADH-GOGAT genes in rice (Table 2). Results reported here for the GS gene family are consistent with the report of Ishiyama et al. [17].
2.4. Statistical analysis
3.2. Expression of GS and GOGAT gene families at different N concentrations
Statistical analysis was conducted using procedures in Excel software (Microsoft Office Excel 2003). Mean comparison was calculated according to Duncan Multiple Range test using the Statistical Analysis System (SPSS 11.5). 3. Results 3.1. The GS and GOGAT gene families in rice In order to establish the genes encoding GS and GOGAT in rice, we identified GS and GOGAT gene families in rice through a complete homologous blast search on the internet (http://www.ncbi.nlm.nih.gov/blast/, http://rgp.dna.affrc.go.jp/ blast/runblast.html, and http://www.ddbj.nig.ac.jp/search/ blast-j.html) with a known gene sequence as a probe and named them as shown in Table 2. The search identified three GS1 genes, a single GS2 gene, two Fd-GOGAT genes and two
Plant GS and GOGAT genes studied previously are very important in N assimilation, but the systematic expression patterns of the GS and GOGAT genes families have not yet been clearly established. Thus, we examined the level of expression of each member of the GS and GOGAT gene families at two different N concentrations using quantitative real-time PCR. Rice plants were grown under two different nutritional conditions. In the first, corresponding to limiting conditions, plants were supplied with 0.1 mM NH4NO3. The second set of plants, cultivated under non-limiting conditions, was fed with 2 mM NH4NO3. The relative and absolute values for gene expression in roots and leaves are shown in Fig. 1. Although we employed several different RT-PCR conditions, we were unable to detect any amplification of OsGln1;3 and OsGlu2 at either N concentration. For the other genes, the
Fig. 1. Expression profiles of individual GS and GOGAT genes. For each gene, the relative amounts of mRNA in roots (open bars) and leaves (black bars) were added and then expressed as a percentage of the sum. Values above the columns were the absolute mRNA contents (amol/mg total RNA) of each individual gene in roots or leaves. After rice plants were grown under full nutrient solutions containing 0.5 mM NH4NO3 for 15 days, rice seedling were treated with limiting (0.1 mM NH4NO3, A) or non-limiting (2 mM NH4NO3, B) N for 10 days. A 25-day-old rice plants (five-leaf stage) were harvested and analyzed.
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levels of transcripts in different organs added were calculated relative to the OsActin gene. The relative amount of mRNA expressed by these genes in each organ is shown in Fig. 1 as a percentage of the resulting sum. When rice plants were cultivated at 0.1 mM NH4NO3 for 10 days, four of the genes, OsGln1;1, OsGln1;2, OsGlt1, and OsGlt2, showed a markedly preferential expression in roots, whereas OsGln2 and OsGlu1 were expressed mainly in leaves (Fig. 1A). When rice plants were cultivated at 2 mM NH4NO3 for 10 days, results similar to those in Fig. 1A were found except for OsGlt1 and OsGlt2 (Fig. 1B). OsGlt1 and OsGlt2 were expressed mainly in leaves at 2 mM NH4NO3 (Fig. 1B) but mainly in roots when the nitrogen supply was decreased to 0.1 mM NH4NO3 (Fig. 1A). Changes of transcriptions about absolute values of mRNA contents (values above different columns in Fig. 1) between 0.1 and 2 mM NH4NO3 are also compared. Two classes of genes can be distinguished by the changes of absolute mRNA contents (Fig. 1). The first class consisted of OsGln1;1 and OsGlt1; the amounts of their mRNAs seemed to be decreased in both roots and leaves from 0.1 to 2 mM NH4NO3. The second, including OsGln1;2, OsGlt2, OsGln2 and OsGlu1, showed a decrease in roots but a increase in leaves from 0.1 to 2 mM NH4NO3.
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those in leaves seemed to be slightly up-regulated by increased N (Fig. 2), which was almost in accordance with the results in Fig. 1. Furthermore, it was interesting that transcription of OsGlt1 was strongly repressed by increased N in both roots and leaves, whereas that of OsGlt2 was significantly induced by increased N in both roots and leaves. According to the results in Fig. 1, expression of OsGlt1 and OsGlt2 should be much higher in leaves than in roots in 2 mM NH4NO3 in Fig. 2, whereas this was not the case in Fig. 2, which was possibly caused by a treatment of 2 mM NH4NO3 for a shorter time (only 6 h) in Fig. 2 while rice seedlings were grown in 2 mM NH4NO3 for 10 days in Fig. 1. 3.4. Influence of NO3 and NH4+ supply on GS and GOGAT gene expression of rice roots
The N supply is a major regulator of GS and GOGAT expression. Fig. 2 shows, for each of the six genes that could be tested, the variations of transcript levels within roots and leaves between treated (with 2 mM NH4NO3) and non-treated (no NH4NO3) rice plants. The mRNA contents of OsGln1;1, OsGln1;2, OsGln2 and OsGlu1 in roots exhibited no significant differences between treated and non-treated conditions, but
NO3 and NH4+ are the two major sources of inorganic N for higher plants. Therefore, we also compared the effect of NO3 to that of NH4+ on expression of GS and GOGAT genes in rice roots by short-time treatments. Rice plants were cultivated under full nutrient solutions for 28 days. After N-starvation for 48 h, some were treated with 1 mM NH4+ in the form of 0.5 mM (NH4)2SO4, and the others were treated with 1 mM NO3 in the form of 1 mM KNO3. Changes of transcription in rice roots with different N sources are illustrated in Fig. 3. Three classes of genes can be distinguished. The first class consisted of OsGln1;1, OsGlt1, OsGlt2 and OsGlu1 genes; all seemed to be significantly down-regulated by both NO3 and NH4+. The second, corresponding to the OsGln1;2 gene, showed significant up-regulation by NH4+ and down-regulation by NO3. The last class, corresponding to the OsGln2 gene, exhibited regulation opposite to that observed with OsGln1;2, i.e., down-regulation by NH4+ and up-regulation by NO3. We could also find that there were important differences in mRNA levels between Figs. 2 and 3, specially for OsGln1;2
Fig. 2. Effects of N conditions on GS and GOGAT gene expression. After rice plants were grown under full nutrient solutions containing 0.5 mM NH4NO3 for 15 days, rice seedling were pre-cultured with 0.1 mM NH4NO3 for 10 days. Finally, 25-day-old rice plants (five-leaf stage) were treated with 2 mM NH4NO3 for 6 h (black bars) or not treated (open bars) and then 25-day-old rice plants were harvested and analyzed. Means of independent triplicate samples and S.D. values (n = 3) are indicated. Different letters above the columns indicate statistically significantly differences at P 0.05.
Fig. 3. Gene expressions of GS and GOGAT in rice roots in response to NO3 and NH4+ supply. Rice plants were grown under full nutrient solutions containing 0.5 mM NH4NO3 for 28 days. After nitrogen-starvation for 48 h, 28-day-old rice seedlings (five to six-leaf stage) were exposed to 1 mM NH4+ (gray bars) or 1 mM NO3 (black bars) or no N (open bars) for 2 h and then rice roots were harvested and analyzed, respectively. Means of independent triplicate samples and S.D. values (n = 3) are indicated. Different letters above the columns indicate statistically significantly differences at P 0.05.
3.3. Influence of increased N levels on GS and GOGAT gene expression
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and OsGln2. Different N nutrition conditions of rice plants in Figs. 1 and 2 possibly resulted in these differences. Compared to the mRNA levels of OsGln1;2 and OsGln2 in Fig. 2, excessive nitrogen-starvation of rice plants may greatly reduce the mRNA levels of OsGln1;2 and OsGln2 in Fig. 3. 4. Discussion It was thought previously that OsGln1;2 (GSr) is the only GS1 isoenzyme expressed in rice roots [28]. However, our study shows that the GS1 gene family in rice is comprised of three different genes located on chromosomes II and III. The GS2 gene in rice is encoded by a single gene located on chromosome IV, as reported by Ishiyama et al. [17]. The results reported here also indicate that both NADH-GOGAT and FdGOGAT are each encoded by two genes. These two genes encoding NADH-GOGAT are located on chromosomes I and V, and both of the genes encoding Fd-GOGAT are located on chromosome VII. In higher plants, the organ specificity of GS and GOGAT gene expression might depend on the species. Most of the previous studies have used the northern technique. Because of the high degree of homology between the paralogs, the northern technique does not allow specific detection of each gene transcript and, moreover, is not sensitive enough to unravel gene expression at very low levels [29]. Real-time PCR provides a very highly sensitive and reproducible technique that can detect gene expression at very low levels. For example, both OsGln1;1 and OsGln1;2 are expressed mainly in roots (Fig. 1), but using real-time PCR, we have found that the absolute mRNA contents of OsGln1;2 (401.50 amol/mg total RNA) is far larger than that of OsGln1;1 (119.32 amol/mg total RNA) in rice roots under low N conditions (Fig. 1). Regardless of limiting or non-limiting N conditions, OsGln1;1 and OsGln1;2 exhibit a strong root preferential pattern of expression, whereas OsGln2 shows a preference for leaves (Fig. 1). This study also suggests that the OsGln1;1 and OsGln1;2 proteins perform their functions in roots, but the OsGln2 protein performs theirs in leaves. GS2 is expressed predominantly in green tissues. It has been proposed that GS2 plays an important role in the assimilation of ammonia from nitrate reduction, and it has also been demonstrated that GS2 is indispensable for re-assimilation of photorespiratory ammonia [30–32]. Similar results for OsGln1;1, OsGln1;2 and OsGln2 were reported by Ishiyama et al. [17]. When rice seedlings were transferred from 0.1 to 2 mM NH4NO3 for 6 h, the mRNA contents of OsGln1;1, OsGln1;2 and OsGln2 exhibited no significant differences in rice roots (Fig. 2), but those in leaves seemed to be up-regulated by increased N (Figs. 1 and 2). All of these suggest that, when outer N concentrations in soil are increased, OsGln1;1, OsGln1;2 and OsGln2 possibly play more important roles in leaves relative to roots. Thus, increased inorganic N in rice roots can be fast transport to rice leaves and assimilated into Gln or other N compounds in rice leaves, and so rice growth and development are improved by this kind of N assimilative mechanism.
The results [17,18] support the importance of OsGln1;1 in promoting rapid conversion of ammonium to Gln at the surface cell layers that are in contact with soils particularly under lowammonium conditions. In contrast to OsGln1;1, the OsGln1;2 transcript accumulates abundantly in the surface cell layers after ammonium treatment. Apparently, OsGln1;1 and OsGln1;2 are regulated oppositely by ammonium in these specific cell types at the mRNA level [17]. In the present study, transcription of OsGln1;1 in rice roots is strongly repressed by nitrate and ammonium (Fig. 3). However, OsGln1;2 is highly up-regulated by ammonium but repressed by nitrate. As for OsGln2, it appears to be repressed by ammonium but slightly up-regulated by nitrate. The OsGln1;1, OsGln1;2 and OsGln2 transcripts show reciprocal responses to nitrate and ammonium supply in rice roots. Plants have already developed an intrinsic regulation mechanism to adapt themselves to uneven Ndistribution environments and utilize mineral nutrition efficiently. The OsGln1;1, OsGln1;2 and OsGln2 transcripts show different responses to nitrate and ammonium supply. Thus, rice plants can efficiently assimilate N in rice roots by this kind of flexible and reciprocal regulative mechanism, and control rice growth and development. With regard to OsGlt1 and OsGlt2, they are expressed in roots under limiting N conditions but in leaves under nonlimiting N conditions (Fig. 1). Therefore, expression of the genes encoding NADH-GOGAT possibly changes with external N conditions. Growth of rice plants can be regulated to be adaptive to outer heterogeneous N conditions in soil by this kind of changes. When N level is higher in soil environments, expression of OsGlt1 and OsGlt2 can be decreased in roots in order to limit N acquisition, but in the opposite side, expression of OsGlt1 and OsGlt2 can be enhanced in roots in order to increase N acquisition. This may be a kind of buffering effects in higher plants. Our results also show that transcription of OsGlt1 is decreased in roots and leaves by increased N levels but transcription of OsGlt2 is increased in roots and leaves (Fig. 2). These results suggest that OsGlt1 possibly performs functions mainly under low N conditions while OsGlt2 primarily under high N conditions. As like OsGln1;1, OsGln1;2 and OsGln2, the OsGlt1 and OsGlt2 transcripts also show reciprocal responses to increased N supply in roots and leaves. This is very useful for plants to adapt outer nutrient heterogeneity in soil. Previous studies show that the mRNA and protein for NADH-GOGAT accumulate markedly in whole roots or the root-tip sections of rice plants within 12 h supplying a low concentration of NH4Cl [15,33,34]. A similar response is seen in rice cells in suspension culture [35]. However, OsGlt1 and OsGlt2 are both repressed in rice roots by a short-time treatment of nitrate and ammonium in the present study (Fig. 3). The present results do not reflect previous reports showing clear ammonium inducibility [15,33–35] of NADH-GOGAT in rice roots. Rice seedlings by previous reports were grown in water for 26 days before treated, but seedlings in the present study were only N-starvation for 48 h before treated. Rice growth for a long time in water must result in exhausting of N in rice
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seedlings and reducing of NADH-GOGAT activity, so, when ammonium is supplied at the moment, the mRNA and protein for NADH-dependent glutamate synthase are increased dramatically in roots of rice seedlings in order to assimilate ammonium. However, in the present study, rice seedlings of Nstarvation for a short time possibly induce expression of genes encoding NADH-GOGAT in order to acquire more N, thus, when ammonium and nitrate are supplied to rice roots, the mRNA contents for NADH-dependent glutamate synthase are decreased dramatically in roots of rice seedlings. The OsGlu1 gene encoding Fd-GOGAT shows a pattern of expression similar to OsGln2. In spite of limiting or nonlimiting N conditions, OsGlu1 is expressed mainly in rice leaves (Fig. 1), and transcription of OsGlu1 in leaves is significantly up-regulated by increased N (Figs. 1 and 2) while it is repressed by nitrate and ammonium in rice roots (Fig. 3). Therefore, Gln can be transferred to 2-oxogluarate to form Glu by the transcription of OsGlu1 in rice leaves. However, in rice roots, the OsGlt1 and OsGlt2 encoding NADH-GOGAT not OsGlu1 possibly perform such functions in the transfer of the amide group of Gln to 2-oxogluarate to form two Glu molecules. In maize roots, Fd-GOGAT and GS2 are induced within 30 min by 10 mM nitrate (with or without cycloheximide) and within 2 h by 10 mM nitrate [36]. In another study using detached maize leaves, GS2, Fd-GOGAT, and NADH-GOGAT are all induced by 16 mM nitrate after 2 h [37]. In tobacco NR mutants, GS1, GS2, and Fd-GOGAT transcripts were found to be more plentiful in plants grown with 12 mM nitrate than in plants grown with 0.2 mM nitrate [38]. However, in the present study, the transcription of OsGlu1 encoding Fd-GOGAT is repressed by nitrate supply. The results presented here in this study provide an unknown role of nitrate as a negative regulator of Fd-GOGAT genes in rice roots. The reason for different regulatory mechanism of Fd-GOGAT in rice should be further studied in the future. It should be pointed out that OsGln1;3 and OsGlu2 were not detected under the conditions used in this study. We suggest that these two genes might be pseudogenes in rice. Lack of amplification of OsGln1;3 in this study confirms previous reports [15]. There are previous reports [39] that Fd-GOGATs are immunologically distinct proteins in rice roots and leaves. Two sets of different primers have been used for OsGlu2 PCR attempts of amplification in the present study (Table 1). However, we still cannot get amplification of OsGlu2 by our efforts. In addition, we also fortunately find that OsGlu2 is marked for pseudogene in rice gene bank (AP004343, http:// www.ncbi.nlm.nih.gov/), and it is indicated to be pseudogene and probably inactive due to including stop codon(s) in cDNA. Such a lot of expression patterns for the genes encoding GS and GOGAT gene families have been examined in the present study. It will be very interesting to use reporter genes or green fluorescent protein to follow the fine distribution of each GS and GOGAT gene at the cellular level. From the results of this preliminary study, it is difficult to speculate on the role of each of these genes in N assimilation. However, it is tempting to associate a particular pattern of expression and a putative function. Work is in progress in our
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laboratory to identify the function of these genes individually by the RNAi technique. Acknowledgements This investigation was supported financially by a grant from the National Natural Science Foundation of China (No. 30390083). We also thank enthusiastically Dr. J. Yan of Eastwin Life Sciences Inc. for technical guidance. References [1] C.M. Lin, S. Koh, G. Stacey, S.M. Yu, T.Y. Lin, Y.F. Tsay, Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice, Plant Physiol. 122 (2000) 379–388. [2] A. Kumar, S.N. Silim, M. Okamoto, M.Y. Siddiqi, A.D.M. Glass, Differential expression of three members of the AMT1 gene family encoding putative high-affinity NH4+ transporters in roots of Oryza sativa subspecies indica, Plant Cell Environ. 26 (2003) 907–914. [3] Y. Sonoda, A. Ikeda, S. Saiki, N.V. Wire´n, T. Yamaya, J. Yamaguchi, Distinct expression and function of three ammonium transporter genes (OsAMT1;1–1;3) in rice, Plant Cell Physiol. 44 (2003) 726–734. [4] A. Suenaga, K. Moriya, Y. Sonoda, A. Ikeda, N.V. Wire´n, T. Hayakawa, J. Yamaguchi, T. Yamaya, Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants, Plant Cell Physiol. 44 (2003) 206–211. [5] P.J. Lea, B.J. Miflin, Alternative route for nitrogen assimilation in higher plants, Nature 251 (1974) 614–616. [6] N. Hirose, T. Yamaya, Okadaic acid mimics nitrogen-stimulated transcription of the NADH-glutamate synthase gene in rice cell cultures, Plant Physiol. 121 (1999) 805–812. [7] J. Harrison, N. Brugiere, B. Phillipson, S. Ferrario-Mery, T. Becker, M.A. Limami, B. Hirel, Manipulating the pathway of ammonia assimilation through genetic engineering and breeding. Consequences on plant physiology and plant development, Plant Soil 221 (2000) 81–93. [8] B.J. Miflin, D.Z. Habash, The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops, J. Exp. Bot. 53 (2002) 979–987. [9] A. Weber, U.I. Flugge, Interaction of cytosolic and plastidic nitrogen metabolism in plants, J. Exp. Bot. 53 (2002) 865–874. [10] M. Lancien, P. Gadal, M. Hodges, Enzyme redundancy and the importance of 2-oxoglutarate in higher plant ammonium assimilation, Plant Physiol. 123 (2000) 817–824. [11] S.J. Temple, C.P. Vance, J.S. Gantt, Glutamate synthase and nitrogen assimilation, Trends Plant Sci. 3 (1998) 51–56. [12] P.J. Lea, B.J. Miflin, Glutamate synthase and the synthesis of glutamate in plants, Plant Physiol. Biochem. 41 (2003) 555–564. [13] A. Suzuki1, D.B. Knaff, Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism, Photosynth. Res. 83 (2005) 191–217. [14] G.B. Trepp, D.W. Plank, J.S. Gantt, C.P. Vance, NADH-glutamate synthase in alfalfa root nodules. Immunocytochemical localization, Plant Physiol. 119 (1999) 829–837. [15] A.K. Tobin, T. Yamaya, Cellular compartmentation of ammonium assimilation in rice and barley, J. Exp. Bot. 52 (2001) 591–604. [16] K. Ishiyama, T. Hayakawa, T. Yamaya, Expression of NADH-dependent glutamate synthase protein in the epidermis and exodermis of rice roots in response to the supply of ammonium ions, Planta 204 (1998) 288–294. [17] K. Ishiyama, E. Inoue, M. Tabuchi, T. Yamaya, H. Takahashi, Biochemical background and compartmentalized function of cytosolic glutamine synthetase for active ammonium assimilation in rice roots, Plant Cell Physiol. 45 (2004) 1640–1647. [18] K. Ishiyama, E. Inoue, A. Watanabe-Takahashi, M. Obara, T. Yamaya, H. Takahashi, Kinetic properties and ammonium-dependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis, J. Biol. Chem. 279 (2004) 16598–16605.
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Expression analysis of the glutamine synthetase and glutamate synthase gene families in young rice (Oryza sativa) seedlings Xue-Qiang Zhao a,b, Wei-Ming Shi a,* a
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China b Graduate School of Chinese Academy of Sciences, Beijing 100081, China Received 10 August 2005; received in revised form 2 November 2005; accepted 12 November 2005 Available online 5 December 2005
Abstract Glutamine synthetase (GS) and glutamate synthase (GOGAT) serve for primary assimilation of N in higher plants. When NH4+ is the major inorganic N source, rice plants must assimilate NH4+ quickly in roots through the GS/GOGAT cycle to ameliorate the toxic effect of excess NH4+. The sequence of the rice genome is almost complete, facilitating the identification of the GS and GOGAT gene families in this species. Thus, we investigated the different members of GS and GOGAT genes, and analyzed the pattern of expression of each gene in young rice seedlings by quantitative real-time PCR, revealing a distinct expression pattern for these genes. OsGln1;1 and OsGln1;2 mainly functions in roots, and OsGln2 and OsGlu1 are preferentially expressed in leaves. However, transcriptions of OsGln1;1, OsGln1;2, OsGln2 and OsGlu1 in leaves are all increased by increased N level while those in roots not influenced or even decreased. OsGlt1 and OsGlt2 are expressed primarily in roots when N is limiting, but in leaves when non-limiting. Transcription of OsGlt1 is decreased, but OsGlt2 is increased by increased N level in roots and leaves. When rice roots are exposed to NO3 and NH4+ for 2 h after N-starvation, transcriptions of OsGln1;1, OsGlt1, OsGlt2 and OsGlu1 are all repressed by NO3 and NH4+. OsGln1;2 expression shows significant up-regulation by NH4+ and down-regulation by NO3 while OsGln2 down-regulation by NH4+ and up-regulation by NO3. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Gene expression; Glutamate synthase; Glutamine synthetase; Real-time PCR; Rice seedlings
1. Introduction Nitrogen is an essential element for plant growth. NH4+ and NO3 are initially taken up by root cells from the soil solution in processes mediated by AMT (ammonium transporter) and NRT (nitrate transporter), and previous reports have described AMT and NRT in rice [1–4]. Then plants assimilate these inorganic N forms into amino acids through the GS/ GOGAT cycle. Either from a direct uptake by plant roots or produced by reduction of nitrate, NH4+ is first assimilated through the GS/ GOGAT cycle defined by Lea and Miflin [5]. GS catalyzes synthesis of Gln from NH4+ and Glu in an ATP-dependent
Abbreviations: GS, glutamine synthetase; GOGAT, glutamate synthase * Corresponding author at: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, East Beijing Road, No. 71, Nanjing 210008, China. Tel.: +86 25 86881566; fax: +86 25 86881000. E-mail address: [email protected] (W.-M. Shi). 0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.11.006
manner. GOGAT catalyzes reductive transfer of the amide group of Gln to 2-oxogluarate to form two Glu molecules [6]. In higher plants, there are two major isoforms of GS: cytosolic GS (GS1), which occurs in the cytosol, and chloroplastic GS (GS2), which, although nuclear encoded, is located in the chloroplasts or plastids [7–9]. In all plants studied, GS2 is encoded by a single gene, whereas GS1 belongs to a small gene family. Distinct roles for these two enzymes have been suggested by a number of studies on organ, tissue, and development [7–10]. Similarly, Fd-GOGAT and NADHGOGAT are two major forms in most species based on whether they use NADH (NADH-GOGAT) or reduced ferredoxin (FdGOGAT) as the electron donor for the (two-electron) conversion of L-glutamine plus 2-oxoglutarate to L-glutamate [7,9–13]. These observations provided the rationale for us to study the role of the GS/GOGAT cycle in rice. Immunocytochemical localization of GS and GOGAT has been studied extensively in most plant species, especially in alfalfa [14], rice [15–17] and Arabidopsis [18]. NADH-
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GOGAT is responsible for synthesis of glutamate from glutamine that is transported from senescing tissues to the spikelets in rice [19]. Transgenic tobacco plants that overexpress alfalfa NADH-GOGAT have increased carbon and N content [20]. Over-production of NADH-GOGAT in transgenic rice advocates that NADH-GOGAT is indeed a key step for N utilization and grain filling in rice [21]. Modification of N assimilation efficiency has been approached recently in trees by overexpression of a cytosolic pine GS in poplar [22]. Plastidic GS2 encoded by the GLN2 gene facilitates NH4+ recovery during photorespiration in both leaf mitochondria and chloroplasts [7,8]. Although these biochemical and molecular biological studies provide a solid groundwork in most plant species, a systematic picture of the N-assimilation process and its regulation in a single plant is still in need. Rice, growing in paddy fields, uses NH4+ as its major source of N. Because of the toxicity of NH4+ in plants, NH4+ must be assimilated quickly in rice roots [15]. Therefore, NH4+ uptake and metabolism in plants must be tightly regulated. The GS/ GOGAT cycle plays an important role in anaerobic amino acid accumulation in rice roots [23]. Although good progress has been made to dissect and better understand both the major steps and the regulation of inorganic N assimilation in higher plants, the role of alternative metabolic pathways that are potentially able to incorporate NH4+ into organic molecules is still not fully understood. Moreover, regulation and control of NH4+ uptake and assimilation by the GS and GOGAT gene families remains perplexing to this day. Furthermore, much less is known about how the expression of these genes is regulated and under what conditions they execute their functions. Considering great amount of basal N fertilizer but lower uptake ability at young rice seedling stage, it is essential for us to explore the molecular basis of N efficiency of young rice seedlings in order to improve N use efficiency of basal N fertilizer and reduce N pollution. Therefore, the expression patterns of GS and GOGAT gene families were studied mainly at young rice seedling stage in the present study. To better understand and to systematically clarify the role and regulation of the GS and GOGAT gene families in young rice seedlings, we investigated different members of the GS and GOGAT genes, and analyzed the pattern of expression of each gene in different plant organs and in plants grown with different sources of N by quantitative real-time PCR.
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2. Materials and methods 2.1. Plant materials and growth conditions Rice (Oryza sativa) plants were cultivated in a growth chamber controlled at 25 8C with 70% relative humidity under a 14 h light and 10 h dark cycle. The light intensity was 300 mmol photon m2 s1. Rice plants were first grown hydroponically in a modified Kimura B nutrient solution [24] before treated, which contained NH4NO3 (0.5 mM), NaH2PO42H2O (0.18 mM), KCl (0.55 mM), CaCl22H2O (0.36 mM), MgSO47H2O (0.6 mM), FeSO47H2O-EDTA (20 mM), H3BO3 (50 mM), MnCl24H2O (9 mM), CuSO45H2O 5H2O (0.3 mM), ZnSO47H2O (0.7 mM), and H2MoO44H2O (0.1 mM). Because co-provision of NH4+ and NO3 could improve rice growth [25,26], NH4NO3 was used for the N form in the pre-culture cultivated stage. Nitrification was inhibited by 5.89 mg L1 C2H4N4 (Shanghai Chemical Co. www.reagent.com.cn). The pH of growth media was maintained at 5.5 by adding diluted NaOH or HCl daily. Three replicates were carried out for each procedure. For RNA extraction, plant materials were harvested, frozen quickly in liquid nitrogen, and stored at 80 8C. 2.2. Total RNA extraction and cDNA synthesis Total RNA was extracted by Guanidine Isothiocyanate (Nanjing Sunshine Biotechnology Co. Ltd.) methods [27]. Five micrograms total RNA was used to synthesize cDNA by reverse transcriptase powerscriptTM (BD Bioscience Clontech www.bdbiosciences.com) following the manufacturer’s protocol. The cDNA samples were used as templates to quantify target gene expression levels. 2.3. Quantitative real-time PCR Quantitative real-time PCR analysis was carried out using gene-specific primers (Table 1). Primers were designed according to the sequences of BAC clones (Table 2). Constitutive expression of OsActin (NCBI/GenBank accession number XM_469569) was determined to confirm the equality of molecular copies. The PCR products were detected as SYBR Green I fluorescence using the DNA Engine Opticon 2 System (MJ,
Table 1 Gene-specific primers used for the real-time PCR analysis Gene
Forward primer
Reverse primer
OsGln1;1 OsGln1;2 OsGln1;3 OsGln2 OsGlt1 OsGlt2 OsGlu1 OsGlu2 OsGlu2 OsActin
50 -CAAGTCCGCCATTGAGAAGC-30 50 -GGTTGGAGGATCGGGCATAG-30 50 -AGCCGATTCCGACGAACAAC-30 50 -ACCAAGAGTATGCGTGAAGA-30 50 -GGAGGGAAATCTAATACAGG-30 50 -AGACAAACAATTTCCCTGAG-30 50 -AAACAGGCAGCGAGAAAGGT-30 50 -TCCAATAGGACCAATACAGA-30 50 -CAAACCGTCCTGTCAATGTG-30 50 -CTTCATAGGAATGGAAGCTGCGGGTA-30
50 -CTTGCCGTTCTGCTCCGTCT-30 50 -TCACCTTGTGGCGTGTAGCA-30 50 -GTAGCGTGCCACCCAGACAT-30 50 -AACCTGTCAACCTCCTTTCA-30 50 -AGTTCATCAGCGTTAGTCAG-30 50 -TAAAGGGTCACTTCCAACAT-30 50 -ACTCGTTCAAACTCGGCACA-30 50 -ATCAACAACATCTCACCACC-30 50 -TGGAACTTGTGGCAGCGTCT-30 50 -CGACCACCTTGATCTTCATGCTGCTA-30
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Table 2 Summary of GS and GOGAT gene families in rice Gene name
Product
BAC clone accession number
Chromosome location
OsGln1;1 OsGln1;2 OsGln1;3 OsGln2 OsGlt1 OsGlt2 OsGlu1 OsGlu2
GS1 GS1 GS1 GS2 NADH-GOGAT NADH-GOGAT Fd-GOGAT Fd-GOGAT
AP004880 AC105364 AC082645 AL662953 AP004363 AC104709 AP003833 AP004343
chr02 chr03 chr03 chr04 chr01 chr05 chr07 chr07
USA, www.mjr.com) for continuous fluorescence detection. Gene copies were determined quantitatively by using a purified cDNA clone as standard.
NADH-GOGAT genes in rice (Table 2). Results reported here for the GS gene family are consistent with the report of Ishiyama et al. [17].
2.4. Statistical analysis
3.2. Expression of GS and GOGAT gene families at different N concentrations
Statistical analysis was conducted using procedures in Excel software (Microsoft Office Excel 2003). Mean comparison was calculated according to Duncan Multiple Range test using the Statistical Analysis System (SPSS 11.5). 3. Results 3.1. The GS and GOGAT gene families in rice In order to establish the genes encoding GS and GOGAT in rice, we identified GS and GOGAT gene families in rice through a complete homologous blast search on the internet (http://www.ncbi.nlm.nih.gov/blast/, http://rgp.dna.affrc.go.jp/ blast/runblast.html, and http://www.ddbj.nig.ac.jp/search/ blast-j.html) with a known gene sequence as a probe and named them as shown in Table 2. The search identified three GS1 genes, a single GS2 gene, two Fd-GOGAT genes and two
Plant GS and GOGAT genes studied previously are very important in N assimilation, but the systematic expression patterns of the GS and GOGAT genes families have not yet been clearly established. Thus, we examined the level of expression of each member of the GS and GOGAT gene families at two different N concentrations using quantitative real-time PCR. Rice plants were grown under two different nutritional conditions. In the first, corresponding to limiting conditions, plants were supplied with 0.1 mM NH4NO3. The second set of plants, cultivated under non-limiting conditions, was fed with 2 mM NH4NO3. The relative and absolute values for gene expression in roots and leaves are shown in Fig. 1. Although we employed several different RT-PCR conditions, we were unable to detect any amplification of OsGln1;3 and OsGlu2 at either N concentration. For the other genes, the
Fig. 1. Expression profiles of individual GS and GOGAT genes. For each gene, the relative amounts of mRNA in roots (open bars) and leaves (black bars) were added and then expressed as a percentage of the sum. Values above the columns were the absolute mRNA contents (amol/mg total RNA) of each individual gene in roots or leaves. After rice plants were grown under full nutrient solutions containing 0.5 mM NH4NO3 for 15 days, rice seedling were treated with limiting (0.1 mM NH4NO3, A) or non-limiting (2 mM NH4NO3, B) N for 10 days. A 25-day-old rice plants (five-leaf stage) were harvested and analyzed.
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levels of transcripts in different organs added were calculated relative to the OsActin gene. The relative amount of mRNA expressed by these genes in each organ is shown in Fig. 1 as a percentage of the resulting sum. When rice plants were cultivated at 0.1 mM NH4NO3 for 10 days, four of the genes, OsGln1;1, OsGln1;2, OsGlt1, and OsGlt2, showed a markedly preferential expression in roots, whereas OsGln2 and OsGlu1 were expressed mainly in leaves (Fig. 1A). When rice plants were cultivated at 2 mM NH4NO3 for 10 days, results similar to those in Fig. 1A were found except for OsGlt1 and OsGlt2 (Fig. 1B). OsGlt1 and OsGlt2 were expressed mainly in leaves at 2 mM NH4NO3 (Fig. 1B) but mainly in roots when the nitrogen supply was decreased to 0.1 mM NH4NO3 (Fig. 1A). Changes of transcriptions about absolute values of mRNA contents (values above different columns in Fig. 1) between 0.1 and 2 mM NH4NO3 are also compared. Two classes of genes can be distinguished by the changes of absolute mRNA contents (Fig. 1). The first class consisted of OsGln1;1 and OsGlt1; the amounts of their mRNAs seemed to be decreased in both roots and leaves from 0.1 to 2 mM NH4NO3. The second, including OsGln1;2, OsGlt2, OsGln2 and OsGlu1, showed a decrease in roots but a increase in leaves from 0.1 to 2 mM NH4NO3.
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those in leaves seemed to be slightly up-regulated by increased N (Fig. 2), which was almost in accordance with the results in Fig. 1. Furthermore, it was interesting that transcription of OsGlt1 was strongly repressed by increased N in both roots and leaves, whereas that of OsGlt2 was significantly induced by increased N in both roots and leaves. According to the results in Fig. 1, expression of OsGlt1 and OsGlt2 should be much higher in leaves than in roots in 2 mM NH4NO3 in Fig. 2, whereas this was not the case in Fig. 2, which was possibly caused by a treatment of 2 mM NH4NO3 for a shorter time (only 6 h) in Fig. 2 while rice seedlings were grown in 2 mM NH4NO3 for 10 days in Fig. 1. 3.4. Influence of NO3 and NH4+ supply on GS and GOGAT gene expression of rice roots
The N supply is a major regulator of GS and GOGAT expression. Fig. 2 shows, for each of the six genes that could be tested, the variations of transcript levels within roots and leaves between treated (with 2 mM NH4NO3) and non-treated (no NH4NO3) rice plants. The mRNA contents of OsGln1;1, OsGln1;2, OsGln2 and OsGlu1 in roots exhibited no significant differences between treated and non-treated conditions, but
NO3 and NH4+ are the two major sources of inorganic N for higher plants. Therefore, we also compared the effect of NO3 to that of NH4+ on expression of GS and GOGAT genes in rice roots by short-time treatments. Rice plants were cultivated under full nutrient solutions for 28 days. After N-starvation for 48 h, some were treated with 1 mM NH4+ in the form of 0.5 mM (NH4)2SO4, and the others were treated with 1 mM NO3 in the form of 1 mM KNO3. Changes of transcription in rice roots with different N sources are illustrated in Fig. 3. Three classes of genes can be distinguished. The first class consisted of OsGln1;1, OsGlt1, OsGlt2 and OsGlu1 genes; all seemed to be significantly down-regulated by both NO3 and NH4+. The second, corresponding to the OsGln1;2 gene, showed significant up-regulation by NH4+ and down-regulation by NO3. The last class, corresponding to the OsGln2 gene, exhibited regulation opposite to that observed with OsGln1;2, i.e., down-regulation by NH4+ and up-regulation by NO3. We could also find that there were important differences in mRNA levels between Figs. 2 and 3, specially for OsGln1;2
Fig. 2. Effects of N conditions on GS and GOGAT gene expression. After rice plants were grown under full nutrient solutions containing 0.5 mM NH4NO3 for 15 days, rice seedling were pre-cultured with 0.1 mM NH4NO3 for 10 days. Finally, 25-day-old rice plants (five-leaf stage) were treated with 2 mM NH4NO3 for 6 h (black bars) or not treated (open bars) and then 25-day-old rice plants were harvested and analyzed. Means of independent triplicate samples and S.D. values (n = 3) are indicated. Different letters above the columns indicate statistically significantly differences at P 0.05.
Fig. 3. Gene expressions of GS and GOGAT in rice roots in response to NO3 and NH4+ supply. Rice plants were grown under full nutrient solutions containing 0.5 mM NH4NO3 for 28 days. After nitrogen-starvation for 48 h, 28-day-old rice seedlings (five to six-leaf stage) were exposed to 1 mM NH4+ (gray bars) or 1 mM NO3 (black bars) or no N (open bars) for 2 h and then rice roots were harvested and analyzed, respectively. Means of independent triplicate samples and S.D. values (n = 3) are indicated. Different letters above the columns indicate statistically significantly differences at P 0.05.
3.3. Influence of increased N levels on GS and GOGAT gene expression
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and OsGln2. Different N nutrition conditions of rice plants in Figs. 1 and 2 possibly resulted in these differences. Compared to the mRNA levels of OsGln1;2 and OsGln2 in Fig. 2, excessive nitrogen-starvation of rice plants may greatly reduce the mRNA levels of OsGln1;2 and OsGln2 in Fig. 3. 4. Discussion It was thought previously that OsGln1;2 (GSr) is the only GS1 isoenzyme expressed in rice roots [28]. However, our study shows that the GS1 gene family in rice is comprised of three different genes located on chromosomes II and III. The GS2 gene in rice is encoded by a single gene located on chromosome IV, as reported by Ishiyama et al. [17]. The results reported here also indicate that both NADH-GOGAT and FdGOGAT are each encoded by two genes. These two genes encoding NADH-GOGAT are located on chromosomes I and V, and both of the genes encoding Fd-GOGAT are located on chromosome VII. In higher plants, the organ specificity of GS and GOGAT gene expression might depend on the species. Most of the previous studies have used the northern technique. Because of the high degree of homology between the paralogs, the northern technique does not allow specific detection of each gene transcript and, moreover, is not sensitive enough to unravel gene expression at very low levels [29]. Real-time PCR provides a very highly sensitive and reproducible technique that can detect gene expression at very low levels. For example, both OsGln1;1 and OsGln1;2 are expressed mainly in roots (Fig. 1), but using real-time PCR, we have found that the absolute mRNA contents of OsGln1;2 (401.50 amol/mg total RNA) is far larger than that of OsGln1;1 (119.32 amol/mg total RNA) in rice roots under low N conditions (Fig. 1). Regardless of limiting or non-limiting N conditions, OsGln1;1 and OsGln1;2 exhibit a strong root preferential pattern of expression, whereas OsGln2 shows a preference for leaves (Fig. 1). This study also suggests that the OsGln1;1 and OsGln1;2 proteins perform their functions in roots, but the OsGln2 protein performs theirs in leaves. GS2 is expressed predominantly in green tissues. It has been proposed that GS2 plays an important role in the assimilation of ammonia from nitrate reduction, and it has also been demonstrated that GS2 is indispensable for re-assimilation of photorespiratory ammonia [30–32]. Similar results for OsGln1;1, OsGln1;2 and OsGln2 were reported by Ishiyama et al. [17]. When rice seedlings were transferred from 0.1 to 2 mM NH4NO3 for 6 h, the mRNA contents of OsGln1;1, OsGln1;2 and OsGln2 exhibited no significant differences in rice roots (Fig. 2), but those in leaves seemed to be up-regulated by increased N (Figs. 1 and 2). All of these suggest that, when outer N concentrations in soil are increased, OsGln1;1, OsGln1;2 and OsGln2 possibly play more important roles in leaves relative to roots. Thus, increased inorganic N in rice roots can be fast transport to rice leaves and assimilated into Gln or other N compounds in rice leaves, and so rice growth and development are improved by this kind of N assimilative mechanism.
The results [17,18] support the importance of OsGln1;1 in promoting rapid conversion of ammonium to Gln at the surface cell layers that are in contact with soils particularly under lowammonium conditions. In contrast to OsGln1;1, the OsGln1;2 transcript accumulates abundantly in the surface cell layers after ammonium treatment. Apparently, OsGln1;1 and OsGln1;2 are regulated oppositely by ammonium in these specific cell types at the mRNA level [17]. In the present study, transcription of OsGln1;1 in rice roots is strongly repressed by nitrate and ammonium (Fig. 3). However, OsGln1;2 is highly up-regulated by ammonium but repressed by nitrate. As for OsGln2, it appears to be repressed by ammonium but slightly up-regulated by nitrate. The OsGln1;1, OsGln1;2 and OsGln2 transcripts show reciprocal responses to nitrate and ammonium supply in rice roots. Plants have already developed an intrinsic regulation mechanism to adapt themselves to uneven Ndistribution environments and utilize mineral nutrition efficiently. The OsGln1;1, OsGln1;2 and OsGln2 transcripts show different responses to nitrate and ammonium supply. Thus, rice plants can efficiently assimilate N in rice roots by this kind of flexible and reciprocal regulative mechanism, and control rice growth and development. With regard to OsGlt1 and OsGlt2, they are expressed in roots under limiting N conditions but in leaves under nonlimiting N conditions (Fig. 1). Therefore, expression of the genes encoding NADH-GOGAT possibly changes with external N conditions. Growth of rice plants can be regulated to be adaptive to outer heterogeneous N conditions in soil by this kind of changes. When N level is higher in soil environments, expression of OsGlt1 and OsGlt2 can be decreased in roots in order to limit N acquisition, but in the opposite side, expression of OsGlt1 and OsGlt2 can be enhanced in roots in order to increase N acquisition. This may be a kind of buffering effects in higher plants. Our results also show that transcription of OsGlt1 is decreased in roots and leaves by increased N levels but transcription of OsGlt2 is increased in roots and leaves (Fig. 2). These results suggest that OsGlt1 possibly performs functions mainly under low N conditions while OsGlt2 primarily under high N conditions. As like OsGln1;1, OsGln1;2 and OsGln2, the OsGlt1 and OsGlt2 transcripts also show reciprocal responses to increased N supply in roots and leaves. This is very useful for plants to adapt outer nutrient heterogeneity in soil. Previous studies show that the mRNA and protein for NADH-GOGAT accumulate markedly in whole roots or the root-tip sections of rice plants within 12 h supplying a low concentration of NH4Cl [15,33,34]. A similar response is seen in rice cells in suspension culture [35]. However, OsGlt1 and OsGlt2 are both repressed in rice roots by a short-time treatment of nitrate and ammonium in the present study (Fig. 3). The present results do not reflect previous reports showing clear ammonium inducibility [15,33–35] of NADH-GOGAT in rice roots. Rice seedlings by previous reports were grown in water for 26 days before treated, but seedlings in the present study were only N-starvation for 48 h before treated. Rice growth for a long time in water must result in exhausting of N in rice
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seedlings and reducing of NADH-GOGAT activity, so, when ammonium is supplied at the moment, the mRNA and protein for NADH-dependent glutamate synthase are increased dramatically in roots of rice seedlings in order to assimilate ammonium. However, in the present study, rice seedlings of Nstarvation for a short time possibly induce expression of genes encoding NADH-GOGAT in order to acquire more N, thus, when ammonium and nitrate are supplied to rice roots, the mRNA contents for NADH-dependent glutamate synthase are decreased dramatically in roots of rice seedlings. The OsGlu1 gene encoding Fd-GOGAT shows a pattern of expression similar to OsGln2. In spite of limiting or nonlimiting N conditions, OsGlu1 is expressed mainly in rice leaves (Fig. 1), and transcription of OsGlu1 in leaves is significantly up-regulated by increased N (Figs. 1 and 2) while it is repressed by nitrate and ammonium in rice roots (Fig. 3). Therefore, Gln can be transferred to 2-oxogluarate to form Glu by the transcription of OsGlu1 in rice leaves. However, in rice roots, the OsGlt1 and OsGlt2 encoding NADH-GOGAT not OsGlu1 possibly perform such functions in the transfer of the amide group of Gln to 2-oxogluarate to form two Glu molecules. In maize roots, Fd-GOGAT and GS2 are induced within 30 min by 10 mM nitrate (with or without cycloheximide) and within 2 h by 10 mM nitrate [36]. In another study using detached maize leaves, GS2, Fd-GOGAT, and NADH-GOGAT are all induced by 16 mM nitrate after 2 h [37]. In tobacco NR mutants, GS1, GS2, and Fd-GOGAT transcripts were found to be more plentiful in plants grown with 12 mM nitrate than in plants grown with 0.2 mM nitrate [38]. However, in the present study, the transcription of OsGlu1 encoding Fd-GOGAT is repressed by nitrate supply. The results presented here in this study provide an unknown role of nitrate as a negative regulator of Fd-GOGAT genes in rice roots. The reason for different regulatory mechanism of Fd-GOGAT in rice should be further studied in the future. It should be pointed out that OsGln1;3 and OsGlu2 were not detected under the conditions used in this study. We suggest that these two genes might be pseudogenes in rice. Lack of amplification of OsGln1;3 in this study confirms previous reports [15]. There are previous reports [39] that Fd-GOGATs are immunologically distinct proteins in rice roots and leaves. Two sets of different primers have been used for OsGlu2 PCR attempts of amplification in the present study (Table 1). However, we still cannot get amplification of OsGlu2 by our efforts. In addition, we also fortunately find that OsGlu2 is marked for pseudogene in rice gene bank (AP004343, http:// www.ncbi.nlm.nih.gov/), and it is indicated to be pseudogene and probably inactive due to including stop codon(s) in cDNA. Such a lot of expression patterns for the genes encoding GS and GOGAT gene families have been examined in the present study. It will be very interesting to use reporter genes or green fluorescent protein to follow the fine distribution of each GS and GOGAT gene at the cellular level. From the results of this preliminary study, it is difficult to speculate on the role of each of these genes in N assimilation. However, it is tempting to associate a particular pattern of expression and a putative function. Work is in progress in our
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