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Journal of Experimental Botany, Vol. 54, No. 387, pp. 1545-1551,
June 1, 2003
© 2003 Oxford University Press
Molecular cloning of the cDNA encoding aspartate aminotransferase from bean root nodules and determination of its role in nodule nitrogen metabolism
Received 10 December 2002; Accepted 27 February 2003
Centro de Investigación sobre Fijación de Nitrógeno, Universidad Nacional Autónoma de México, Apartado Postal 565-A, Cuernavaca, Morelos, México
1 To whom correspondence should be addressed. Fax: +52 777 317 5581. E-mail: lara{at}cifn.unam.mx
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Abstract |
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A cDNA clone encoding aspartate aminotransferase (PVAAT-2) (EC 2.6.1.1 [EC] ) was isolated from the common bean Phaseolus vulgaris nodule cDNA library. The nucleotide sequence analysis of the full-length cDNA allowed its identification by comparison with sequence databases. The amino acid sequence of the bean PvAAT-2 showed high similarity with the AAT-2 isoforms described in other leguminous plants. The amino-terminal region of the PvAAT-2 contains a sequence, which shares common features of plastid transit peptides. Southern blot analysis showed that the PvAAT-2 clone is encoded by a single gene in the P. vulgaris genome. Analysis of the PvAAT-2 mRNA levels suggests that the expression of this gene is nodule enhanced. The PvAAT-2 transcript is more abundant in nodules with increased synthesis of amides and is down-regulated in conditions where ureides accumulate. When plants were supplemented with ureides or with amides, PvAAT-2 expression was reduced, while it was not affected when plants were treated with allopurinol, an inhibitor of ureide synthesis. On the other hand, the expression of asparagine synthetase (another enzyme involved in the synthesis of amides) is not affected either by ureides or amides. These data suggest a role for AAT-2 in the mechanism involved in the synthesis of nitrogen compounds in bean nodules.
Key words: Aspartate aminotransferase, bean, cDNA, molecular cloning, nodules, Phaseolus vulgaris.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Aspartate aminotransferase (AAT) plays an important role in nitrogen and carbon metabolism, particularly in legume root nodules and leaves of C4 species. It catalyses the formation of 2-oxoglutarate and aspartate via reversible amino group transfer from glutamate to oxalacetate (Givan, 1980). Several functions have been attributed to AAT. These include catabolism and biosynthesis of aspartate (Bryan, 1980), conversion of tricarboxylic acid cycle intermediates to amino acids (Ryan and Fottrell, 1974) and the assimilation of fixed nitrogen into asparagine in amide exporting nodules (Farnham et al., 1990). This enzyme is also believed to control the redistribution of nitrogen and carbon pools between plant cell cytoplasm and other compartments, and between microbial symbiont and host cytoplasm, as has been proposed in models involving a metabolic shuttle system, such as a malate–aspartate shuttle (Appels and Haaker, 1991; Wallsgrove et al., 1983).
AAT from several legume and non-legume species has been characterized and shown to be a dimeric enzyme composed of two subunits of approximately 40 kDa.
Two isoforms of AAT (AAT-1 and AAT-2) have been described in legumes such as soybean (Ryan et al., 1972), lupin (Reynolds and Farnden, 1979; Reynolds et al., 1992), and alfalfa nodules (Griffith and Vance, 1989). They differ in their kinetic properties and expression characteristics throughout nodule development. The plastidic AAT-2 form was shown to be responsible for the nodule-located activity (Vance et al., 1994).
AATs are present in all organisms usually as multiple isozymes, which are tissue or organelle specific (Cooper and Meister, 1985; Jensen and Calhoun, 1981). Individual AAT isoenzymes respond differently to environmental conditions, and to C and N compounds suggesting that they have specific and non-overlapping functions (Schultz et al., 1998; Taniguchi et al., 1995).
cDNA and genomic clones encoding AAT have been isolated from legumes such as alfalfa (Gantt et al., 1992; Gregerson et al., 1993, 1994; Udvardi and Kahn, 1991), lupin (Reynolds et al., 1992; Winifield et al., 1994) and soybean (Wadsworth et al., 1993, 1994) as well as from non-legumes such as Arabidopsis (Schultz and Coruzzi, 1995; Wilkie et al., 1995), carrot (Turano et al., 1992) and Panicum (Taniguchi et al., 1992).
Although the role of AAT in legumes that synthesize and transport amides is clear, less is known about the role of this enzyme in the nodules that export ureides. In order to clarify the role of AAT in ureide transporting nodules, a cDNA encoding the nodule enhanced AAT (PvAAT-2) has been isolated from common bean (Phaseolus vulgaris) and the regulation of its expression in response to various nitrogen compounds that are synthesized and transported in N2-fixing nodules has been investigated.
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Materials and methods |
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Plant growth and treatments
Bean (Phaseolus vulgaris L. cv. Negro Jamapa) seeds were surface-sterilized in 10% (v/v) commercial sodium hypochlorite for 10 min and then rinsed several times with sterile distilled water. The seeds were placed on moist paper towels in trays and germinated in the dark. Three days post-imbibition, the seedlings were sown in vermiculite pots and inoculated with Rhizobium etli CE3 and CFN037 strains. This treatment allowed the selection of plants of the same size and a homogeneous nodule sample to be obtained. Plants were grown in a greenhouse under natural light. Plants were watered daily and, in addition, irrigated with N-free Hoagland solution twice a week.
When the plants were 17-d-old, one-half of the plants received no irrigation (drought plants), while the remaining plants received water continuously (control plants) for a further period of 4 d.
Experimental treatments to assess the effects of different N compounds in AAT-2 regulation were imposed on 17-d-old plants. The potted plants were grouped randomly into six treatment sets and each pot was irrigated daily with 200 ml Hoagland solution containing asparagine (10 mM), glutamine (10 mM), allantoin (10 mM), xanthine (10 mM) or allopurinol (2 mM) for 4 d. Control plants were supplemented only with Hoagland solution. Nodules for RNA extraction were harvested, immediately frozen in liquid nitrogen and stored at –80 °C until used.
Assay of nitrogenase activity
Nitrogenase activity was measured by acetylene reduction assay as described by Dart et al. (1972).
Analysis of ureides in xylem exudates and plant tissues
Xylem exudates were collected for 1 h at midday from the cut stems of the plants, the volume was determined and the sap was stored at –20 °C as reported by Silvente et al. (2002). After collecting the xylem sap, nodules and leaves were also collected for the determination of ureides. Plant tissues were ground in liquid nitrogen, extracted in 0.4 M potassium phosphate buffer (pH 7.4) and the concentration of ureides was measured using the differential analysis method of Vogels and Van der Drifft (1970).
RNA isolation and northern hybridization
Total RNA was isolated from 0.2 g of frozen cotyledons, stem, leaf, root, and nodule tissues using Trizol (Gibco BRL Life Technologies, Inc., Grand Island, NY) and 20 µg was electrophoresed on a 1.2% formaldehyde agarose gel and blotted onto a nylon membrane (Hybond-N+, Amersham Life Sciences, UK) according to the manufacturer’s instructions, without blocking reagent. After 30 min prehybridization in a solution containing 0.3 M NaH2PO4, pH 7.2, 10 mM EDTA, and 7% (w/v) SDS, the blot was hybridized for 24 h at 65 °C with a 32P-labelled AAT-2 probe. After stringency washing, radioactive membranes were exposed to X-Ray film (Kodak) overnight at –80 °C.
Isolation of the P. vulgaris AAT-2 cDNA clone
A nodule-specific 
Zap cDNA library from Phaseolus vulgaris root nodules was screened for full-length AAT clones. The library was probed with an AAT-2 specific 675 bp fragment amplified by PCR with a forward primer (5'-GGCATACCTATGG CTCCTCCT) and a reverse primer (5'-GAAGCTGCATCTTCATCAAG) derived from a conserved portion of AAT sequences deposited in DNA databases. The AAT-2 cDNA inserts from positive clones were subcloned into pBluescript SK+ plasmid (Stratagene, La Jolla, USA) and transformed into the ‘SOLR’ E. coli strain (Stratagene). A clone containing the longest insert was sequenced (Medigenomix, Germany), and was found to contain the entire AAT-2 coding region based on sequence analysis using the GCG software package. The sequence of the cDNA clone was deposited in the Genbank under accession no. AF315376
[GenBank]
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Plant DNA extraction and Southern analysis
Genomic DNA was isolated from leaves of 5-d-old seedlings using a DNA isolation kit (GENTRA Systems, Minneapolis, MN). For Southern blot analysis, genomic DNA was digested with the indicated enzymes, separated on a 0.8% agarose gel (20 µg lane–1) and transferred to Hybond-N+ filters (Amersham, Life Sciences, UK). The gene-specific DNA fragment from the entire coding region of AAT-2 cDNA was labelled with 32P by random priming (multiprime DNA-labelling system kit, Amersham). The blot was prehybridized at 65 °C for 30 min in a solution of 0.3 M NaH2PO4, pH 7.2, 10 mM EDTA, and 7% (w/v) SDS. Hybridization was carried out in the same buffer at 65 °C for 24 h. The hybridized filter was washed with 2, 1, and 0.1x SSC with 0.1% (w/v) SDS at 65 °C for 30 min each, and exposed to Kodak X Omat film.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Isolation and characterization of a cDNA clone encoding AAT-2
Using an AAT-specific probe, several plaques were isolated from a bean nodule cDNA library. One clone with about 1.8 kb insert was selected, sequenced and designated as AAT-2 because of the close identity with AAT-2 of other legumes. The nucleotide sequence of the AAT-2 clone (Fig. 1) contained an open reading frame of 1386 bp flanked by 173 bp and 338 bp at the untranslated 5' and 3' regions, respectively. A sequence (AACAAA) with homology to the putative adenylation sequence of plants (Joshi, 1987) was found 72 bp upstream of the poly A residue.
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The deduced amino acid sequence of the bean AAT-2 showed higher identity (87–91%) with legume AAT-2 (Fig. 2) than with non-legume AAT (55% Arabidopsis thaliana and 82% Panicum milaceum). The deduced protein sequence of AAT-2 showed 55% and 56% identity with the putative cytosolic isozyme AAT-1 from alfalfa and AAT-P1 from lupin, respectively.
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A sequence of about 57 amino acids at the N terminal region could represent a leader sequence of the AAT-2 protein. This leader region with 22% of serine residues agrees with the observation that the hydrophobic nature and high serine content are important characteristics for plastid transport peptides (Keegstra et al., 1989). Analysis of this sequence by the PSORT (Predicted Protein Sorting Signals Coded in Amino Acid Sequences) corroborates that this sequence is a putative plastid transport peptide.
Gene copy number
Southern analysis was performed in order to elucidate the presence of different isoforms of AAT-2 in bean genome. When the blot was hybridized with the 32P-labelled PvAAT-2 cDNA probe containing the entire coding sequence, only one band was observed with genomic DNA when digested with EcoRV, XmaI, XbaI, and PstI restriction endonucleases, while two bands were seen with HindIII (Fig. 3). The appearance of two bands when digested with HindIII is due to the presence of a HindIII restriction site within the gene. The banding pattern obtained with the EcoRV, XmaI, XbaI, and PstI digests clearly suggests the presence of a single copy of PvAAT-2 gene in the bean genome.
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PvAAT-2 expression and synthesis of nitrogen compounds in bean nodules
RNA gel blot analysis was performed using total RNA extracted from the leaf, stem, cotyledon, root, and nodule tissues. As shown in the Fig. 4A and B, AAT-2 transcript was detected almost exclusively in nodules, although a weak expression was observed in young leaves. This indicated that PvAAT-2 is a nodule-enhanced gene. In order to explore the role of AAT-2 in the synthesis and transport of nitrogen compounds, expression of PvAAT-2 during symbiosis was investigated in R. etli CE3 (wild-type strain) and in R. etli CFN037 (mutant strain) induced nodules, which were shown to accumulate predominantly ureide or amide compounds, respectively (Silvente et al., 2002). In the CFN037 nodules, the PvAAT-2 transcript was expressed maximally between 11–21 d after inoculation (DAI), and at higher levels than in the CE3 nodules, except at 13 d. The duration of the expression of AAT-2 in CFN037 nodules coincided with the period of high amide synthesis (Silvente et al., 2002), and this suggests that AAT-2 is involved in the regulation of the synthesis and transport of nitrogen compounds, mainly ureides or amides, which are synthesized in bean nodules.
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Relationship between the acetylene reduction assay, AAT-2 and ureide concentration
The decline in ARA activity following drought treatment was shown to coincide with an increased ureide concentration in the shoots (de Silva et al., 1996; Purcell et al., 1998), which was suggested to trigger the accumulation of an intermediate compound (Serraj et al., 1999) leading to a feedback inhibition of N2 fixation. In order to know if this increase in ureide concentration during drought would affect the PvAAT-2 expression, plants were grown without irrigation for 4 d and analysed for the expression of PvAAT-2. As shown in Fig. 5, water deficit reduced ARA by about 22% compared to the control plants, and simultaneously resulted in a slight increase in ureide concentration in nodules and a 4-fold increase in young leaves. Concomitantly with these results, PvAAT transcript abundance decreased, while the expression of asparagine synthetase (PvAS-2) remained unaffected and uricase increased during the drought conditions (Fig. 5).
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Regulation of the nodule PvAAT-2 expression by nitrogen compounds
In order to know if the reduction in PvAAT-2 expression during drought is mediated by ureides or some other nitrogen compounds, and to assess the relationship between ureide concentration and the PvAAT-2 expression, plants were irrigated for 4 d with different nitrogenous compounds, and with allopurinol, which is an inhibitor of ureide synthesis. As is shown in Fig. 6, the expression levels of the AAT-2 mRNA was down-regulated with asparagine, glutamine and allantoin in a similar manner as with drought. Xanthine, a precursor for the synthesis of ureides, also inhibited the expression of PvAAT-2 transcripts. However, allopurinol had no negative effect on the expression of PvAAT-2 mRNA. By contrast, the expression of (PvAS-2) was up-regulated in all the conditions tested.
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Discussion |
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In alfalfa (Udvardi and Kahn, 1991; Gantt et al., 1992) and lupin (Winifield et al., 1994; Reynolds et al., 1992) two distinct AAT genes, a cytosolic form and a plastidic form called AAT1 and AAT-2, have been identified. the isolation, cloning and characterization of a nodule-enhanced aspartate aminotransferase cDNA from developing root nodules of bean is reported here. This nodule-enhanced PvAAT-2 is highly homologous to the plastid form (AAT-2) at the nucleotide levels within the coding region (90% and 85%). In addition, the presence of a putative plastid targeting sequence at the N-terminal suggests that bean AAT-2 may be localized in the plastid, like AAT-2 and AAT-P2 of alfalfa and lupin, respectively (Gantt et al., 1992; Reynolds et al., 1992). This transit sequence shared some characteristics with known plastid transit peptides, such as high serine content, and hydrophobic nature (Keegstra et al., 1989).
Genomic southern analysis revealed a single band with most of the restriction enzymes tested indicating that the PvAAT-2 gene is present as a single copy in the bean genome. Northern blot analysis revealed that PvAAT-2 is a nodule-enhanced gene, which is expressed predominantly in the nodules.
The higher and persistent PvAAT-2 mRNA levels in the nodules induced by R. etli CFN037, which transport less ureides and more amides than the ureide producing nodules induced by the wild-type R. etli CE3 strain (Silvente et al., 2002), suggest that the regulation mechanisms of PvAAT-2 of bean may be under the control of nitrogen compounds that are synthesized and transported in nodules. Amide transporter legumes (where AAT-2 is highly expressed) were shown to have N2 fixation that is relatively drought tolerant (Sinclair and Serraj, 1995). It has been proposed that changes in the glutamine content in the phloem sap (Neo and Layzell, 1977) and an increase in the ureide concentration in the plant as a result of water deficit (Serraj et al., 1999) may trigger the inhibition of nodule metabolism and nitrogenase activity.
To characterize the role of bean AAT-2 in ureide transport, its expression under water deficit conditions was examined. During water stress, a decline in ARA activity was accompanied by a sharp decline in PvAAT-2 transcript levels and a concomitant increase in ureide content. This appears to be specific for PvAAT-2, because mRNA levels of PvAS2 and uricase were much less affected by drought. These results argue for an inhibitory role of ureides in the regulation of the expression of PvAAT-2 in bean. These results support this view, not only because ureides (allantoin) reduced PvAAT-2 expression but also because, in the presence of allopurinol which is an inhibitor of ureide synthesis, PvAAT-2 expression is not affected. In addition, amides (Asn and Gln) reduced the expression of PvAAT-2 in nodules. It is interesting to note that although AS and AAT genes code for the enzymes belonging to a common metabolic pathway, their expression is regulated very differently in all the conditions tested (Fig. 6). The putative localization of AAT-2 in plastids where purine and asparagine synthesis take place (Boland et al, 1982), the higher expression of PvAAT-2 in nodules that produce more amides than ureides, and the negative effect of ureides on the expression of PvAAT-2, suggest that AAT-2, but not AS, may be acting as an important switching enzyme in driving the metabolic flow of nitrogen through amide or ureide synthesis in bean nodules.
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Acknowledgements |
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We are grateful to Dr Pallavolu M Reddy for critically reviewing the manuscript and MC Lourdes Blanco for providing the
Zap and gt-22a libraries. We also thank A Sánchez for technical support in the greenhouse and P Gaytan and E López for oligonucleotide synthesis. This project was supported partially by the Consejo Nacional de Ciencia y Tecnología (CONACyT) grant G31751
[GenBank]
-B.
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