- Email: [email protected]
Asparagine synthetase gene TaASN1 from wheat is up-regulated by salt stress, osmotic stress and ABA
ARTICLE IN PRESS Journal of Plant Physiology 162 (2005) 81—89
www.elsevier.de/jplph
Asparagine synthetase gene TaASN1 from wheat is up-regulated by salt stress, osmotic stress and ABA Huabo Wanga,b, Dongcheng Liua, Jiazhu Suna, Aimin Zhanga,b, a
Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, People’s Republic of China b Graduate School of the Chinese Academy of Sciences, People’s Republic of China Received 2 June 2004; accepted 27 July 2004
KEYWORDS ABA; Asparagine synthetase; Differential display; Osmotic stress; Salt stress; TaASN1; TaASN2
Summary Differences in gene expression between salinity stressed and normally grown wheat seedlings were compared by the differential display (DD) technique. One DD-derived cDNA clone was characterized as a partial sequence of the wheat asparagine synthetase (AS) gene by sequence analysis and homology search of GenBank databases. Two AS genes of wheat, TaASN1 and TaASN2, were further isolated by the RT-PCR approach. Comparison of the deduced polypeptide of TaASN1 and TaASN2 with AS proteins from other organisms revealed several homologous regions, in particular, the conserved glutamine binding sites and Class-II Glutamine amidotransferases domain. The functionality of TaASN1 was demonstrated by complementing an Escherichia coli asparagine auxotroph. TaASN1 transcripts were detected in roots, shoots, anthers and young spikes by RT-PCR analysis. Abundance of TaASN1 mRNA in young spikes and anthers was higher than that in shoots and roots under normal growth conditions. TaASN1 was dramatically induced by salinity, osmotic stress and exogenous abscisic acid (ABA) in wheat seedlings. TaASN2 transcripts were very low in all detected tissues and conditions and were only slightly induced by ABA in roots. & 2004 Elsevier GmbH. All rights reserved.
Introduction Soil salinity limits agricultural production throughout the world. The United Nations Environment
Program estimates that approximately 20% of agricultural land and 50% of cropland in the world is salt-stressed (Zhu, 2001). As one of the most important crops, the biomass of wheat (Triticum
Abbreviations: ABA, abscisic acid; Asn, asparagine; AS, asparagine synthetase; DD, differential display; IPTG, isopropyl bthiogalactoside Corresponding author. Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, People’s Republic of China. Tel.: +86-10-64889347; fax: +86-1064854467. E-mail address: [email protected] (A. Zhang). 0176-1617/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.07.006
ARTICLE IN PRESS 82 aestivum) is reduced by over 90% in 50% seawater (250 mM NaCl) (Kingsbury and Epstein, 1984), and grain yield is reduced by 50–90% in only 50 mM NaCl (Marschner, 1995). Clearly, there is considerable scope for improvement in yield of wheat grown in a salt-stressed environment. Salt stress affects plant growth in many ways, such as the Na ionic toxicity and hyperosmotic stress, which cause plant nutritional imbalance and oxidation stress. In order to adapt to a high-salt environment, plants change gene expression patterns, metabolic activity, and ion and water transport to minimize stress damage and to reestablish ion and water homeostasis. Glutamine-dependent asparagine synthetase (AS: EC 6.3.5.4) catalyses the transfer of an amide group from glutamine to aspartate-forming asparagine (Asn) in an ATP-dependent reaction. In most plant species, AS seems to be encoded by a small gene family. Three AS genes have been identified in Arabidopsis (Lam et al., 1998) and sunflower (Herrera-Rodriguez et al., 2002), but in most species only one or two genes are known. Asn plays a prominent role in nitrogen transport and storage in plants. High concentrations of Asn can also be found in various plant tissues under stress conditions, such as mineral deficiencies, salt stress or drought (Moller et al., 2003). In barley, soybean and Coleus blumei, the Asn pool increased markedly when the plants were subjected to severe water stress or salt stress (Gilbert et al., 1998). The accumulation of Asn may be caused by stimulated synthesis, inhibited degradation of amino acids, impaired protein synthesis, and/or enhanced protein degradation (Ranieri et al., 1989), but much information about these processes is still unknown. In this paper, using a differential display (DD) technique, we isolated and characterized one saltinducible gene, TaASN1, which encodes the glutamine-dependent AS. The functional activity of TaASN1 was tested by a complementation assay. The expression patterns of TaASN1 in different tissues of wheat (shoots, roots, spikes, anthers) and different stress conditions (ABA, osmotic, salt) were also investigated.
Materials and methods Plant growth condition and NaCl stress treatments Seeds of Triticum aestivum L. cv. Keyi26 were germinated in a Petri dish, then were cultivated hydroponically in full-strength Hoagland solution (Elberse et al., 2003) in a greenhouse (16/8 h daily
H. Wang et al. light period, 25 1C temperature (night/day) and 60–70% relative humidity). Two-week-old seedlings were transferred to fresh Hoagland medium supplemented with 250 mM NaCl, 5.0% (w/v) mannitol or 20 mM abscisic acid (ABA). After exposure to the stress treatments for 3, 7 or 24 h, shoots and roots were collected in liquid nitrogen and stored at 80 1C until further processing.
Differential display The roots of NaCl-treated and untreated control seedlings were used for DD RT-PCR. Total RNA was extracted with Trizol reagent (GIBCO-BRL, USA) and was digested with DNAase I (Promega, Madison, WI). DD was performed according to the procedures described previously (von der Kammer et al., 1999) with modification. Three reverse transcription reactions per sample are set for the first strand cDNA synthesis. 2 mg RNA from roots of control and stressed seedlings was used for reverse transcription with either one-base anchor oligonucleotides HT1 (50 -TGC CGA AGC TTT TTT TTT TTA-30 ), HT2 (50 TGC CGA AGC TTT TTT TTT TTG-30 ) or HT3 (50 -TGC CGA AGC TTT TTT TTT TTC-30 ) and 200 units M-MLV reverse transcriptase (Promega) in a volume of 20 mL according to the manufacturer’s instructions. The reverse-transcription reactions were diluted fivefold, and 2 mL was initially subjected to PCR employing the corresponding one-base anchor oligonucleotide (1 mM) along with either one of the DD random primers (von der Kammer et al., 1999), 1.5 mM MgCl2, 0.2 mM dNTPs, 1 buffer, and 0.2 mL Tag DNA polymerase (TaKaRa, Dalian, China). Reactions were performed in a thermal cycler (PCR system 9700, Applied Biosystems, USA) with the following cycling conditions: one round at 94 1C for 4 min for denaturing; cooling 40 1C for 4 min for low-stringency annealing of primer; and heating 72 1C for 1 min for extension. This round was followed by 35 high-stringency cycles: 94 1C for 45 s; 60 1C for 2 min; and 72 1C for 1 min. One final step at 72 1C for 10 min was added to the last cycle. The denature loading buffer was added to PCR products and heated to 94 1C for 5 min and stored on ice prior to loading on 6% denaturing polyacrylamide DNA sequencing gel (BIO-RAD, USA). Electrophoresis was performed at a constant 85 W for 3 h and then the gel was subjected to silver staining (Bassam et al., 1991). The differential cDNA fragments were recovered from the sequencing gel and eluted. The elusions were used as templates for reamplification using the same sets of primer pairs. The reamplified fragments were
ARTICLE IN PRESS Asparagine synthetase gene from wheat purified from agarose gels and cloned into pGEM-T easy vector (Promega) for sequencing.
Cloning TaASN1 and TaASN2 Primers TaASN1F (50 -TGT TGC CGT CGA TCC AGG AAA ATG-30 ) and TaASN1R (50 -GGC AAG CAG GAC AGG ACA CCA TCA AC-30 ), TaASN2F (50 -ACG AGG CTG GCT GTA GAG GAC-30 ) and TaASN2R (50 -AAC CGT TCT TAT TTG CCT TTA GTAG-30 ) based the Genbank sequence BT009245 and BT009049, respectively, were used to amplify the cDNA of TaASN1 and TaASN2. First strand cDNA synthesis was performed on 2 mg of total RNA with M-MLV Reverse Transcriptase enzyme (Promega), and an oligo dT18 primer. The 20 mL PCR reaction mixture contained 2 mL of fivefold diluted cDNA, 0.2 mM of each primer, 1.5 mM MgCl2, and 0.2 mM dNTPs, 1 buffer and 0.2 mL high-fidelity Pyrobest polymerase (Takara). Reactions were performed in the following cycling conditions: 94 1C 5 min, then 35 cycles of 94 1C 0.5 min; 54 1C 0.5 min; and 72 1C 2 min; after cycling a final step of 72 1C 10 min. The PCR products were purified and cloned into pGEM-T easy vector (Promega) to generate pTaASN1 and pTaASN2 for sequencing.
83
RT-PCR analysis Protocols for total RNA extraction and synthesis of cDNA were described as above. The reversetranscription reactions were diluted tenfold, and 2 mL was initially subjected to PCR reaction. A fragment of a-tubulin gene (GenBank accession No.U76558) was amplified with specific primers TUBF (50 -ACC GCC AGC TCT TCC ACC CT-30 ) and TUBR (50 -TCA CTG GGG CAT AGG AGG AA-30 ) as the internal control for RT-PCR analysis. The PCR was performed under the following temperature profile: 94 1C 5 min;; then 28 cycles of 94 1C 0.5 min; 57 1C 0.5 min; and 72 1C 0.5 min, after cycling a final step of 72 1C 7 min. Specific primer pair TaASN1RTF (50 -GAG CAT CTC CCA GCA ACC ATC ATG-30 ) and TaASN1R were designed to amplify 111 bp TaASN1 fragment and TaASN2RTF (50 -CAC GGT CGC TGT GGG AGG TAG-30 ) and TaASN2R were designed to amplify a 128 bp TaASN2 fragment. The reaction is 94 1C 5 min, then 31 cycles of 94 1C 0.5 min; 57 1C 0.5 min; 72 1C 0.5 min; after cycling a final step of 72 1C 5 min. To test the specificity of the primers, RT-PCR products were cloned into pGEM-T easy vector (Promega) for sequencing. For genomic contamination tests, samples without reverse-transcription reactions were also used as control.
Complementation of an E. coli Asn auxotroph Nucleotide sequence analysis The full-length coding regions of TaASN1 and TaASN2 were obtained by amplification of plasmids pTaASN1 and pTaASN2 using the Pyrobest polymerase (Takara). Primers ASN1-E (50 -CCG GAA TTC TAA TGT GCG GCA TAC TGG CG-30 ) and ASN1R-X (50 -AGT CTA GAC AGG ACA GGA CAC CAT CAA C-30 ) were used for amplifying EcoRI-XbaI fragment of TaASN1, then cloned into EcoRI-XbaI sites of pGEX-KG to generate p-KG-TaASN1. Primers ASN2F-B (50 -GCC GGA TCC TGA GCA TGT GCG GCA TAC TAG-30 ) and ASN2R-X (50 -GAT CTA GAA TCA AGT CTC AAT GGC AAC-30 ) were used for amplifying BamHI-XbaI fragment of TaASN2 then cloned into BamHI-XbaI sites to generate p-KG-TaASN2. The resulting plasmids and the empty pGEX-KG were introduced into the E. coli Asn auxotroph strain ER (asnA, asnB, thi1, relA, spoT1) gifted from the Genetic Stock Center (New Haven, CI, USA). For growth tests, the plasmid-containing E. coli ER were grown in liquid M9 medium overnight. The cultures were diluted 1–100 in fresh M9 medium containing 100 mg/mL ampicillin and 1 mM IPTG, supplemented with100 mg/mL Asn when needed. The optical density was measured at 600 nm after 3, 6, 9, 24, 36, and 48 h of growth.
Nucleotide sequences were determined on both strands using an automatic sequencer, ABI PRISMTM 310 (Applied Biosystems, USA). Alignment analysis was performed using CLUSTALW (www.ebi.ac.uk/ clustalw) and BOXSHADE (http://www.ch.embnet.org/software/BOX-form.html). The nucleotide sequence of the TaASN1 has been deposited in the GenBank database under the accession number AY621539.
Accession numbers The sequence accession numbers for TaASN1 and the homologs shown in Figs. 2 and 3 are as follows: HvAS1, GenBank AAK49456; HvAS2, GenBank AAO39048.1; PEA ASN2, SW P19252; LOTJA ASN1, SW P49092; ASN2 LOTJA, SW P49093; At ASN3, GenBank AAC72836; TRIVS ASNS, SW O24661; SANAU ASNS, SW O24338; ASPOF ASNS, SW P31752; HUMAN ASNS, SW P08243; MOUSE ASNS, SW Q61024; YEAST ASN2, SW P49090; YEAST ASN1, SW P49089; ECOLI ASNB, SW P22106; At ASN1, SW P49078; At ASN2, GenBank AAC72837; ASPOF ASNS,
ARTICLE IN PRESS 84 SW P31752; SANAU_ASNS, SW O24338; TRIVS ASNS, SW O24661.ORYSA ASNS, SW Q43011.
Results Differential display Differential display (DD) was performed to detect changes in the transcripts of wheat roots under salt-stress conditions. Three one-base anchored oligo-dT primers (HT1, HT2 and HT3) and twenty arbitrary oligonucleotide primers (von der Kammer et al., 1999) were used for DD-PCR amplification. Of the 28 differential bands displayed on the sequencing gel, 10 were up-regulated and 18 were down-regulated by salt stress. One up-regulated fragment (Fig. 1) was further characterized. Sequence analysis revealed that this fragment was 1110 bp in length and was similar with two Genbank unannotated mRNA sequences (99% Accession No. BT009245 and 85% identify with BT009049). The BLASTX searches of GenBank databases reveal that they encode plant glutamine-dependent AS. There-
H. Wang et al. fore, we named the two gene TaASN1 and TaASN2, respectively. Based on BT009245 and BT009049, we performed RT-PCR to obtain the full-coding cDNA sequences of TaASN1 and TaASN2. There are 12 bp differences between TaASN1and BT009245, which lead to three deduced amino acid residue differences (M161–V161, V219–I 219 and V428–M428) (Fig. 2 region III). The open reading frame of the cDNA of TaASN1 encodes a 585 amino acid residue polypeptide with a calculated mass of 65.4 kD, whereas TaASN2 encodes a 581 amino acid residue polypeptide (65.0 kD). The two deduced amino acid sequences of TaASN1 and TaASN2 show an overall sequence identity of 88.7%. The conserved amino acid residues for substrate binding and catalytic activity were found in wheat AS by sequence alignment among AS from Arabidopsis, maize and E. coli (Fig. 2). The Nterminal part of the deduced amino acid sequences are characterized by the presence of a Class-II Glutamine amidotransferases domain (x-C-[GS][IV]-[LIVMFYW]-[AG]) (Van Heeke and Schuster, 1989), indicated by asterisks in Fig. 2, region I. Essential residues for binding of aspartate and ATP, T316, T317, R319 and C523 (Boehlein et al., 1997a, b), are also conserved in both TaASN1 and TaASN2. The deduced polypeptide sequences of TaASN1 and TaASN2 were aligned with AS amino acid sequences from plants, yeast, mammals., and E. coli to create a dendrogram (Fig. 3) that divides the plant AS into two groups. Both TaASN1 and TaASN2 belongs to class I, the majority of plant AS peptides, and closed to AS from Hordeum vulgare HvAS1 and HvAS2.
Functional analysis of TaASN1 by complementation of an E. coli Asn auxotroph
Figure 1. Analysis of mRNA in wheat root used by DD RTPCR. Lanes 1–3 indicate control, salt treated for 3 h, salt treated for 24 h, respectively. The arrow indicates the amplified fragment of TaASN1.
For the instability of the AS, the functional activities of TaASN1 and TaASN2 were tested by complementation of an E. coli Asn auxotroph. The regions encoding the proteins TaASN1 and TaASN2 were inserted inframe in the pGEX-KG expression vector (Guan and Dixon, 1991). The new constructs (pGEX-KG-TaASN1 and pGEX-KG-TaASN2) were transformed into the E. coli auxotroph ER strain (asnA, asnB, thi-1, relA, spoT1) lacking AS activity (Felton et al., 1980). As expected, growth of E. coli ER transformed with the empty vector was very poor when cultured in a medium without Asn. Higher growth levels were obtained in the same medium when the ER strain was transformed with pGEX-KG-TaASN1, but the growth level was not significantly elevated with pGEX-KG-TaASN2 (Fig. 4).
ARTICLE IN PRESS Asparagine synthetase gene from wheat
85
Figure 2. Alignment of the amino acid sequences of TaASN1, TaASN2, AtASN1, MAIZE_ASNS and ECOLI_ASNB. Multiple sequence alignment was determined using the ClustalW program and residues were shaded using BoxShade 3.21. Identical residues are shown on a black background, and conservative substitutions are shown on a gray background. I, Class-II Glutamine amidotransferases domain; II, aspartate and ATP binding sites; III, different amino acids residues between TaASN1 and BT009245.
ARTICLE IN PRESS 86
H. Wang et al.
Figure 3. Phylogenetic distances among 22 different AS proteins shown in a dendogram constructed following the ClustalW method for multiple sequence comparison. The AS proteins from plant are separated into two groups (class I and class II), where the TaASN1 and TaASN2 from wheat belong to class I as the majority of AS peptides. Only the ASN2 and ASN3 from Arabidopsis, the rice, the maize and belong to class II. At, A. thaliana; Os, Oryza sativa; Hv, H. vulgare; LOTJA, Lotus japonicus; ASPOF, Asparagus officinalis; SANAU, Sandersonia aurantiaca; TRIVS, Triphysaria versicolor.
Expression and regulation of AS genes in wheat 4
3
A600
To study expression patterns of TaASN1 and TaASN2 in wheat, total RNA was extracted from shoots and roots of two-week-old seedlings, young spikes and anthers and then subjected to RT-PCR analysis using the specific primers (considering that the high homology between TaASN1 and TaASN2, the specificity of the primers to TaASN1 and TaASN2 was determined by sequencing of RT-PCR products). Abundance of TaASN1 mRNA in young spikes and anthers was higher than that in shoots and roots (Fig. 5A) under normal growth conditions. To determine the effects of various environmental stresses on the expression of TaASN1 and TaASN2 in wheat seedlings, a time-course experiment was performed. Salinity stress caused a
p-KG-TaASN1 -ASN p-KG-TaASN1 +ASN p-KG-TaASN2 -ASN p-KG-TaASN2 +ASN pGEX-KG -ASN pGEX-KG +ASN
2
1
Figure 4. Complementation of an E. coli asparagine auxotroph. The auxotroph strain ER was transformed with the plasmid p-KG-TaASN1 (dot), p-KG-TaASN2 (triangle) or pGEX-KG (square). The transformants were cultured at 28 C in M9 minimum medium containing ampicillin (100 mg/mL) and 0.1 mM IPTG, with asparagine (open symbols) or without asparagine (filled symbols). Three independent experiments were performed.
0 0HR
3HR
6HR
9HR
Time
24HR
36HR
48HR
ARTICLE IN PRESS Asparagine synthetase gene from wheat
87
Figure 5. RT-PCR analysis of TaASN1 and TaASN2 expression. Expression pattern of TaPdxK in different tissues (root, shoot, spike and anther) of T. aestivum (A.). Time course experiment of TaASN1 and TaASN2 expression pattern in the roots (B) and shoots (C) wheat seedlings under 250 mM NaCl 5.0% (w/v) mannitol and 20 mM ABA.
rapid accumulation of the TaASN1 transcript both in shoots and roots, and its level reached a maximum after 3 h, and then kept the level (Fig. 5A). Under osmotic stress condition, its mRNA level in shoots increased with stressed time, but in roots its expression rapidly reached a peak after 3 h, then declined to an unstressed level at 24 h. ABA is implicated in plant stress responses. Exogenous application of ABA also up-regulated the expression of TaASN1, which in roots accumulated more rapidly than in shoots. The expression of TaASN2 is different from TaASN1, which is only significantly up-regulated by ABA in roots at 24 h.
Discussion The glutamine-dependent AS enzyme is now generally accepted as the major route for Asn biosynthesis in plants. In the present study, a cDNA-encoded AS, TaASN1, was isolated from wheat roots by the DD technique. By homology
searches of the GenBank databases, we identified and cloned another wheat AS gene, TaASN2. The two AS genes from wheat are predicted to encode proteins of 585 and 581 amino acids, respectively, with 88.7% homology at the amino acid level. Both AS proteins have conservative amino acid residues involved in glutamine, ATP, and aspartate binding. Alignment analysis reveals that the deduced sequences of TaASN1 and TaASN2 polypeptides were very similar to two AS genes of barley (TaASN1 is 99% identified with HvAS1 and TaASN2 has 97.5% identity with HvAS2). It suggests that the two AS genes may be developed before barley and wheat diverged. The effects of stress on free amino acid accumulation have been frequently studied in a variety of monocots and dicots. Under severe drought stress, free amino acids, especially proline and Asn, increased markedly in soybean leaf (Fukutoku and Yamada, 1984). Salt stress also leads to an increase of Asn and other amino acids in C. blumai (Gilbert et al., 1998) and in barley seedling (Moller et al., 2003). Although it has been well documented that Asn is implicated in the response of plants to abiotic
ARTICLE IN PRESS 88 stresses, the definite function of Asn in stress response remains unclear. Results from some studies suggested that Asn may act as an ammonia detoxification production, produced when abiotic stresses lead to ammonia accumulation in plants. In Arabidopsis, NaCl and cold stress increase cellularfree ammonium and ASN2 mRNA levels in a coordinated manner supporting this notion (Wong et al., 2004). The accumulation of Asn was partially due to de novo synthesis under stressed conditions (Gilbert et al., 1998). The de novo synthesis could be the consequences of the induction of AS genes or the activities of these enzymes. Results from our experiments showed that expression of the TaASN1 gene in wheat seedlings is dramatically induced by salinity and osmotic stresses (Fig. 5), suggesting that regulation at a transcriptional level plays an important role in the activation of the AS gene under stress conditions. The up-regulation of AS genes by salt and other abiotic stresses were also reported in maize (Chevalier et al., 1996) and Arabidopsis (Wong et al., 2004). ABA is known to be involved in responses of plants to various environmental stresses, and most stress-inducible genes are induced by exogenous application of ABA. Our results showed that expression of the TaASN1 gene is also affected by ABA (Fig. 5), suggesting the induction of the TaASN1 gene by salinity and osmotic stresses may be through ABA-dependent pathways. TaASN2 expression is very low in the investigated tissues and only slightly up-regulated in root after 24 h treatment with ABA. In contrast to the complete complementation function of TaASN1, TaASN2 only slightly elevated the growth of E. coli Asn auxotroph. mRNA sequence of TaASN2 (BT009049) belongs to Genbank UniGene Cluster Ta.6223. In this cluster, 22 out 24 EST sequences were from seed embryos or development seeds and one from cold-stressed seedlings. We assume that the expression of TaASN2 may be involved in the seed embryo development. The data suggest that TaASN2 may function in different conditions when compared with TaASN1.
Acknowledgments We thank E. coli Genetic Stock Center (CGSC) for kindly providing the E. coli strains ER. This work was supported by National Specific Program for Research and Industrialization of Transgenic Plant Grant No. JY03A12.
H. Wang et al.
References Bassam BJ, Caetano-Anolles G, Gresshoff PM. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 1991;196:80–3. Boehlein SK, Walworth ES, Richards NG, Schuster SM. Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by E. coli asparagine synthetase B. J Biol Chem 1997;272: 12384–92. Boehlein SK, Walworth ES, Schuster SM. Identification of cysteine-523 in the aspartate binding site of E. coli asparagine synthetase B. Biochemistry 1997;36: 10168–77. Chevalier C, Bourgeois E, Just D, Raymond P. Metabolic regulation of asparagine synthetase gene expression in maize (Zea mays L.) root tips. Plant J 1996;9:1–11. Elberse IAM, Damme JMMv, Tienderen PHv. Plasticity of growth characteristics in wild barley (Hordeum spontaneum) in response to nutrient limitation. J Ecol 2003;91:371–82. Felton J, Michaelis S, Wright A. Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli. J Bacteriol 1980;142:221–8. Fukutoku Y, Yamada Y. Sources of proline-nitrogen in water-stressed soybean (Glycine max). II. Fate of 15N-labelled protein. Physiol Plant 1984;61:622–8. Gilbert GA, Gadush MV, Wilson C, Madore MA. Amino acid accumulation in sink and source tissues of Coleus blumei Benth. during salinity stress. J Exp Bot 1998;49:107–14. Guan KL, Dixon JE. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem 1991;192:262–7. Herrera-Rodriguez MB, Carrasco-Ballesteros S, Maldonado JM, Pineda M, Aguilar M, Perez-Vicente R. Three genes showing distinct regulatory patterns encode the asparagine synthetase of sunflower (Helianthus annuus). New Phytol 2002;155:33–45. Kingsbury R, Epstein E. Selection for salt-resistant spring wheat. Crop Sci 1984;24:310–5. Lam HM, Hsieh MH, Coruzzi G. Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana. Plant J 1998;16: 345–53. Marschner H. Mineral Nutrition of Higher Plants. London, San Diego: Academic Press; 1995. Moller MG, Taylor C, Rasmussen SK, Holm PB. Molecular cloning and characterisation of two genes encoding asparagine synthetase in barley (Hordeum vulgare L.). Biochim Biophys Acta 2003;1628:123–32. Ranieri A, Bernardi R, Lanese P, Slodatini G. Changes in free amino acid content and protein pattern of maize seedlings under water stress. Journal of Experimental Botany 1989;29:351–7. Van Heeke G, Schuster SM. Expression of human asparagine synthetase in E. coli. J Biol Chem 1989;264:5503–9.
ARTICLE IN PRESS Asparagine synthetase gene from wheat von der Kammer H, Albrecht C, Mayhaus M, Hoffmann B, Stanke G, Nitsch RM. Identification of genes regulated by muscarinic acetylcholine receptors: application of an improved and statistically comprehensive mRNA differential display technique. Nucleic Acids Res 1999;27:2211–8.
89 Wong HK, Chan HK, Coruzzi GM, Lam HM. Correlation of ASN2 gene expression with ammonium metabolism in Arabidopsis. Plant Physiol 2004;134: 332–8. Zhu JK. Plant salt tolerance. Trends Plant Sci 2001;6: 66–71.
www.elsevier.de/jplph
Asparagine synthetase gene TaASN1 from wheat is up-regulated by salt stress, osmotic stress and ABA Huabo Wanga,b, Dongcheng Liua, Jiazhu Suna, Aimin Zhanga,b, a
Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, People’s Republic of China b Graduate School of the Chinese Academy of Sciences, People’s Republic of China Received 2 June 2004; accepted 27 July 2004
KEYWORDS ABA; Asparagine synthetase; Differential display; Osmotic stress; Salt stress; TaASN1; TaASN2
Summary Differences in gene expression between salinity stressed and normally grown wheat seedlings were compared by the differential display (DD) technique. One DD-derived cDNA clone was characterized as a partial sequence of the wheat asparagine synthetase (AS) gene by sequence analysis and homology search of GenBank databases. Two AS genes of wheat, TaASN1 and TaASN2, were further isolated by the RT-PCR approach. Comparison of the deduced polypeptide of TaASN1 and TaASN2 with AS proteins from other organisms revealed several homologous regions, in particular, the conserved glutamine binding sites and Class-II Glutamine amidotransferases domain. The functionality of TaASN1 was demonstrated by complementing an Escherichia coli asparagine auxotroph. TaASN1 transcripts were detected in roots, shoots, anthers and young spikes by RT-PCR analysis. Abundance of TaASN1 mRNA in young spikes and anthers was higher than that in shoots and roots under normal growth conditions. TaASN1 was dramatically induced by salinity, osmotic stress and exogenous abscisic acid (ABA) in wheat seedlings. TaASN2 transcripts were very low in all detected tissues and conditions and were only slightly induced by ABA in roots. & 2004 Elsevier GmbH. All rights reserved.
Introduction Soil salinity limits agricultural production throughout the world. The United Nations Environment
Program estimates that approximately 20% of agricultural land and 50% of cropland in the world is salt-stressed (Zhu, 2001). As one of the most important crops, the biomass of wheat (Triticum
Abbreviations: ABA, abscisic acid; Asn, asparagine; AS, asparagine synthetase; DD, differential display; IPTG, isopropyl bthiogalactoside Corresponding author. Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, People’s Republic of China. Tel.: +86-10-64889347; fax: +86-1064854467. E-mail address: [email protected] (A. Zhang). 0176-1617/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.07.006
ARTICLE IN PRESS 82 aestivum) is reduced by over 90% in 50% seawater (250 mM NaCl) (Kingsbury and Epstein, 1984), and grain yield is reduced by 50–90% in only 50 mM NaCl (Marschner, 1995). Clearly, there is considerable scope for improvement in yield of wheat grown in a salt-stressed environment. Salt stress affects plant growth in many ways, such as the Na ionic toxicity and hyperosmotic stress, which cause plant nutritional imbalance and oxidation stress. In order to adapt to a high-salt environment, plants change gene expression patterns, metabolic activity, and ion and water transport to minimize stress damage and to reestablish ion and water homeostasis. Glutamine-dependent asparagine synthetase (AS: EC 6.3.5.4) catalyses the transfer of an amide group from glutamine to aspartate-forming asparagine (Asn) in an ATP-dependent reaction. In most plant species, AS seems to be encoded by a small gene family. Three AS genes have been identified in Arabidopsis (Lam et al., 1998) and sunflower (Herrera-Rodriguez et al., 2002), but in most species only one or two genes are known. Asn plays a prominent role in nitrogen transport and storage in plants. High concentrations of Asn can also be found in various plant tissues under stress conditions, such as mineral deficiencies, salt stress or drought (Moller et al., 2003). In barley, soybean and Coleus blumei, the Asn pool increased markedly when the plants were subjected to severe water stress or salt stress (Gilbert et al., 1998). The accumulation of Asn may be caused by stimulated synthesis, inhibited degradation of amino acids, impaired protein synthesis, and/or enhanced protein degradation (Ranieri et al., 1989), but much information about these processes is still unknown. In this paper, using a differential display (DD) technique, we isolated and characterized one saltinducible gene, TaASN1, which encodes the glutamine-dependent AS. The functional activity of TaASN1 was tested by a complementation assay. The expression patterns of TaASN1 in different tissues of wheat (shoots, roots, spikes, anthers) and different stress conditions (ABA, osmotic, salt) were also investigated.
Materials and methods Plant growth condition and NaCl stress treatments Seeds of Triticum aestivum L. cv. Keyi26 were germinated in a Petri dish, then were cultivated hydroponically in full-strength Hoagland solution (Elberse et al., 2003) in a greenhouse (16/8 h daily
H. Wang et al. light period, 25 1C temperature (night/day) and 60–70% relative humidity). Two-week-old seedlings were transferred to fresh Hoagland medium supplemented with 250 mM NaCl, 5.0% (w/v) mannitol or 20 mM abscisic acid (ABA). After exposure to the stress treatments for 3, 7 or 24 h, shoots and roots were collected in liquid nitrogen and stored at 80 1C until further processing.
Differential display The roots of NaCl-treated and untreated control seedlings were used for DD RT-PCR. Total RNA was extracted with Trizol reagent (GIBCO-BRL, USA) and was digested with DNAase I (Promega, Madison, WI). DD was performed according to the procedures described previously (von der Kammer et al., 1999) with modification. Three reverse transcription reactions per sample are set for the first strand cDNA synthesis. 2 mg RNA from roots of control and stressed seedlings was used for reverse transcription with either one-base anchor oligonucleotides HT1 (50 -TGC CGA AGC TTT TTT TTT TTA-30 ), HT2 (50 TGC CGA AGC TTT TTT TTT TTG-30 ) or HT3 (50 -TGC CGA AGC TTT TTT TTT TTC-30 ) and 200 units M-MLV reverse transcriptase (Promega) in a volume of 20 mL according to the manufacturer’s instructions. The reverse-transcription reactions were diluted fivefold, and 2 mL was initially subjected to PCR employing the corresponding one-base anchor oligonucleotide (1 mM) along with either one of the DD random primers (von der Kammer et al., 1999), 1.5 mM MgCl2, 0.2 mM dNTPs, 1 buffer, and 0.2 mL Tag DNA polymerase (TaKaRa, Dalian, China). Reactions were performed in a thermal cycler (PCR system 9700, Applied Biosystems, USA) with the following cycling conditions: one round at 94 1C for 4 min for denaturing; cooling 40 1C for 4 min for low-stringency annealing of primer; and heating 72 1C for 1 min for extension. This round was followed by 35 high-stringency cycles: 94 1C for 45 s; 60 1C for 2 min; and 72 1C for 1 min. One final step at 72 1C for 10 min was added to the last cycle. The denature loading buffer was added to PCR products and heated to 94 1C for 5 min and stored on ice prior to loading on 6% denaturing polyacrylamide DNA sequencing gel (BIO-RAD, USA). Electrophoresis was performed at a constant 85 W for 3 h and then the gel was subjected to silver staining (Bassam et al., 1991). The differential cDNA fragments were recovered from the sequencing gel and eluted. The elusions were used as templates for reamplification using the same sets of primer pairs. The reamplified fragments were
ARTICLE IN PRESS Asparagine synthetase gene from wheat purified from agarose gels and cloned into pGEM-T easy vector (Promega) for sequencing.
Cloning TaASN1 and TaASN2 Primers TaASN1F (50 -TGT TGC CGT CGA TCC AGG AAA ATG-30 ) and TaASN1R (50 -GGC AAG CAG GAC AGG ACA CCA TCA AC-30 ), TaASN2F (50 -ACG AGG CTG GCT GTA GAG GAC-30 ) and TaASN2R (50 -AAC CGT TCT TAT TTG CCT TTA GTAG-30 ) based the Genbank sequence BT009245 and BT009049, respectively, were used to amplify the cDNA of TaASN1 and TaASN2. First strand cDNA synthesis was performed on 2 mg of total RNA with M-MLV Reverse Transcriptase enzyme (Promega), and an oligo dT18 primer. The 20 mL PCR reaction mixture contained 2 mL of fivefold diluted cDNA, 0.2 mM of each primer, 1.5 mM MgCl2, and 0.2 mM dNTPs, 1 buffer and 0.2 mL high-fidelity Pyrobest polymerase (Takara). Reactions were performed in the following cycling conditions: 94 1C 5 min, then 35 cycles of 94 1C 0.5 min; 54 1C 0.5 min; and 72 1C 2 min; after cycling a final step of 72 1C 10 min. The PCR products were purified and cloned into pGEM-T easy vector (Promega) to generate pTaASN1 and pTaASN2 for sequencing.
83
RT-PCR analysis Protocols for total RNA extraction and synthesis of cDNA were described as above. The reversetranscription reactions were diluted tenfold, and 2 mL was initially subjected to PCR reaction. A fragment of a-tubulin gene (GenBank accession No.U76558) was amplified with specific primers TUBF (50 -ACC GCC AGC TCT TCC ACC CT-30 ) and TUBR (50 -TCA CTG GGG CAT AGG AGG AA-30 ) as the internal control for RT-PCR analysis. The PCR was performed under the following temperature profile: 94 1C 5 min;; then 28 cycles of 94 1C 0.5 min; 57 1C 0.5 min; and 72 1C 0.5 min, after cycling a final step of 72 1C 7 min. Specific primer pair TaASN1RTF (50 -GAG CAT CTC CCA GCA ACC ATC ATG-30 ) and TaASN1R were designed to amplify 111 bp TaASN1 fragment and TaASN2RTF (50 -CAC GGT CGC TGT GGG AGG TAG-30 ) and TaASN2R were designed to amplify a 128 bp TaASN2 fragment. The reaction is 94 1C 5 min, then 31 cycles of 94 1C 0.5 min; 57 1C 0.5 min; 72 1C 0.5 min; after cycling a final step of 72 1C 5 min. To test the specificity of the primers, RT-PCR products were cloned into pGEM-T easy vector (Promega) for sequencing. For genomic contamination tests, samples without reverse-transcription reactions were also used as control.
Complementation of an E. coli Asn auxotroph Nucleotide sequence analysis The full-length coding regions of TaASN1 and TaASN2 were obtained by amplification of plasmids pTaASN1 and pTaASN2 using the Pyrobest polymerase (Takara). Primers ASN1-E (50 -CCG GAA TTC TAA TGT GCG GCA TAC TGG CG-30 ) and ASN1R-X (50 -AGT CTA GAC AGG ACA GGA CAC CAT CAA C-30 ) were used for amplifying EcoRI-XbaI fragment of TaASN1, then cloned into EcoRI-XbaI sites of pGEX-KG to generate p-KG-TaASN1. Primers ASN2F-B (50 -GCC GGA TCC TGA GCA TGT GCG GCA TAC TAG-30 ) and ASN2R-X (50 -GAT CTA GAA TCA AGT CTC AAT GGC AAC-30 ) were used for amplifying BamHI-XbaI fragment of TaASN2 then cloned into BamHI-XbaI sites to generate p-KG-TaASN2. The resulting plasmids and the empty pGEX-KG were introduced into the E. coli Asn auxotroph strain ER (asnA, asnB, thi1, relA, spoT1) gifted from the Genetic Stock Center (New Haven, CI, USA). For growth tests, the plasmid-containing E. coli ER were grown in liquid M9 medium overnight. The cultures were diluted 1–100 in fresh M9 medium containing 100 mg/mL ampicillin and 1 mM IPTG, supplemented with100 mg/mL Asn when needed. The optical density was measured at 600 nm after 3, 6, 9, 24, 36, and 48 h of growth.
Nucleotide sequences were determined on both strands using an automatic sequencer, ABI PRISMTM 310 (Applied Biosystems, USA). Alignment analysis was performed using CLUSTALW (www.ebi.ac.uk/ clustalw) and BOXSHADE (http://www.ch.embnet.org/software/BOX-form.html). The nucleotide sequence of the TaASN1 has been deposited in the GenBank database under the accession number AY621539.
Accession numbers The sequence accession numbers for TaASN1 and the homologs shown in Figs. 2 and 3 are as follows: HvAS1, GenBank AAK49456; HvAS2, GenBank AAO39048.1; PEA ASN2, SW P19252; LOTJA ASN1, SW P49092; ASN2 LOTJA, SW P49093; At ASN3, GenBank AAC72836; TRIVS ASNS, SW O24661; SANAU ASNS, SW O24338; ASPOF ASNS, SW P31752; HUMAN ASNS, SW P08243; MOUSE ASNS, SW Q61024; YEAST ASN2, SW P49090; YEAST ASN1, SW P49089; ECOLI ASNB, SW P22106; At ASN1, SW P49078; At ASN2, GenBank AAC72837; ASPOF ASNS,
ARTICLE IN PRESS 84 SW P31752; SANAU_ASNS, SW O24338; TRIVS ASNS, SW O24661.ORYSA ASNS, SW Q43011.
Results Differential display Differential display (DD) was performed to detect changes in the transcripts of wheat roots under salt-stress conditions. Three one-base anchored oligo-dT primers (HT1, HT2 and HT3) and twenty arbitrary oligonucleotide primers (von der Kammer et al., 1999) were used for DD-PCR amplification. Of the 28 differential bands displayed on the sequencing gel, 10 were up-regulated and 18 were down-regulated by salt stress. One up-regulated fragment (Fig. 1) was further characterized. Sequence analysis revealed that this fragment was 1110 bp in length and was similar with two Genbank unannotated mRNA sequences (99% Accession No. BT009245 and 85% identify with BT009049). The BLASTX searches of GenBank databases reveal that they encode plant glutamine-dependent AS. There-
H. Wang et al. fore, we named the two gene TaASN1 and TaASN2, respectively. Based on BT009245 and BT009049, we performed RT-PCR to obtain the full-coding cDNA sequences of TaASN1 and TaASN2. There are 12 bp differences between TaASN1and BT009245, which lead to three deduced amino acid residue differences (M161–V161, V219–I 219 and V428–M428) (Fig. 2 region III). The open reading frame of the cDNA of TaASN1 encodes a 585 amino acid residue polypeptide with a calculated mass of 65.4 kD, whereas TaASN2 encodes a 581 amino acid residue polypeptide (65.0 kD). The two deduced amino acid sequences of TaASN1 and TaASN2 show an overall sequence identity of 88.7%. The conserved amino acid residues for substrate binding and catalytic activity were found in wheat AS by sequence alignment among AS from Arabidopsis, maize and E. coli (Fig. 2). The Nterminal part of the deduced amino acid sequences are characterized by the presence of a Class-II Glutamine amidotransferases domain (x-C-[GS][IV]-[LIVMFYW]-[AG]) (Van Heeke and Schuster, 1989), indicated by asterisks in Fig. 2, region I. Essential residues for binding of aspartate and ATP, T316, T317, R319 and C523 (Boehlein et al., 1997a, b), are also conserved in both TaASN1 and TaASN2. The deduced polypeptide sequences of TaASN1 and TaASN2 were aligned with AS amino acid sequences from plants, yeast, mammals., and E. coli to create a dendrogram (Fig. 3) that divides the plant AS into two groups. Both TaASN1 and TaASN2 belongs to class I, the majority of plant AS peptides, and closed to AS from Hordeum vulgare HvAS1 and HvAS2.
Functional analysis of TaASN1 by complementation of an E. coli Asn auxotroph
Figure 1. Analysis of mRNA in wheat root used by DD RTPCR. Lanes 1–3 indicate control, salt treated for 3 h, salt treated for 24 h, respectively. The arrow indicates the amplified fragment of TaASN1.
For the instability of the AS, the functional activities of TaASN1 and TaASN2 were tested by complementation of an E. coli Asn auxotroph. The regions encoding the proteins TaASN1 and TaASN2 were inserted inframe in the pGEX-KG expression vector (Guan and Dixon, 1991). The new constructs (pGEX-KG-TaASN1 and pGEX-KG-TaASN2) were transformed into the E. coli auxotroph ER strain (asnA, asnB, thi-1, relA, spoT1) lacking AS activity (Felton et al., 1980). As expected, growth of E. coli ER transformed with the empty vector was very poor when cultured in a medium without Asn. Higher growth levels were obtained in the same medium when the ER strain was transformed with pGEX-KG-TaASN1, but the growth level was not significantly elevated with pGEX-KG-TaASN2 (Fig. 4).
ARTICLE IN PRESS Asparagine synthetase gene from wheat
85
Figure 2. Alignment of the amino acid sequences of TaASN1, TaASN2, AtASN1, MAIZE_ASNS and ECOLI_ASNB. Multiple sequence alignment was determined using the ClustalW program and residues were shaded using BoxShade 3.21. Identical residues are shown on a black background, and conservative substitutions are shown on a gray background. I, Class-II Glutamine amidotransferases domain; II, aspartate and ATP binding sites; III, different amino acids residues between TaASN1 and BT009245.
ARTICLE IN PRESS 86
H. Wang et al.
Figure 3. Phylogenetic distances among 22 different AS proteins shown in a dendogram constructed following the ClustalW method for multiple sequence comparison. The AS proteins from plant are separated into two groups (class I and class II), where the TaASN1 and TaASN2 from wheat belong to class I as the majority of AS peptides. Only the ASN2 and ASN3 from Arabidopsis, the rice, the maize and belong to class II. At, A. thaliana; Os, Oryza sativa; Hv, H. vulgare; LOTJA, Lotus japonicus; ASPOF, Asparagus officinalis; SANAU, Sandersonia aurantiaca; TRIVS, Triphysaria versicolor.
Expression and regulation of AS genes in wheat 4
3
A600
To study expression patterns of TaASN1 and TaASN2 in wheat, total RNA was extracted from shoots and roots of two-week-old seedlings, young spikes and anthers and then subjected to RT-PCR analysis using the specific primers (considering that the high homology between TaASN1 and TaASN2, the specificity of the primers to TaASN1 and TaASN2 was determined by sequencing of RT-PCR products). Abundance of TaASN1 mRNA in young spikes and anthers was higher than that in shoots and roots (Fig. 5A) under normal growth conditions. To determine the effects of various environmental stresses on the expression of TaASN1 and TaASN2 in wheat seedlings, a time-course experiment was performed. Salinity stress caused a
p-KG-TaASN1 -ASN p-KG-TaASN1 +ASN p-KG-TaASN2 -ASN p-KG-TaASN2 +ASN pGEX-KG -ASN pGEX-KG +ASN
2
1
Figure 4. Complementation of an E. coli asparagine auxotroph. The auxotroph strain ER was transformed with the plasmid p-KG-TaASN1 (dot), p-KG-TaASN2 (triangle) or pGEX-KG (square). The transformants were cultured at 28 C in M9 minimum medium containing ampicillin (100 mg/mL) and 0.1 mM IPTG, with asparagine (open symbols) or without asparagine (filled symbols). Three independent experiments were performed.
0 0HR
3HR
6HR
9HR
Time
24HR
36HR
48HR
ARTICLE IN PRESS Asparagine synthetase gene from wheat
87
Figure 5. RT-PCR analysis of TaASN1 and TaASN2 expression. Expression pattern of TaPdxK in different tissues (root, shoot, spike and anther) of T. aestivum (A.). Time course experiment of TaASN1 and TaASN2 expression pattern in the roots (B) and shoots (C) wheat seedlings under 250 mM NaCl 5.0% (w/v) mannitol and 20 mM ABA.
rapid accumulation of the TaASN1 transcript both in shoots and roots, and its level reached a maximum after 3 h, and then kept the level (Fig. 5A). Under osmotic stress condition, its mRNA level in shoots increased with stressed time, but in roots its expression rapidly reached a peak after 3 h, then declined to an unstressed level at 24 h. ABA is implicated in plant stress responses. Exogenous application of ABA also up-regulated the expression of TaASN1, which in roots accumulated more rapidly than in shoots. The expression of TaASN2 is different from TaASN1, which is only significantly up-regulated by ABA in roots at 24 h.
Discussion The glutamine-dependent AS enzyme is now generally accepted as the major route for Asn biosynthesis in plants. In the present study, a cDNA-encoded AS, TaASN1, was isolated from wheat roots by the DD technique. By homology
searches of the GenBank databases, we identified and cloned another wheat AS gene, TaASN2. The two AS genes from wheat are predicted to encode proteins of 585 and 581 amino acids, respectively, with 88.7% homology at the amino acid level. Both AS proteins have conservative amino acid residues involved in glutamine, ATP, and aspartate binding. Alignment analysis reveals that the deduced sequences of TaASN1 and TaASN2 polypeptides were very similar to two AS genes of barley (TaASN1 is 99% identified with HvAS1 and TaASN2 has 97.5% identity with HvAS2). It suggests that the two AS genes may be developed before barley and wheat diverged. The effects of stress on free amino acid accumulation have been frequently studied in a variety of monocots and dicots. Under severe drought stress, free amino acids, especially proline and Asn, increased markedly in soybean leaf (Fukutoku and Yamada, 1984). Salt stress also leads to an increase of Asn and other amino acids in C. blumai (Gilbert et al., 1998) and in barley seedling (Moller et al., 2003). Although it has been well documented that Asn is implicated in the response of plants to abiotic
ARTICLE IN PRESS 88 stresses, the definite function of Asn in stress response remains unclear. Results from some studies suggested that Asn may act as an ammonia detoxification production, produced when abiotic stresses lead to ammonia accumulation in plants. In Arabidopsis, NaCl and cold stress increase cellularfree ammonium and ASN2 mRNA levels in a coordinated manner supporting this notion (Wong et al., 2004). The accumulation of Asn was partially due to de novo synthesis under stressed conditions (Gilbert et al., 1998). The de novo synthesis could be the consequences of the induction of AS genes or the activities of these enzymes. Results from our experiments showed that expression of the TaASN1 gene in wheat seedlings is dramatically induced by salinity and osmotic stresses (Fig. 5), suggesting that regulation at a transcriptional level plays an important role in the activation of the AS gene under stress conditions. The up-regulation of AS genes by salt and other abiotic stresses were also reported in maize (Chevalier et al., 1996) and Arabidopsis (Wong et al., 2004). ABA is known to be involved in responses of plants to various environmental stresses, and most stress-inducible genes are induced by exogenous application of ABA. Our results showed that expression of the TaASN1 gene is also affected by ABA (Fig. 5), suggesting the induction of the TaASN1 gene by salinity and osmotic stresses may be through ABA-dependent pathways. TaASN2 expression is very low in the investigated tissues and only slightly up-regulated in root after 24 h treatment with ABA. In contrast to the complete complementation function of TaASN1, TaASN2 only slightly elevated the growth of E. coli Asn auxotroph. mRNA sequence of TaASN2 (BT009049) belongs to Genbank UniGene Cluster Ta.6223. In this cluster, 22 out 24 EST sequences were from seed embryos or development seeds and one from cold-stressed seedlings. We assume that the expression of TaASN2 may be involved in the seed embryo development. The data suggest that TaASN2 may function in different conditions when compared with TaASN1.
Acknowledgments We thank E. coli Genetic Stock Center (CGSC) for kindly providing the E. coli strains ER. This work was supported by National Specific Program for Research and Industrialization of Transgenic Plant Grant No. JY03A12.
H. Wang et al.
References Bassam BJ, Caetano-Anolles G, Gresshoff PM. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 1991;196:80–3. Boehlein SK, Walworth ES, Richards NG, Schuster SM. Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by E. coli asparagine synthetase B. J Biol Chem 1997;272: 12384–92. Boehlein SK, Walworth ES, Schuster SM. Identification of cysteine-523 in the aspartate binding site of E. coli asparagine synthetase B. Biochemistry 1997;36: 10168–77. Chevalier C, Bourgeois E, Just D, Raymond P. Metabolic regulation of asparagine synthetase gene expression in maize (Zea mays L.) root tips. Plant J 1996;9:1–11. Elberse IAM, Damme JMMv, Tienderen PHv. Plasticity of growth characteristics in wild barley (Hordeum spontaneum) in response to nutrient limitation. J Ecol 2003;91:371–82. Felton J, Michaelis S, Wright A. Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli. J Bacteriol 1980;142:221–8. Fukutoku Y, Yamada Y. Sources of proline-nitrogen in water-stressed soybean (Glycine max). II. Fate of 15N-labelled protein. Physiol Plant 1984;61:622–8. Gilbert GA, Gadush MV, Wilson C, Madore MA. Amino acid accumulation in sink and source tissues of Coleus blumei Benth. during salinity stress. J Exp Bot 1998;49:107–14. Guan KL, Dixon JE. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem 1991;192:262–7. Herrera-Rodriguez MB, Carrasco-Ballesteros S, Maldonado JM, Pineda M, Aguilar M, Perez-Vicente R. Three genes showing distinct regulatory patterns encode the asparagine synthetase of sunflower (Helianthus annuus). New Phytol 2002;155:33–45. Kingsbury R, Epstein E. Selection for salt-resistant spring wheat. Crop Sci 1984;24:310–5. Lam HM, Hsieh MH, Coruzzi G. Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana. Plant J 1998;16: 345–53. Marschner H. Mineral Nutrition of Higher Plants. London, San Diego: Academic Press; 1995. Moller MG, Taylor C, Rasmussen SK, Holm PB. Molecular cloning and characterisation of two genes encoding asparagine synthetase in barley (Hordeum vulgare L.). Biochim Biophys Acta 2003;1628:123–32. Ranieri A, Bernardi R, Lanese P, Slodatini G. Changes in free amino acid content and protein pattern of maize seedlings under water stress. Journal of Experimental Botany 1989;29:351–7. Van Heeke G, Schuster SM. Expression of human asparagine synthetase in E. coli. J Biol Chem 1989;264:5503–9.
ARTICLE IN PRESS Asparagine synthetase gene from wheat von der Kammer H, Albrecht C, Mayhaus M, Hoffmann B, Stanke G, Nitsch RM. Identification of genes regulated by muscarinic acetylcholine receptors: application of an improved and statistically comprehensive mRNA differential display technique. Nucleic Acids Res 1999;27:2211–8.
89 Wong HK, Chan HK, Coruzzi GM, Lam HM. Correlation of ASN2 gene expression with ammonium metabolism in Arabidopsis. Plant Physiol 2004;134: 332–8. Zhu JK. Plant salt tolerance. Trends Plant Sci 2001;6: 66–71.